Integrin-linked kinase and its use

Methods for isolating ILK genes are provided. The ILK nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as identification of cell type based on expression, and the like.

INTRODUCTION 
BACKGROUND 
Proteins of the extracellular matrix (ECM) act to influence fundamental 
cell and tissue behaviors. ECM regulates cell structure, growth, survival, 
differentiation, motility and, at the organismal level, proper 
development. ECM proteins interact with cells via a class of cell membrane 
spanning receptors called integrins. ECM acts as a biological signal, 
where the integrin receptor is a specific transducer across the cell's 
plasma membrane of this signal. Integrins are also important in 
proliferative disorders, mediating such processes as wound healing and 
inflammation, angiogenesis, as well as tumor migration and invasion. 
A major biochemical response to ECM integrin interactions is elevation of 
an enzymatic activity known as protein phosphorylation. Phosphorylation is 
important in signal transduction mediated by receptors for extracellular 
biological signals such as growth factors or hormones. For example, many 
cancer causing genes (oncogenes) are protein kinases, enzymes that 
catalyze protein phosphorylation reactions, or are specifically regulated 
by phosphorylation. In addition, a kinase can have its activity regulated 
by one or more distinct protein kinases, resulting in specific signaling 
cascades. 
Research on signal transduction over the years has clearly established the 
importance of direct, protein-protein interactions in the cytoplasm as a 
major mechanism underlying the specification of signaling pathways. These 
interactions can, in part, be those between a receptor and a cytoplasmic 
protein kinase, or between a protein kinase and its substrate molecule(s). 
A number of known protein kinases, such as mitogen-activated kinase (MAPK), 
focal adhesion kinase (FAK), and protein kinase C (PKC), have their kinase 
activity stimulated by integrin-ECM interaction. For example, see Maguire 
et al. (1995) J Exp Med 182:2079-2090; Richardson and Parsons (1995) 
Bioessays 17:229-236; Morino et al. (1995) J. Biol. Chem. 270:269-273; and 
Nojima et al. (1995) J Biol Chem 270:15398-15402. However, no cellular 
protein kinase has been identified to date that has been demonstrated to 
bind to an integrin molecule under physiological conditions. As such is 
the case, the direct molecular connection between integrins and the 
ECM-induced phosphorylation of cellular proteins is unclear. As such is 
the case, if the direct molecular connection between integrins and the 
ECM-induced phosphorylation of cellular proteins were determined, products 
which modulated that connection would be useful therapeutics. These 
products could be used to modulate cell growth, cell adhesion, cell 
migration and cell invasion. 
It is known that kinases can form complex signaling cascades, where the 
activation of one kinase causes it to activate or de-activate another 
kinase, and so forth through several iterations. One advantage to this 
type of pathway is that a single "second messenger" can affect a number of 
different processes, depending on the specific kinase expression pattern 
in a cell. A particularly interesting second messenger in this respect is 
phosphatidylinositol 3,4,5 triphosphate [Ptdlns(3,4,5)P.sub.3 ]. 
[Ptdlns(3,4,5)P.sub.3 ] acts on pathways that control cell proliferation, 
cell survival and metabolic changes--often through protein kinases. This 
lipid can be produced by PI3 kinases, a family of related proteins 
(Vanhaesebroeck et al. (1997) TIBS 22:267; Toker and Cantley (1997) Nature 
387:673676). One downstream effector is protein kinase B (PKB/AKT) 
(Downward (1998) Science 279:673-674). PKB contains a pleckstrin homology 
(PH) domain, to which the [Ptdlns(3,4,5)P.sub.3 ] signaling molecule 
binds. In addition, PKB itself is phosphorylated when 
[Ptdlns(3,4,5)P.sub.3 ] is present, by two different protein kinases, one 
of which has been cloned (Stephens et al. (1998) Science 279:710-714; 
Alessi et al. (1997) Curr. Biol. 7:776). The molecular identity of the 
other kinase has not previously been established. The determination of 
this kinase, as well as its substrates and modulators, is of great 
interest for providing a point of intervention in this pathway. 
If it were determined that a specific kinase regulates integrin function, 
products that regulate the activity of that kinase could be used for the 
treatment of cancer, leukemia, solid tumors, chronic inflammatory disease, 
restenosis, diabetes, neurological disorders, arthritis and osteoporosis, 
among other indications. 
Relevant Literature 
A review of integrin mediated signal transduction in oncogenesis may be 
found in Dedhar(1995) Cancer Metastasis Rev 14:165-172. Hannigan et al. 
(1995) 86th Annual Meeting of the American Institute for Cancer Research, 
provide a brief abstract directed to the cloning of a novel protein kinase 
associated with betal integrin cytoplasmic tails. Hannigan et al. (1995) 
Molecular Biology of the Cell suppl. 6, p. 2244, is an abstract directed 
to the effect of overexpression of a novel integrin linked kinase (ILK) in 
induction of a transformed phenotype and cyclin D1 expression. Rosales et 
al. (1995) Biochim Biophys Acta 1242:77-98 reviews signal transduction by 
cell adhesion receptors. Signaling by cell adhesion receptors may, involve 
aspects that impinge on previously known signaling pathways including the 
RTK/Ras pathway and serpentine receptor/G protein pathways. A possible 
signaling role for the Syk tyrosine kinase is described in Lin et al. 
(1995) J Biol Chem 270:16189-16197. 
Miyamoto et al. (1995) Science 267:883-885 compare the roles of receptor 
occupancy and aggregation on integrin receptor mediation of cell adhesion, 
signal transduction, and cytoskeletal organization. An EST sequence is 
provided by EMBL sequence DNA library accession no. p H70160, the Wash. 
U.--Merck EST project. 
The sequences of a number of kinases are known in the art, including human 
protein kinase B (Coffer and Woodgett(1991) Eur. J. Biochem. 201:475-481). 
PI3 kinases have been characterized, including phosphatidylinositol 
3-kinase gamma polypeptide, (OMIM 601232); phosphatidylinositol 3-kinase 
alpha polypeptide (OMIM 171834); phosphatidylinositol 3-kinase regulatory 
subunit (OMIM 171833); mouse PI3 kinase (Genbank M60651); rat PI3 kinase 
(Genbank D78486, D64045). Glycogen synthase kinase 3 sequences can be 
accessed at Genbank; the human cDNA sequence has the accession number 
L40027. 
SUMMARY OF THE INVENTION 
Isolated nucleotide compositions and sequences are provided for integrin 
linked kinase (ILK) genes. The ILK nucleic acid compositionsfind use in 
identifying homologous or related genes; for production of the encoded 
kinase; in producing compositions that modulate the expression or function 
of its encoded protein; for gene therapy; mapping functional regions of 
the protein; and in studying associated physiological pathways. In 
addition, modulation of the gene activity in vivo is used for prophylactic 
and therapeutic purposes, such as treatment of cancer, identification of 
cell type based on expression, and the like.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Nucleic acid compositions encoding integrin linked kinase (ILK) are 
provided. They are used in identifying homologous or related genes; in 
producing compositions that modulate the expression or function of its 
encoded protein; for gene therapy; mapping functional regions of the 
protein; and in studying associated physiological pathways. The ILK gene 
product (herein p59ILK) is a serine threonine kinase having two functional 
domains, identified by comparison of the ILK sequence against those found 
in current protein databases. These are the catalytic domain, responsible 
for phosphotransferase activity (kinase domain), and a non-overlapping 
domain in the amino terminus, comprised of four contiguous ankyrin-like 
repeats. 
Modulation of ILK gene activity in vivo is used for prophylactic and 
therapeutic purposes, such as treatment of cancer, investigation of 
integrin signaling pathway function, identification of cell type based on 
expression, and the like. The protein is useful as an immunogen for 
producing specific antibodies, in screening for biologically active agents 
that act in the integrin signaling pathway and for therapeutic and 
prophylactic purposes. 
The present invention demonstrates a physical linkage between integrin and 
ILK. Dysregulated expression of ILK protein modulates the function of 
integrins, thus providing a biological link between ILK and integrin. 
Dysregulated expression of ILK modulates cell growth, cell adhesion, cell 
migration and cell invasion. Hence, products that modulate the expression 
and/or activity of ILK have a therapeutic effect in the treatment of 
cancer, leukemia, solid tumors, chronic or acute inflammatory disease, 
restenosis, diabetes, neurological disorders, arthritis and osteoporosis, 
among other indications. 
Characterization of ILK 
The human gene sequence of ILK is provided as SEQ ID NO:1, the encoded 
polypeptide product as SEQ ID NO:2. The ILK protein is encoded by a 1.8 
kilobase pair messenger RNA (1.8 kb mRNA). The sequence of this mRNA was 
used to deduce the primary amino acid sequence of the protein, which has a 
predicted molecular weight of 50 kiloDaltons (kDa). The recombinant 
protein migrates on analytical polyacrylamide electrophoresis gels with an 
apparent molecular weight of 59 kDa, in rough agreement with the predicted 
size. p59ILK is a serine threonine kinase having two functional domains, 
identified by comparison of the ILK sequence against those found in 
current protein databases. These are the catalytic domain, responsible for 
phosphotransferase activity (kinase domain), and a non-overlapping domain 
in the amino terminus, comprised of four contiguous ankyrin-like repeats. 
The function of ankyrin repeats in ILK is to mediate protein-protein 
interactions. The ILK ankyrin repeat domain is not required for the 
binding of p59.sup.ILK to integrin, and it is predicted to mediate the 
interaction of p59.sup.ILK with other cellular protein(s). Thus, 
p59.sup.ILK bridges integrin in the plasma membrane with intracellular 
proteins active regulating the cell's response to ECM signals. These 
proteins are likely to be located in the cytoplasm, or as part of the 
cell's structural framework (cytoskeleton). 
ILK has novel structural and functional features. The molecular 
architecture is unusual, in that a protein kinase and an ankyrin repeat 
domain are contained within the same protein. The kinase domain has a high 
degree of similarity to other kinase sequences in existing databases, and 
can be divided into typical subdomains (I through XI) based on this 
conserved structure. However one amino acid in subdomain VIb of all other 
protein kinase domains is not present in ILK. Despite this unique 
structural feature, ILK clearly acts as a protein kinase, and thus 
represents a prototype member of a new subfamily of protein kinase 
molecules. 
ILK regulates integrin extracellular activity (ECM interactions) from 
inside the cell via its direct interaction with the integrin subunit 
(colloquially known as inside-out signaling). Interfering with ILK 
activity allows the specific targeting of integrin function, while leaving 
other essential signaling pathways intact. Moreover, increasing the levels 
of cellular ILK activity short circuits the normal requirement for 
adhesion to ECM (i.e. integrin function) in regulating cell growth. Thus, 
inhibiting ILK activity inhibits anchorage-independent (i.e. cancerous) 
cell growth. 
The amino acid sequence of ILK contains a sequence motif found in 
pleckstrin homology (PH) domains (Klarulund et al. (1997) Science 
275:1927-1930). This motif has been shown to be involved in the binding of 
phosphatidylinositol phosphates (Lemmon et al. (1996) Cell 85:621-624). 
Amino acids critical to the binding of such lipids to the PH domain are 
completely conserved in ILK. The phosphatidylinositol 3,4,5, triphosphate 
binding sites are the lysines at positions 162 and 209 (SEQ ID NO:2). The 
PH motifs are comprised of residues 158-165 and 208-212 (SEQ ID NO:2). 
There is a high degree of sequence identity within this motif between ILK 
and other PH-domain containing proteins such as cytohesin-1 (a .beta.2 
integrin cytoplasmic domain interacting protein) and GRP-1. It was 
determined that ILK activity is influenced by the presence of 
phosphatidylinositol3,4,5, triphosphate, and interacts with other kinase 
proteins in this pathway. 
ILK activity can be stimulated by phosphatidylinositol 3,4,5 trisphosphate 
in vitro. Both insulin and fibronectin can rapidly stimulate ILK activity 
in a phosphoinositide-3OH kinase (PI(3)K)-dependent manner. In addition, 
constitutively active PI(3)K activates ILK. The activated ILK can then 
inhibit the activity of glycogen synthase kinase-3 (GSK-3), contributing 
to ILK induced nuclear translocation of .beta.-catenin. ILK can also 
phosphorylate protein kinase B (PKB/AKT) on serine-473, resulting in its 
activation, demonstrating that ILK is involved in agonist stimulated 
PI(3)K-dependent PKB/AKT activation. 
The ILK chromosomal locus is mapped to region 11p15. A subset of breast 
carcinomas displays LOH for markers in chromosomal region 11p15.5. This 
region has also been implicated in an inherited form of cardiac arrythmia, 
the long QT syndrome. A high level of expression of ILK mRNA indicates an 
integrin-independent function for ILK in cardiac tissue. 
In untransformed intestinal epithelial cells, the kinase activity of ILK is 
inhibited upon cell-extracellular matrix interactions, and overexpression 
of constitutively active ILK results in anchorage-independent growth and 
tumorigenicity in nude mice. A consequence of elevation of ILK levels is a 
disruption of cell-cell interactions and manifestation of fibroblastic 
cell morphology and phenotypic properties, which include formation of a 
fibronectin matrix and invasion of collagen gels. 
Overexpression of ILK results in a downregulation of E-cadherin expression, 
formation of a complex between .beta.-catenin and the HMG transcription 
factor, LEF-1, translocation of .beta.-catenin to the nucleus, and 
transcriptional activation by this LEF-1/.beta.-catenin complex. LEF-1 
protein expression is rapidly modulated by cell detachment from the 
extracellular matrix, and LEF-1 protein levels are constitutively 
upregulated upon ILK overexpression. These effects are specific for ILK. 
Overexpression of ILK stimulates fibronectin matrix assembly in epithelial 
cells. The integrin-linked kinase activity is involved in transducing 
signals leading to the up-regulation of fibronectin matrix assembly, as 
overexpression of a kinase-inactive ILK mutant fails to enhance the matrix 
assembly. The increase in fibronectin matrix assembly is accompanied by a 
substantial reduction in cellular E-cadherin. The increased fibronectin 
matrix assembly is associated with an increased potential for tumor growth 
in vitro and in vivo. 
Identification of ILK Sequences 
Homologs of ILK are identified by any of a number of methods. A fragment of 
the provided cDNA may be used as a hybridization probe against a cDNA 
library from the target organism of interest, where low stringency 
conditions are used. The probe may be a large fragment, or one or more 
short degenerate primers. Nucleic acids having sequence similarity are 
detected by hybridization under low stringency conditions, for example, at 
50.degree. C. and 10.times.SSC (0.9 M saline/0.09 M sodium citrate) and 
remain bound when subjected to washing at 55.degree. C. in 1.times.SSC. 
Sequence identity may be determined by hybridization under stringent 
conditions, for example, at 50.degree. C. or higher and 0.1.times.SSC (9 
mM saline/0.9 mM sodium citrate). Nucleic acids that are substantially 
identical to the provided ILK sequences, e.g. allelic variants, 
genetically altered versions of the gene, etc., bind to the provided ILK 
sequences under stringent hybridization conditions. By using probes, 
particularly labeled probes of DNA sequences, one can isolate homologous 
or related genes. The source of homologous genes may be any species, e.g. 
primate species, particularly human; rodents, such as rats and mice, 
canines, felines, bovines, ovines, equines, yeast, nematodes, etc. 
Between mammalian species, e.g. human and mouse, homologs have substantial 
sequence similarity, i.e. at least 75% sequence identity between 
nucleotide sequences. Sequence similarity is calculated based on a 
reference sequence, which may be a subset of a larger sequence, such as a 
conserved motif, coding region, flanking region, etc. A reference sequence 
will usually be at least about 18 nt long, more usually at least about 30 
nt long, and may extend to the complete sequence that is being compared. 
Algorithms for sequence analysis are known in the art, such as BLAST, 
described in Altschul et al. (1990) J Mol Biol 215:403-10. The sequences 
provided herein are essential for recognizing ILK related and homologous 
proteins in database searches. 
ILK Nucleic Acid Compositions 
Nucleic acids encoding ILK may be cDNA or genomic DNA or a fragment 
thereof. The term ILK gene shall be intended to mean the open reading 
frame, encoding specific ILK polypeptides, introns, as well as adjacent 5 
and 3 non-coding nucleotide sequences involved in the regulation of 
expression, up to about 20 kb beyond the coding region, but possibly 
further in either direction. The gene may be introduced into an 
appropriate vector for extrachromosomal maintenance or for integration 
into a host genome. The term cDNA as used herein is intended to include 
all nucleic acids that share the arrangement of sequence elements found in 
native mature mRNA species, where sequence elements are exons and 3 and 5 
non-coding regions. Normally mRNA species have contiguous exons, with the 
intervening introns, when present, removed by nuclear RNA splicing, to 
create a continuous open reading frame encoding a ILK protein. 
A genomic sequence of interest comprises the nucleic acid present between 
the initiation codon and the stop codon, as defined in the listed 
sequences, including all of the introns that are normally present in a 
native chromosome. It may further include the 3 and 5 untranslated regions 
found in the mature mRNA. It may further include specific transcriptional 
and translational regulatory sequences, such as promoters, enhancers, 
etc., including about 1 kb, but possibly more, of flanking genomic DNA at 
either the 5 or 3 end of the transcribed region. The genomic DNA may be 
isolated as a fragment of 100 kbp or smaller; and substantially free of 
flanking chromosomal sequence. The genomic DNA flanking the coding region, 
either 3' or 5', or internal regulatory sequences as sometimes found in 
introns, contains sequences required for proper tissue and stage specific 
expression. 
The sequence of the 5' flanking region may be utilized for promoter 
elements, including enhancer binding sites, that provide for developmental 
regulation in tissues where ILK is expressed. The tissue specific 
expression is useful for determining the pattern of expression, and for 
providing promoters that mimic the native pattern of expression. Naturally 
occurring polymorphisms in the promoter region are useful for determining 
natural variations in expression, particularly those that may be 
associated with disease. 
Alternatively, mutations may be introduced into the promoter region to 
determine the effect of altering expression in experimentally defined 
systems. Methods for the identification of specific DNA motifs involved in 
the binding of transcriptional factors are known in the art, e.g. sequence 
similarity to known binding motifs, gel retardation studies, etc. For 
examples, see Blackwell et al. (1995) Mol Med 1:194-205; Mortlock et al. 
(1996) Genome Res. 6:327-33; and Joulin and Richard-Foy (1995) Eur J. 
Biochem 232:620-626. 
The regulatory sequences may be used to identify cis acting sequences 
required for transcriptional or translational regulation of ILK 
expression, especially in different tissues or stages of development, and 
to identify cis acting sequences and trans acting factors that regulate or 
mediate ILK expression. Such transcription or translational control 
regions may be operably linked to a ILK gene in order to promote 
expression of wild type or altered ILK or other proteins of interest in 
cultured cells, or in embryonic, fetal or adult tissues, and for gene 
therapy. 
The nucleic acid compositions of the subject invention may encode all or a 
part of the subject polypeptides. Double or single stranded fragments may 
be obtained of the DNA sequence by chemically synthesizing 
oligonucleotides in accordance with conventional methods, by restriction 
enzyme digestion, by PCR amplification, etc. For the most part, DNA 
fragments will be of at least 15 nt, usually at least 18 nt or 25 nt, and 
may be at least about 50 nt. Such small DNA fragments are useful as 
primers for PCR, hybridization screening probes, etc. Larger DNA 
fragments, i.e. greater than 100 nt are useful for production of the 
encoded polypeptide. Regions of the provided sequence that are of interest 
as fragments include the 5' end of the gene, i.e. a portion of the 
sequence set forth in SEQ ID NO:1, nucleotides 1 to 1100. 
For use in amplification reactions, such as PCR, a pair of primers will be 
used. The exact composition of the primer sequences is not critical to the 
invention, but for most applications the primers will hybridize to the 
subject sequence under stringent conditions, as known in the art. It is 
preferable to choose a pair of primers that will generate an amplification 
product of at least about 50 nt, preferably at least about 100 nt. 
Algorithms for the selection of primer sequences are generally known, and 
are available in commercial software packages. Amplification primers 
hybridize to complementary strands of DNA, and will prime towards each 
other. 
The ILK genes are isolated and obtained in substantial purity, generally as 
other than an intact chromosome. Usually, the DNA will be obtained 
substantially free of other nucleic acid sequences that do not include a 
ILK sequence or fragment thereof generally being at least about 50%, 
usually at least about 90% pure and are typically recombinant, i.e. 
flanked by one or more nucleotides with which it is not normally 
associated on a naturally occurring chromosome. 
The DNA may also be used to identify expression of the gene in a biological 
specimen. The manner in which one probes cells for the presence of 
particular nucleotide sequences, as genomic DNA or RNA, is well 
established in the literature and does not require elaboration here. DNA 
or mRNA is isolated from a cell sample. The mRNA may be amplified by 
RT-PCR, using reverse transcriptase to form a complementary DNA strand, 
followed by polymerase chain reaction amplification using primers specific 
for the subject DNA sequences. Alternatively, the mRNA sample is separated 
by gel electrophoresis, transferred to a suitable support, e.g. 
nitrocellulose, nylon, etc., and then probed with a fragment of the 
subject DNA as a probe. Other techniques, such as oligonucleotide ligation 
assays, in situ hybridizations, and hybridization to DNA probes arrayed on 
a solid chip may also find use. Detection of mRNA hybridizing to the 
subject sequence is indicative of ILK gene expression in the sample. 
The sequence of a ILK gene, including flanking promoter regions and coding 
regions, may be mutated in various ways known in the art to generate 
targeted changes in promoter strength, sequence of the encoded protein, 
etc. The DNA sequence or protein product of such a mutation will usually 
be substantially similar to the sequences provided herein, i.e. will 
differ by at least one nucleotide or amino acid, respectively, and may 
differ by at least two but not more than about ten nucleotides or amino 
acids. The sequence changes may be substitutions, insertions or deletions. 
Deletions may further include larger changes, such as deletions of a 
domain or exon. Other modifications of interest include epitope tagging, 
e.g. with the FLAG system, HA, etc. For studies of subcellular 
localization, fusion proteins with green fluorescent proteins (GFP) may be 
used. 
Techniques for in vitro mutagenesis of cloned genes are known. Examples of 
protocols for site specific mutagenesis may be found in Gustin et al. 
(1993) Biotechniques 14:22; Barany (1985) Gene 37:111-23; Colicelli et al. 
(1985) Mol Gen Genet 199:537; and Prentki et al. (1984) Gene 29:303-13. 
Methods for site specific mutagenesis can be found in Sambrook et al., 
Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; 
Weiner et al., Gene 126:35-41 (1993); Sayers et al. Biotechniques 13:592-6 
(1992); Jones and Winistorfer, Biotechniques 12:528-30 (1992); Barton et 
al., Nucleic Acids Res 18:7349-55 (1990); Marotti and Tomich, Gene Anal 
Tech 6:67-70 (1989); and Zhu, Anal Biochem 177:1204 (1989). Such mutated 
genes may be used to study structure-function relationships of ILK, or to 
alter properties of the protein that affect its function or regulation. 
ILK Polypeptides 
The subject gene may be employed for producing all or portions of ILK 
polypeptides. For expression, an expression cassette may be employed. The 
expression vector will provide a transcriptional and translational 
initiation region, which may be inducible or constitutive, where the 
coding region is operably linked under the transcriptional control of the 
transcriptional initiation region, and a transcriptional and translational 
termination region. These control regions may be native to an ILK gene, or 
may be derived from exogenous sources. 
The peptide may be expressed in prokaryotes or eukaryotes in accordance 
with conventional ways, depending upon the purpose for expression. For 
large scale production of the protein, a unicellular organism, such as E. 
coli, B. subtilis, S. cerevisiae, insect cells in combination with 
baculovirus vectors, or cells of a higher organism such as vertebrates, 
particularly mammals, e.g. COS 7 cells, may be used as the expression host 
cells. In some situations, it is desirable to express the ILK gene in 
eukaryotic cells, where the ILK protein will benefit from native folding 
and post-translational modifications. Small peptides can also be 
synthesized in the laboratory. Peptides that are subsets of the complete 
ILK sequence may be used to identify and investigate parts of the protein 
important for function, such as the GTPase binding domain, or to raise 
antibodies directed against these regions. Peptides may be from about 8 
amino acids in length, usually at least about 12 amino acids in length, or 
20 amino acids in length, and up to complete domains, e.g. the ankyrin 
domains, or a substantially complete protein, i.e. 90 to 95% of the mature 
polypeptide. 
With the availability of the protein or fragments thereof in large amounts, 
by employing an expression host, the protein may be isolated and purified 
in accordance with conventional ways. A lysate may be prepared of the 
expression host and the lysate purified using HPLC, exclusion 
chromatography, gel electrophoresis, affinity chromatography, or other 
purification technique. The purified protein will generally be at least 
about 80% pure, preferably at least about 90% pure, and may be up to and 
including 100% pure. Pure is intended to mean free of other proteins, as 
well as cellular debris. 
The expressed ILK polypeptides are useful for the production of antibodies, 
where short fragments provide for antibodies specific for the particular 
polypeptide, and larger fragments or the entire protein allow for the 
production of antibodies over the surface of the polypeptide. Antibodies 
may be raised to the wild-type or variant forms of ILK. Antibodies may be 
raised to isolated peptides corresponding to these domains, or to the 
native protein. 
Antibodies are prepared in accordance with conventional ways, where the 
expressed polypeptide or protein is used as an immunogen, by itself or 
conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other 
viral or eukaryotic proteins, or the like. Various adjuvants may be 
employed, with a series of injections, as appropriate. For monoclonal 
antibodies, after one or more booster injections, the spleen is isolated, 
the lymphocytes immortalized by cell fusion, and then screened for high 
affinity antibody binding. The immortalized cells, i.e. hybridomas, 
producing the desired antibodies may then be expanded. For further 
description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and 
Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 
1988. If desired, the mRNA encoding the heavy and light chains may be 
isolated and mutagenized by cloning in E. coli, and the heavy and light 
chains mixed to further enhance the affinity of the antibody. Alternatives 
to in vivo immunization as a method of raising antibodies include binding 
to phage display libraries, usually in conjunction with in vitro affinity 
maturation. 
Modulation of ILK Activity 
ILK activity is upregulated by the presence of the lipid 
[Ptdlns(3,4,5)P.sub.3 ]. The activity of ILK is manipulated by agents that 
affect cellular levels of [Ptdlns(3,4,5)P.sub.3 ], or that block the 
binding of [Ptdlns(3,4,5)P.sub.3 ] to ILK. This lipid binds to specific 
amino acid residues in ILK. The amino acid sequence of ILK contains a 
sequence motif found in pleckstrin homology (PH) domains, which are 
involved in the binding of phosphatidylinositol phosphates. The 
[Ptdlns(3,4,5)P.sub.3 ] binding sites are the lysines at positions 162 and 
209 (SEQ ID NO:2). The PH motifs are comprised of residues 158-165 and 
208-212 (SEQ ID NO:2). 
Agents of interest for down-regulating ILK activity include direct blocking 
of [Ptdlns(3,4,5)P.sub.3 ] binding sites through competitive binding, 
steric hindrance, etc. Of particular interest are antibodies that bind to 
the PH domains, thereby blocking the site. Antibodies include fragments, 
e.g. F(Ab), F(Ab)', and other mimetics of the binding site. Such 
antibodies can be raised by immunization with the protein or the specific 
domain. Mimetics are identified by screening methods, as described herein. 
Analogs of [Ptdlns(3,4,5)P.sub.3 ] that compete for binding sites but do 
not result in activation of ILK are also of interest. 
The activity of ILK is also down-regulated by inhibiting the activity of 
PI(3) kinase, thereby decreasing cellular levels of [Ptdlns(3,4,5)P.sub.3 
]. Phosphatidylinositol 3-kinase (EC 2.7.1.137) is composed of 85-kD and 
110-kD subunits. The 85-kD subunit lacks PI3-kinase activity and acts as 
an adaptor, coupling the 110-kD subunit (p110) to activated protein 
tyrosine kinases. p110 may require a complex with p85-alpha for catalytic 
activity. The genetic and amino acid sequence of p110 subunits for human 
PI(3) kinase can be obtained from Genbank, accession numbers Z29090, 
X83368. 
Agents of interest include inhibitors of PI(3) kinase, e.g. wortmannin, 
LY294002, etc. Physiologically effective levels of wortmannin range from 
about 10 to 1000 nM, usually from about 100 to 500 nM, and optimally at 
about 200 nM. Physiologically effective levels of LY294002 range from 
about 1 to 500 .mu.M, usually from about 25 to 100 .mu.M, and optimally at 
about 50 .mu.M. The inhibitors are administered in vivo or in vitro at a 
dose sufficient to provide for these concentrations in the target tissue. 
Other inhibitors of PI(3) kinase include anti-sense reagents, as described 
for ILK, which are specific for PI(3) kinase. Of particular interest are 
anti-sense molecules derived from the human PI(3) kinase sequence, 
particularly the catalytic p110 subunit, using the publicly available 
sequence. Alternatively, antibodies, antibody fragments and analogs or 
other blocking agents are used to bind to the PI(3) kinase in order to 
reduce the activity. 
Agents that block ILK activity provide a point of intervention in an 
important signaling pathway. As described in other sections of the instant 
application, numerous agents are useful in reducing ILK activity, 
including agents that directly modulate ILK expression, e.g. expression 
vectors, anti-sense specific for ILK, ILK specific antibodies and analogs 
thereof, small organic molecules that block ILK catalytic activity, etc.; 
and agents that affect ILK activity through direct or indirect modulation 
of [Ptdlns(3,4,5)P.sub.3 ] levels in a cell. 
ILK phosphorylates protein kinase B (PKB/AKT) at amino acid residue 473, 
which is a serine. The sequence of PKB may be found in Genbank, accession 
number X61037. By modulating ILK activity, the phosphorylation of PKB 
ser473 is manipulated, either increasing or decreasing the level. The 
ser473 phosphorylation increased the catalytic activity of PKB. Modulating 
the activity of PKB affects the activity of GSK-3, which is inactivated by 
phosphorylation at ser9 (Genbank L40027). The inactivation of GSK-3 may 
also be directly affected by ILK. Once inactivated, GSK-3 results in the 
nuclear translocation of .beta.-catenin and activation of 
Lef-1/.beta.-catenin transcriptional activity. 
Formulations 
The compounds of this invention can be incorporated into a variety of 
formulations for therapeutic administration. Particularly, agents that 
modulate ILK activity, or ILK polypeptides and analogs thereof are 
formulated for administration to patients for the treatment of ILK 
dysfunction, where the ILK activity is undesirably high or low, e.g. to 
reduce the level of ILK in cancer cells. More particularly, the compounds 
of the present invention can be formulated into pharmaceutical 
compositions by combination with appropriate, pharmaceutically acceptable 
carriers or diluents, and may be formulated into preparations in solid, 
semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, 
granules, ointments, solutions, suppositories, injections, inhalants, 
gels, microspheres, and aerosols. As such, administration of the compounds 
can be achieved in various ways, including oral, buccal, rectal, 
parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., 
administration. The ILK may be systemic after administration or may be 
localized by the use of an implant that acts to retain the active dose at 
the site of implantation. 
The compounds of the present invention can be administered alone, in 
combination with each other, or they can be used in combination with other 
known compounds. In pharmaceutical dosage forms, the compounds may be 
administered in the form of their pharmaceutically acceptable salts, or 
they may also be used alone or in appropriate association, as well as in 
combination with other pharmaceutically active compounds. The following 
methods and excipients are merely exemplary and are in no way limiting. 
For oral preparations, the compounds can be used alone or in combination 
with appropriate additives to make tablets, powders, granules or capsules, 
for example, with conventional additives, such as lactose, mannitol, corn 
starch or potato starch; with binders, such as crystalline cellulose, 
cellulose derivatives, acacia, corn starch or gelatins; with 
disintegrators, such as corn starch, potato starch or sodium 
carboxymethylcellulose; with lubricants, such as talc or magnesium 
stearate; and if desired, with diluents, buffering agents, moistening 
agents, preservatives and flavoring agents. 
The compounds can be formulated into preparations for injections by 
dissolving, suspending or emulsifying them in an aqueous or nonaqueous 
solvent, such as vegetable or other similar oils, synthetic aliphatic acid 
glycerides, esters of higher aliphatic acids or propylene glycol; and if 
desired, with conventional additives such as solubilizers, isotonic 
agents, suspending agents, emulsifying agents, stabilizers and 
preservatives. 
The compounds can be utilized in aerosol formulation to be administered via 
inhalation. The compounds of the present invention can be formulated into 
pressurized acceptable propellants such as dichlorodifluoromethane, 
propane, nitrogen and the like. 
Furthermore, the compounds can be made into suppositories by mixing with a 
variety of bases such as emulsifying bases or water-soluble bases. The 
compounds of the present invention can be administered rectally via a 
suppository. The suppository can include vehicles such as cocoa butter, 
carbowaxes and polyethylene glycols, which melt at body temperature, yet 
are solidified at room temperature. 
Unit dosage forms for oral or rectal administration such as syrups, 
elixirs, and suspensions may be provided wherein each dosage unit, for 
example, teaspoonful, tablespoonful, tablet or suppository, contains a 
predetermined amount of the composition containing one or more compounds 
of the present invention. Similarly, unit dosage forms for injection or 
intravenous administration may comprise the compound of the present 
invention in a composition as a solution in sterile water, normal saline 
or another pharmaceutically acceptable carrier. 
Implants for sustained release formulations are well-known in the art. 
Implants are formulated as microspheres, slabs, etc. with biodegradable or 
non-biodegradable polymers. For example, polymers of lactic acid and/or 
glycolic acid form an erodible polymer that is well-tolerated by the host. 
The implant is placed in proximity to the site of infection, so that the 
local concentration of active agent is increased relative to the rest of 
the body. 
The term "unit dosage form," as used herein, refers to physically discrete 
units suitable as unitary dosages for human and animal subjects, each unit 
containing a predetermined quantity of compounds of the present invention 
calculated in an amount sufficient to produce the desired effect in 
association with a pharmaceutically acceptable diluent, carrier or 
vehicle. The specifications for the novel unit dosage forms of the present 
invention depend on the particular compound employed and the effect to be 
achieved, and the pharmacodynamics associated with each compound in the 
host. 
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, 
carriers or diluents, are readily available to the public. Moreover, 
pharmaceutically acceptable auxiliary substances, such as pH adjusting and 
buffering agents, tonicity adjusting agents, stabilizers, wetting agents 
and the like, are readily available to the public. 
Typical dosages for systemic administration range from 0.1 .mu.g to 100 
milligrams per kg weight of subject per administration. A typical dosage 
may be one tablet taken from two to six times daily, or one time-release 
capsule or tablet taken once a day and containing a proportionally higher 
content of active ingredient. The time-release effect may be obtained by 
capsule materials that dissolve at different pH values, by capsules that 
release slowly by osmotic pressure, or by any other known means of 
controlled release. 
Those of skill will readily appreciate that dose levels can vary as a 
function of the specific compound, the severity of the symptoms and the 
susceptibility of the subject to side effects. Some of the specific 
compounds are more potent than others. Preferred dosages for a given 
compound are readily determinable by those of skill in the art by a 
variety of means. A preferred means is to measure the physiological 
potency of a given compound. 
The use of liposomes as a delivery vehicle is one method of interest. The 
liposomes fuse with the cells of the target site and deliver the contents 
of the lumen intracellularly. The liposomes are maintained in contact with 
the cells for sufficient time for fusion, using various means to maintain 
contact, such as isolation, binding agents, and the like. In one aspect of 
the invention, liposomes are designed to be aerosolized for pulmonary 
administration. Liposomes may be prepared with purified proteins or 
peptides that mediate fusion of membranes, such as Sendai virus or 
influenza virus, etc. The lipids may be any useful combination of known 
liposome forming lipids, including cationic lipids, such as 
phosphatidylcholine. The remaining lipid will normally be neutral lipids, 
such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the 
like. 
For preparing the liposomes, the procedure described by Kato et al. (1991) 
J. Biol. Chem. 266:3361 may be used. Briefly, the lipids and lumen 
composition containing the nucleic acids are combined in an appropriate 
aqueous medium, conveniently a saline medium where the total solids will 
be in the range of about 1-10 weight percent. After intense agitation for 
short periods of time, from about 5-60 sec., the tube is placed in a warm 
water bath, from about 25-40.degree. C. and this cycle repeated from about 
5-10 times. The composition is then sonicated for a convenient period of 
time, generally from about 1-10 sec. and may be further agitated by 
vortexing. The volume is then expanded by adding aqueous medium, generally 
increasing the volume by about from 1-2 fold, followed by shaking and 
cooling. This method allows for the incorporation into the lumen of high 
molecular weight molecules. 
Diagnostic Uses 
DNA-based reagents derived from the sequence of ILK, e.g. PCR primers, 
oligonucleotide or cDNA probes, as well as antibodies against p59ILK, are 
used to screen patient samples, e.g. biopsy-derived tumors, inflammatory 
samples such as arthritic synovium, etc., for amplified ILK DNA, or 
increased expression of ILK mRNA or protein. DNA-based reagents are 
designed for evaluation of chromosomal loci implicated in certain diseases 
e.g. for use in loss-of-heterozygosity (LOH) studies, or design of primers 
based on ILK coding sequence. 
The subject nucleic acid and/or polypeptide compositions may be used to 
analyze a patient sample for the presence of polymorphisms associated with 
a disease state or genetic predisposition to a disease state. Biochemical 
studies may be performed to determine whether a sequence polymorphism in 
an ILK coding region or control regions is associated with disease, 
particularly cancers and other growth abnormalities. Diseases of interest 
may also include restenosis, diabetes, neurological disorders, etc. 
Disease associated polymorphisms may include deletion or truncation of the 
gene, mutations that alter expression level, that affect the binding 
activity of the protein to integrin, kinase activity domain, etc. 
Changes in the promoter or enhancer sequence that may affect expression 
levels of ILK can be compared to expression levels of the normal allele by 
various methods known in the art. Methods for determining promoter or 
enhancer strength include quantitation of the expressed natural protein; 
insertion of the variant control element into a vector with a reporter 
gene such as .beta.-galactosidase, luciferase, chloramphenicol 
acetyltransferase, etc. that provides for convenient quantitation; and the 
like. 
A number of methods are available for analyzing nucleic acids for the 
presence of a specific sequence, e.g. a disease associated polymorphism. 
Where large amounts of DNA are available, genomic DNA is used directly. 
Alternatively, the region of interest is cloned into a suitable vector and 
grown in sufficient quantity for analysis. Cells that express ILK may be 
used as a source of mRNA, which may be assayed directly or reverse 
transcribed into cDNA for analysis. The nucleic acid may be amplified by 
conventional techniques, such as the polymerase chain reaction (PCR), to 
provide sufficient amounts for analysis. The use of the polymerase chain 
reaction is described in Saiki, et al. (1985) Science 239:487, and a 
review of techniques may be found in Sambrook, et al. Molecular Cloning: A 
Laboratory Manual, CSH Press 1989, pp.14.2-14.33. Alternatively, various 
methods are known in the art that utilize oligonucleotide ligation as a 
means of detecting polymorphisms, for examples see Riley et al. (1990) 
N.A.R. 18:2887-2890; and Delahunty et al. (1996) Am. J. Hum. Genet. 
58:1239-1246. 
A detectable label may be included in an amplification reaction. Suitable 
labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), 
rhodamine, Texas Red, phycoerythrin, 
allophycocyanin,6-carboxyfluorescein(6-FAM),2,7-dimethoxy-4,5-dichloro-6-c 
arboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 
6-carboxy-2,4,7,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein 
(5-FAM) or N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive 
labels, e.g. .sup.32 P, .sup.35 S, .sup.3 H; etc. The label may be a two 
stage system, where the amplified DNA is conjugated to biotin, haptens, 
etc. having a high affinity binding partner, e.g. avidin, specific 
antibodies, etc., where the binding partner is conjugated to a detectable 
label. The label may be conjugated to one or both of the primers. 
Alternatively, the pool of nucleotides used in the amplification is 
labeled, so as to incorporate the label into the amplification product. 
The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by 
one of a number of methods known in the art. The nucleic acid may be 
sequenced by dideoxy or other methods, and the sequence of bases compared 
to a wild-type ILK sequence. Hybridization with the variant sequence may 
also be used to determine its presence, by Southern blots, dot blots, etc. 
The hybridization pattern of a control and variant sequence to an array of 
oligonucleotide probes immobilised on a solid support, as described in 
U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of 
detecting the presence of variant sequences. Single strand conformational 
polymorphism (SSCP) analysis, denaturing gradient gel 
electrophoresis(DGGE), and heteroduplex analysis in gel matrices are used 
to detect conformational changes created by DNA sequence variation as 
alterations in electrophoretic mobility. Alternatively, where a 
polymorphism creates or destroys a recognition site for a restriction 
endonuclease, the sample is digested with that endonuclease, and the 
products size fractionated to determine whether the fragment was digested. 
Fractionation is performed by gel or capillary electrophoresis, 
particularly acrylamide or agarose gels. 
Screening for mutations in ILK may be based on the functional or antigenic 
characteristics of the protein. Protein truncation assays are useful in 
detecting deletions that may affect the biological activity of the 
protein. Various immunoassays designed to detect polymorphisms in ILK 
proteins may be used in screening. Where many diverse genetic mutations 
lead to a particular disease phenotype, functional protein assays have 
proven to be effective screening tools. The activity of the encoded ILK 
protein in kinase assays, binding of integrins, etc., may be determined by 
comparison with the wild-type protein. 
Antibodies specific for a ILK may be used in staining or in immunoassays. 
Samples, as used herein, include biological fluids such as semen, blood, 
cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like; 
organ or tissue culture derived fluids; and fluids extracted from 
physiological tissues. Also included in the term are derivatives and 
fractions of such fluids. The cells may be dissociated, in the case of 
solid tissues, or tissue sections may be analyzed. Alternatively a lysate 
of the cells may be prepared. 
Diagnosis may be performed by a number of methods to determine the absence 
or presence or altered amounts of normal or abnormal ILK in patient cells. 
For example, detection may utilize staining of cells or histological 
sections, performed in accordance with conventional methods. Cells are 
permeabilized to stain cytoplasmic molecules. The antibodies of interest 
are added to the cell sample, and incubated for a period of time 
sufficient to allow binding to the epitope, usually at least about 10 
minutes. The antibody may be labeled with radioisotopes, enzymes, 
fluorescers, chemiluminescers, or other labels for direct detection. 
Alternatively, a second stage antibody or reagent is used to amplify the 
signal. Such reagents are well known in the art. For example, the primary 
antibody may be conjugated to biotin, with horseradish 
peroxidase-conjugated avidin added as a second stage reagent. 
Alternatively, the secondary antibody conjugated to a flourescent 
compound, e.g. flourescein rhodamine, Texas red, etc. Final detection uses 
a substrate that undergoes a color change in the presence of the 
peroxidase. The absence or presence of antibody binding may be determined 
by various methods, including flow cytometry of dissociated cells, 
microscopy, radiography, scintillation counting, etc. 
Diagnostic screening may also be performed for polymorphisms that are 
genetically linked to a disease predisposition, particularly through the 
use of microsatellite markers or single nucleotide polymorphisms. 
Frequently the microsatellite polymorphism itself is not phenotypically 
expressed, but is linked to sequences that result in a disease 
predisposition. However, in some cases the microsatellite sequence itself 
may affect gene expression. Microsatellite linkage analysis may be 
performed alone, or in combination with direct detection of polymorphisms, 
as described above. The use of microsatellite markers for genotyping is 
well documented. For examples, see Mansfield et al. (1994) Genomics 
24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib et al., supra. 
Modulation of Gene Expression 
From a therapeutic point of view, inhibiting ILK activity has a therapeutic 
effect on a number of proliferative disorders, including inflammation, 
restenosis, and cancer. Inhibition is achieved in a number of ways. 
Antisense ILK sequences may be administered to inhibit expression. 
Pseudo-substrate inhibitors, for example, a peptide that mimics a 
substrate for ILK may be used to inhibit activity. Other inhibitors are 
identified by screening for biological activity in an ILK-based functional 
assay, e.g. in vitro or in vivo ILK kinase activity. 
The ILK genes, gene fragments, or the encoded protein or protein fragments 
are useful in gene therapy to treat disorders associated with ILK defects. 
Expression vectors may be used to introduce the ILK gene into a cell. Such 
vectors generally have convenient restriction sites located near the 
promoter sequence to provide for the insertion of nucleic acid sequences. 
Transcription cassettes may be prepared comprising a transcription 
initiation region, the target gene or fragment thereof, and a 
transcriptional termination region. The transcription cassettes may be 
introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. 
lentivirus; adenovirus; and the like, where the vectors are able to 
transiently or stably be maintained in the cells, usually for a period of 
at least about one day, more usually for a period of at least about 
several days to several weeks. 
The gene or ILK protein may be introduced into tissues or host cells by any 
number of routes, including viral infection, microinjection, or fusion of 
vesicles. Jet injection may also be used for intramuscular administration, 
as described by Furth et al. (1992) Anal Biochem 205:365-368. The DNA may 
be coated onto gold microparticles, and delivered intradermally by a 
particle bombardment device, or "gene gun" as described in the literature 
(see, for example, Tang et al. (1992) Nature 356:152-154), where gold 
microprojectiles are coated with the ILK or DNA, then bombarded into skin 
cells. 
Antisense molecules can be used to down-regulate expression of ILK in 
cells. The anti-sense reagent may be antisense oligonucleotides (ODN), 
particularly synthetic ODN having chemical modifications from native 
nucleic acids, or nucleic acid constructs that express such anti-sense 
molecules as RNA. The antisense sequence is complementary to the mRNA of 
the targeted gene, and inhibits expression of the targeted gene products. 
Antisense molecules inhibit gene expression through various mechanisms, 
e.g. by reducing the amount of mRNA available for translation, through 
activation of RNAse H, or steric hindrance. One or a combination of 
antisense molecules may be administered, where a combination may comprise 
multiple different sequences. 
Antisense molecules may be produced by expression of all or a part of the 
target gene sequence in an appropriate vector, where the transcriptional 
initiation is oriented such that an antisense strand is produced as an RNA 
molecule. Alternatively, the antisense molecule is a synthetic 
oligonucleotide. Antisense oligonucleotides will generally be at least 
about 7, usually at least about 12, more usually at least about 20 
nucleotides in length, and not more than about 500, usually not more than 
about 50, more usually not more than about 35 nucleotides in length, where 
the length is governed by efficiency of inhibition, specificity, including 
absence of cross-reactivity, and the like. It has been found that short 
oligonucleotides, of from 7 to 8 bases in length, can be strong and 
selective inhibitors of gene expression (see Wagner et al. (1996) Nature 
Biotechnology 14:840-844). 
A specific region or regions of the endogenous sense strand mRNA sequence 
is chosen to be complemented by the antisense sequence. Selection of a 
specific sequence for the oligonucleotide may use an empirical method, 
where several candidate sequences are assayed for inhibition of expression 
of the target gene in vitro or in an animal model. A combination of 
sequences may also be used, where several regions of the mRNA sequence are 
selected for antisense complementation. 
Antisense oligonucleotides may be chemically syn the sized by methods known 
in the art (see Wagner et al. (1993) supra. and Milligan et al., supra.) 
Preferred oligonucleotides are chemically modified from the native 
phosphodiester structure, in order to increase their intracellular 
stability and binding affinity. A number of such modifications have been 
described in the literature, which alter the chemistry of the backbone, 
sugars or heterocyclic bases. 
Among useful changes in the backbone chemistry are phosphorothioates; 
phosphorodithioates, where both of the non-bridging oxygens are 
substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and 
boranophosphates. Achiral phosphate derivatives include 
3'-O-5'-S-phosphorothioate, 3'-S'-5O-phosphorothioate, 3'-CH.sub.2 
-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids 
replace the entire ribose phosphodiester backbone with a peptide linkage. 
Sugar modifications are also used to enhance stability and affinity. The 
.alpha.-anomer of deoxyribose may be used, where the base is inverted with 
respect to the natural .beta.-anomer. The 2'-OH of the ribose sugar may be 
altered to form 2'-O-methyl or 2'-O-allyl sugars, which provides 
resistance to degradation without comprising affinity. Modification of the 
heterocyclic bases must maintain proper base pairing. Some useful 
substitutions include deoxyuridine for deoxythymidine; 
5'-methyl-2'-deoxycytidine and 5'-bromo-2'-deoxycytidine for 
deoxycytidine. 5'-propynyl-2'-deoxyuridine and 
5'-propynyl-2'-deoxycytidine have been shown to increase affinity and 
biological activity when substituted for deoxythymidine and deoxycytidine, 
respectively. 
As an alternative to anti-sense inhibitors, catalytic nucleic acid 
compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to 
inhibit gene expression. Ribozymes may be synthesized in vitro and 
administered to the patient, or may be encoded on an expression vector, 
from which the ribozyme is synthesized in the targeted cell (for example, 
see International patent application WO 9523225, and Beigelman et al. 
(1995) Nucl. Acids Res 23:4434-42). Examples of oligonucleotides with 
catalytic activity are described in WO 9506764. Conjugates of anti-sense 
ODN with a metal complex, e.g. terpyridylCu(II), capable of mediating mRNA 
hydrolysis are described in Bashkin et al. (1995) Appl Biochem Biotechnol 
54:43-56. 
Genetically Altered Cell or Animal Models for ILK Function 
The subject nucleic acids can be used to generate transgenic animals or 
site specific gene modifications in cell lines. Transgenic animals may be 
made through homologous recombination, where the normal ILK locus is 
altered. Alternatively, a nucleic acid construct is randomly integrated 
into the genome. Vectors for stable integration include plasmids, 
retroviruses and other animal viruses, YACs, and the like. 
The modified cells or animals are useful in the study of ILK function and 
regulation. For example, a series of small deletions and/or substitutions 
may be made in the ILK gene to determine the role of different exons in 
integrin binding, kinase activity, oncogenesis, signal transduction, etc. 
Of interest are the use of ILK to construct transgenic animal models for 
cancer, where expression of ILK is specifically reduced or absent. 
Specific constructs of interest include anti-sense ILK, which will block 
ILK expression and expression of dominant negative ILK mutations. A 
detectable marker, such as lac Z may be introduced into the ILK locus, 
where upregulation of ILK expression will result in an easily detected 
change in phenotype. 
One may also provide for expression of the ILK gene or variants thereof in 
cells or tissues where it is not normally expressed or at abnormal times 
of development. By providing expression of ILK protein in cells in which 
it is not normally produced, one can induce changes in cell behavior, e.g. 
through ILK mediated LEK-1 activity. 
DNA constructs for homologous recombination will comprise at least a 
portion of the ILK gene with the desired genetic modification, and will 
include regions of homology to the target locus. DNA constructs for random 
integration need not include regions of homology to mediate recombination. 
Conveniently, markers for positive and negative selection are included. 
Methods for generating cells having targeted gene modifications through 
homologous recombination are known in the art. For various techniques for 
transfecting mammalian cells, see Keown et al. (1990) Methods in 
Enzymology 185:527-537. 
For embryonic stem (ES) cells, an ES cell line may be employed, or 
embryonic cells may be obtained freshly from a host, e.g. mouse, rat, 
guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder 
layer or grown in the presence of leukemia inhibiting factor (LIF). When 
ES or embryonic cells have been transformed, they may be used to produce 
transgenic animals. After transformation, the cells are plated onto a 
feeder layer in an appropriate medium. Cells containing the construct may 
be detected by employing a selective medium. After sufficient time for 
colonies to grow, they are picked and analyzed for the occurrence of 
homologous recombination or integration of the construct. Those colonies 
that are positive may then be used for embryo manipulation and blastocyst 
injection. Blastocysts are obtained from 4 to 6 week old superovulated 
females. The ES cells are trypsinized, and the modified cells are injected 
into the blastocoel of the blastocyst. After injection, the blastocysts 
are returned to each uterine horn of pseudopregnant females. Females are 
then allowed to go to term and the resulting offspring screened for the 
construct. By providing for a different phenotype of the blastocyst and 
the genetically modified cells, chimeric progeny can be readily detected. 
The chimeric animals are screened for the presence of the modified gene and 
males and females having the modification are mated to produce homozygous 
progeny. If the gene alterations cause lethality at some point in 
development, tissues or organs can be maintained as allogeneic or congenic 
grafts or transplants, or in culture. The transgenic animals may be any 
non-human mammal, such as laboratory animals, domestic animals, etc. The 
transgenic animals may be used in functional studies, drug screening, 
etc., e.g. to determine the effect of a candidate drug on oncogenesis, 
down regulation of E-cadherin, up regulation of LEF-1, etc. 
In Vitro Models for ILK Function 
The availability of a number of components in the integrin signaling 
pathway allows in vitro reconstruction of the pathway. Two or more of the 
components may be combined in vitro, and the behavior assessed in terms of 
activation of transcription of specific target sequences; modification of 
protein components, e.g. proteolytic processing, phosphorylation, 
methylation, etc.; ability of different protein components to bind to each 
other; utilization of GTP, etc. The components may be modified by sequence 
deletion, substitution, etc. to determine the functional role of specific 
domains. 
Drug screening may be performed using an in vitro model, a genetically 
altered cell or animal, or purified ILK protein. One can identify ligands 
or substrates that bind to, modulate or mimic the action of ILK. Areas of 
investigation include the development of treatments for 
hyper-proliferative disorders, e.g. cancer, restenosis, osteoarthritis, 
metastasis, etc. 
Drug screening identifies agents that modulate ILK function. Agents that 
mimic its function are predicted to activate the process of cell division 
and growth. Conversely, agents that reverse ILK function may inhibit 
transformation. Of particular interest are screening assays for agents 
that have a low toxicity for human cells. A wide variety of assays may be 
used for this purpose, including labeled in vitro protein-protein binding 
assays, electrophoretic mobility shift assays, immunoassays for protein 
binding, and the like. Knowledge of the 3-dimensional structure of ILK, 
derived from crystallization of purified recombinant ILK protein, leads to 
the rational design of small drugs that specifically inhibit ILK activity. 
These drugs may be directed at specific domains of ILK, e.g. the kinase 
catalytic domain, ankyrin repeat domains, pleckstrin homology domains, 
etc. 
Among the agents of interest for drug screening are those that interfere 
with the binding of [Ptdlns(3,4,5)P.sub.3 ] to the PH domains of ILK and 
agents that inhibit the production of [Ptdlns(3,4,5)P.sub.3 ] by PI(3) 
kinase. Such assays may monitor the ILK activity in the presence of 
[Ptdlns(3,4,5)P.sub.3 ] and a candidate agent, as described in the 
examples. 
The term "agent" as used herein describes any molecule, e.g. protein or 
pharmaceutical, with the capability of altering or mimicking the 
physiological function of ILK. Generally a plurality of assay mixtures are 
run in parallel with different agent concentrations to obtain a 
differential response to the various concentrations. Typically one of 
these concentrations serves as a negative control, i.e. at zero 
concentration or below the level of detection. 
Candidate agents encompass numerous chemical classes, though typically they 
are organic molecules, preferably small organic compounds having a 
molecular weight of more than 50 and less than about 2,500 daltons. 
Candidate agents comprise functional groups necessary for structural 
interaction with proteins, particularly hydrogen bonding, and typically 
include at least an amine, carbonyl, hydroxyl or carboxyl group, 
preferably at least two of the functional chemical groups. The candidate 
agents often comprise cyclical carbon or heterocyclic structures and/or 
aromatic or polyaromatic structures substituted with one or more of the 
above functional groups. Candidate agents are also found among 
biomolecules including peptides, saccharides, fatty acids, steroids, 
purines, pyrimidines, derivatives, structural analogs or combinations 
thereof. 
Candidate agents are obtained from a wide variety of sources including 
libraries of synthetic or natural compounds. For example, numerous means 
are available for random and directed synthesis of a wide variety of 
organic compounds and biomolecules, including expression of randomized 
oligonucleotides and oligopeptides. Alternatively, libraries of natural 
compounds in the form of bacterial, fungal, plant and animal extracts are 
available or readily produced. Additionally, natural or synthetically 
produced libraries and compounds are readily modified through conventional 
chemical, physical and biochemical means, and may be used to produce 
combinatorial libraries. Known pharmacological agents may be subjected to 
directed or random chemical modifications, such as acylation, alkylation, 
esterification, amidification, etc. to produce structural analogs. 
Where the screening assay is a binding assay, one or more of the molecules 
may be joined to a label, where the label can directly or indirectly 
provide a detectable signal. Various labels include radioisotopes, 
fluorescers, chemiluminescers, enzymes, specific binding molecules, 
particles, e.g. magnetic particles, and the like. Specific binding 
molecules include pairs, such as biotin and streptavidin, digoxin and 
antidigoxin, etc. For the specific binding members, the complementary 
member would normally be labeled with a molecule that provides for 
detection, in accordance with known procedures. 
A variety of other reagents may be included in the screening assay. These 
include reagents like salts, neutral proteins, e.g. albumin, detergents, 
etc that are used to facilitate optimal protein-protein binding and/or 
reduce non-specific or background interactions. Reagents that improve the 
efficiency of the assay, such as protease inhibitors, nuclease inhibitors, 
anti-microbial agents, etc. may be used. The mixture of components are 
added in any order that provides for the requisite binding. Incubations 
are performed at any suitable temperature, typically between 4 and 
40.degree. C. Incubation periods are selected for optimum activity, but 
may also be optimized to facilitate rapid high-throughput screening. 
Typically between 0.1 and 1 hours will be sufficient. 
Other assays of interest detect agents that mimic ILK function, such 
integrin binding, kinase activity, down regulation of E-cadherin, up 
regulation of LEF-1, binding properties, etc. For example, an expression 
construct comprising a ILK gene may be introduced into a cell line under 
conditions that allow expression. The level of ILK activity is determined 
by a functional assay, as previously described. In one screening assay, 
candidate agents are added, and the formation of fibronectin matrix is 
detected. In another assay, the ability of candidate agents to enhance ILK 
function is determined. A functional assay of interest detects the 
stimulation of cyclin D1 and/or cyclin A expression. Alternatively, 
candidate agents are added to a cell that lacks functional ILK, and 
screened for the ability to reproduce ILK in a functional assay. 
The compounds having the desired pharmacological activity may be 
administered in a physiologically acceptable carrier to a host for 
treatment of cancer, etc. The compounds may also be used to enhance ILK 
function in wound healing, cell growth, etc. The inhibitory agents may be 
administered in a variety of ways, orally, topically, parenterally e.g. 
subcutaneously, intraperitoneally, by viral infection, intravascularly, 
etc. Topical treatments are of particular interest. Depending upon the 
manner of introduction, the compounds may be formulated in a variety of 
ways. The concentration of therapeutically active compound in the 
formulation may vary from about 0.1-10 wt %. 
Experimental 
The following examples are put forth so as to provide those of ordinary 
skill in the art with a complete disclosure and description of how to make 
and use the subject invention, and are not intended to limit the scope of 
what is regarded as the invention. Efforts have been made to ensure 
accuracy with respect to the numbers used (e.g. amounts, temperature, 
concentrations, etc.) but some experimental errors and deviations should 
be allowed for. Unless otherwise indicated, parts are parts by weight, 
molecular weight is average molecular weight, temperature is in degrees 
centigrade; and pressure is at or near atmospheric. 
EXAMPLE 1 
Isolation of ILK cDNA 
A partial cDNA, BIT-9, was isolated in a two-hybrid screen using a bait 
plasmid expressing the cytoplasmic domain of the, integrin subunit. The 
BIT-9 insert was used to isolate clones from a human placental cDNA 
library. A 1.8 kb clone, Plac5, was found to contain a high degree of 
similarity to cDNAs encoding protein kinases (FIG. 1a-c), and recognized a 
widely expressed transcript of 1.8 kb in Northern blots (FIG. 1d). Deduced 
amino acid residues 186-451 from Plac5 comprise a domain which is highly 
homologous with the catalytic domains of a large number of protein 
tyrosine and serine/threonine kinases (FIG. 1b). Residues 33-164 comprise 
four repeats of a motif originally identified in erythrocyte ankyrin (FIG. 
1c), likely defining a domain involved in mediating additional 
protein-protein interactions. Affinity-purified anti-ILK antibodies (see 
methods described in Example 3) were used in Western blot analyses of 
mammalian cell extracts, and detected a conserved protein of apparent Mr 
of 59 kDa (p59ILK, FIG. 1e). 
FIG. 1 shows yeast two-hybrid cloning, characterization, and expression of 
ILK. (a) The full length ILK cDNA, Plac5, was isolated from a human 
placental library using the BIT-9 insert. Plac5 contains a 1509 bp open 
reading frame, with a presumptive initiator Met at nt 157, and an AAUAAA 
signal 11 bp upstream of the polyadenylation site. In vitro transcription 
and translation of Plac5 in rabbit reticulocyte lysates yielded a protein 
of apparent Mr of 59 kDa (not shown). (b) A search of the PIR protein 
database indicated homology with protein kinase subdomains I to XI, as 
identified by Hanks et al. We note sequence variations in the ILK 
subdomains I, VIb, and VII, relative to catalytic domains of known protein 
kinases. Subdomain I (residues 199-213), does not have the typical GXGXXG 
motif, although this region in ILK is Gly-rich. In subdomain Vlb, Asp328 
of ILK may compensate for the lack of the otherwise conserved Asp319. In 
subdomain VII, the DFG triplet is absent in ILK. The integrin binding site 
maps to amino acid residues 293-451 (BIT-9). The ILK kinase domain is most 
highly related to the CTR1 kinase of Arabidopsis thaliana (30% identity, 
P&lt;10). The CTR1, B-raf, Yes and Csk kinase domains are aligned with Plac5. 
(c) Amino acid residues 33-164 comprise four contiguous ankyrin repeats, 
as defined by Lux et. al. (d) BIT-9 was used to probe a blot of poly A+ 
selected RNA (MTN I, Clontech) from various human tissues. (e) Whole cell 
lysates of mouse, rat and human cell lines (10 .mu.g/lane) were analyzed 
by Western blotting with the affinity-purified 92-2 antibody (see 
description of methods in Example 3). The ILK sequence data are available 
from GenBank under accession number U40282. 
In order to construct integrin `bait` plasmids, sequences encoding amino 
acid residues 738-798 of the .beta..sub.1, and residues 1022-1049 of the 
.alpha..sub.5 integrin subunits were amplified from full-length cDNAs. The 
primers used were (a) 5' amplification 
5'-GGCCGAATTCGCTGGAATTGTTCTTATTGGC-3' and (b) 3' amplification 
5'-GGCCGGATCCTCATTTTCCCTCATACTTCGG-3'. PCR products were directionally 
cloned into pEG202, creating the LexA fusion bait plasmids, pEG202.beta.hd 
1INT and pEG202.alpha..sub.5 INT. pEG202.beta..sub.1 INT and 
pEG202.alpha..sub.5 INT repressed .beta.-gal expression from the pJK101 
reporter by 50-60% and 70-75%, respectively, in host strain EGY48 
(MAT.alpha., his3, trp1, ura3-52, LEU2::pLEU2-LexAop6, constructed by 
Erica Golemis, Massachussetts General Hospital), confirming nuclear 
expression of the LexA fusions. Co-transformation of baits with the 
pSH18-34 reporter verified they were transcriptionally inert. A 
galactose-inducible HeLa cDNA interactor library was present on the TRP+ 
vector, pJG4-5 (constructed by Jeno Gyuris, MGH). For the .beta..sub.1 
interaction trap, EGY48 was transformed sequentially with 
pEG202.beta..sub.1 INT, pSH18-34 and pJG4-5, using the lithium acetate 
protocol (transformation efficiency=5-6.times.10.sup.4 /.mu.g). 
2.times.10.sup.6 primary transformants were screened, of which forty-nine 
interacting clones were confirmed. The most frequent isolate (31/49) was a 
700 bp insert, BIT-9. Retransformation of EGY48 with the BIT-9, pSH18-34, 
and pEG202.beta..sub.1 INT plasmids resulted in strong 
.beta.-galactosidase expression, confirming the interaction. An identical 
screen, using pEG202.alpha..sub.5 INT as bait, resulted in the isolation 
of 16 positives, none of which were represented in the set of 49 
.beta..sub.1 interactors. Trapped inserts were used to screen WM35 human 
melanoma .lambda.gt10, and human placental .lambda.gt11 cDNA libraries, 
using standard procedures. cDNA sequencing of multiple clones from each 
library was done using the dideoxy chain termination method (Sequenase 
2.0, U.S. Biochemical). For data analysis we used the Genetics Computer 
Group software package (version 7.0), and database searches were 
accomplished via the BLAST server at the National Center for Biotechnology 
Information. 
EXAMPLE 2 
Analysis of ILK In Vitro 
For analysis of kinase activity in vitro, a bacterially-expressed fusion 
protein, GST-ILK.sup.132, was SDS-PAGE band purified, and incubated with 
[.gamma.-.sup.32 P]ATP in the presence or absence of the exogenous 
substrate myelin basic protein (FIG. 2). GST-ILK.sup.132 
autophosphorylated and labeled MBP efficiently in these assays (FIG. 2a). 
Anti-GST-ILK.sup.132 (antibody 91-3) immunoprecipitates of PC3 cell 
lysates were incubated with [.gamma.-.sup.32 P]ATP, similar to experiments 
performed with purified recombinant GST-ILK.sup.132. ILK immune complexes 
labeled a protein of apparent Mr of 59 kDa (FIG. 2b), corresponding to 
p59.sup.ILK, as well as cellular proteins of apparent Mr 32 kDa and 70 
kDa, which may be endogenous ILK substrates (FIG. 2b). Cellular 
phosphoproteins (serine/threonine) of approximately 32 kDa and 70 kDa, 
were also seen in .beta..sub.1 integrin-specific immune complex kinase 
assays. 
In ILK immune complex kinase assays a synthetic peptide representing the 
.beta..sub.3 cytoplasmic domain was phosphorylated, while a similar 
peptide representing the .beta..sub.3 cytoplasmic domain was not 
detectably labeled by p59.sup.ILK. The .beta..sub.1 peptide selectively 
inhibited autophosphorylation of ILK in these reactions (FIG. 2b), further 
indicating a differential interaction of the peptides with ILK. The 
results demonstrating phosphorylation of synthetic .beta. peptides by 
endogenous ILK are identical to those seen with recombinant 
GST-ILK.sup.132, and indicate the potential substrate preference of ILK 
for the .beta..sub.1 cytoplasmic tail. This does not, however, necessarily 
rule out an interaction between ILK and the .beta..sub.3 integrin 
cytoplasmic domain. Phosphoamino acid analyses of labeled p59.sup.ILK and 
MBP from the immune complex kinase assays detected only phosphoserine in 
both substrates (FIG. 2c), as was the case for phosphorylation of these 
substrates by GST-ILK.sup.132. The .beta..sub.1 peptide was labeled on 
serine and threonine residues, with approximately equal stoichiometry 
(FIG. 2). As a control, anti-FAK immune complexes from the same lysates 
were analyzed for phosphorylation of MBP, and phosphotyrosine was readily 
detected. 
FIG. 2 shows in vitro and immune-complex kinase assays. a, In vitro kinase 
reactions containing 2 .mu.g of gel-purified GST-ILK132, with and without 
5 .mu.g of myelin basic protein (MBP, Upstate Biotechnologies, Inc.), were 
analyzed by 10% SDS-PAGE. b, Immune complexes were generated from PC3 
whole cell lysates, using affinity-purified 91-3 antibody. Complexes were 
assayed for kinase activity, with and without addition of 5 .mu.g/reaction 
of synthetic peptides, representing .beta..sub.1 or .beta..sub.3 integrin 
cytoplasmic domains or MBP. Products were analyzed by 15% SDS-PAGE (kDa 
markers at left), and migration of peptides confirmed by Coomassie Blue 
staining. c, .sup.32 P-labeled products from the anti-ILK immune complex 
kinase reactions shown in b, were isolated and analyzed for phosphoamino 
acid content. Anti-FAK immune complex kinase assays demonstrated 
phosphotyrosine on MBP. 
Protein kinase assays were performed in 50 .mu.l kinase reaction buffer (50 
mM HEPES pH 7.0, 10 mM MnC.sub.2, 10 mM MgCl.sub.2, 2 mM NaF, 1 mM 
Na.sub.3 VO.sub.4), containing 10 .mu.Ci [.gamma.-.sup.32 P]ATP. Reactions 
were incubated at 30.degree. C. for 20 min, and stopped by the addition of 
SDS-PAGE sample buffer. For assay of recombinant ILK activity, 
GST-ILK.sup.132 was adsorbed from bacterial lysates onto 
glutathione-agarose beads, or GST-ILK.sup.132 was band-purified from 10% 
SDS-PAGE gels. For immune complex kinase assays, affinity-purified 91-3 
anti-ILK antibody (FIG. 3) was used to generate immunoprecipitates from 
NP-40 lysates (150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodium 
deoxycholate, 50 mM HEPES pH 7.5, 1 .mu.g/ml each leupeptin and aprotinin, 
50 .mu.g/ml phenyl-methylsulfonyl flouride) of PC3 cells. Kinase reaction 
products were resolved on 10-15% SDS-PAGE gels, transferred to PVDF, and 
phosphoamino acid analysis performed according to a published protocol. 
EXAMPLE 3 
Association of ILK and .beta. integrin in Mammalian Cells 
Immunofluorescence experiments indicated that ILK and .beta. integrin 
co-localize in focal plaques. In order to test further for this 
association in intact mammalian cells, we performed co-immunoprecipitation 
assays in lysates of PC3 cells, in which integrin expression has been 
well-characterized. PC3 cell lysates were immunoprecipitated with specific 
anti-integrin antibodies, and immune complexes analyzed by Western 
blotting with the anti-ILK antibody, 92-2. The specificities of the 
anti-ILK antibodies were tested by immunoprecipitation and Western 
blotting (FIG. 3a, b). We detected p59.sup.ILK in immune complexes 
obtained with anti-fibronectin receptor (FNR, .alpha..sub.5 /.alpha..sub.3 
.beta. integrin), and anti-vitronectin receptor (VNR, .alpha..sub.v 
.beta..sub.3 /.beta..sub.5 integrin) antibodies, but not in those obtained 
with non-immune serum (FIG. 3c). Three anti-.beta..sub.1 monoclonal 
antibodies also co-precipitated p59.sup.ILK from PC3 lysates, confirming 
the .beta. integrin specificity of p59.sup.ILK interaction (FIG. 3d). The 
detection of p59.sup.ILK in anti-VNR immune complexes suggests that ILK 
may also interact with the .beta..sub.3 and/or .beta..sub.5 integrin 
subunit(s). 
FIG. 3 shows that antibodies to GST-ILK.sup.132 recognize p59.sup.ILK in 
integrin co-immunoprecipitations. a, Unfractionated polyclonal anti-ILK 
sera 91-3 (shown) and 92-2 specifically recognize a .sup.35 S-methionine, 
metabolically-labeled cellular protein, of apparent Mr of 59 kDa. A 
fluorograph is shown (En.sup.3 Hance, NEN). b, Affinity-purified 92-2 
antibody was adsorbed with 165 .mu.g of agarose-coupled GST-ILK.sup.132, 
or agarose-GST, which preparations were used in parallel Western blots 
containing 10 .mu.g/lane of whole cell lysates of PC3 cells, Jurkat 
T-lymphoblasts, or the 60 kDa GST-ILK.sup.132. c, Polyclonal anti-integrin 
antibodies, specific for the fibronectin and vitronectin receptors, were 
used to precipitate surface-biotinylated integrins from PC3 cells, and 
immune complexes were then analyzed for the presence of p59.sup.ILK, by 
Western blotting with affinity-purified, biotin-labeled 92-2 antibody. 
This result is representative of six independent experiments. d, 
Anti-.beta..sub.1 monoclonal antibodies were used in co-precipitation 
analyses of NP-40 lysates of PC3: lane 1, A.sub.II B.sub.2 ; lane 2, 
anti-CD29; lane 3, 3S3. Western blotting of anti-.beta..sub.1 immune 
complexes with affinity-purified, biotinylated 92-2 antibody (left). This 
blot was stripped and reprobed with the same concentration of biotinylated 
92-2, adsorbed against an excess of GST-ILK.sup.132 beads (right). We 
observe co-precipitation of p59.sup.ILK using a panel of 11 
anti-.beta..sub.1 monoclonals, but not with an anti-CD44 monoclonal 
antibody. The migration of p59.sup.ILK was confirmed in parallel lanes 
containing PC3 whole cell NP-40 lysates. Markers at left, in kDa. 
Amino acid residues 132-451 of ILK were expressed as a GST fusion protein, 
in E. coli. Recombinant GST-ILK.sup.132 protein was purified and used to 
inject two rabbits. The resulting antisera, 91-3 and 92-2 (raised by 
Research Genetics, Inc.), were affinity-purified over a column of 
CNBr-Sepharose coupled GST-ILK.sup.132. PC3 cells were metabolically 
labeled with 100 .mu.Ci/ml [.sup.35 S]methionine/[.sup.35 S]cysteine 
([.sup.35 S] ProMix, 1000 Ci/mmol, Amersham), for 18 hours in 
cysteine/methionine-free MEM. For co-immunoprecipitation experiments PC3 
cells were surface-labeled with sulfo-NHS-biotin (Pierce Chemicals), prior 
to lysis in NP-40 buffer. Polyclonal anti-fibronectin receptor (anti-FNR, 
Telios A108), and anti-vitronectin receptor (anti-VNR, Telios A109) 
antibodies were purchased from Gibco/BRL. 1-2 mg of NP-40 lysate was 
incubated at 4.degree. C., with 2-3 .mu.l/ml anti-FNR or anti-VNR 
antiserum, or 2 .mu.g/ml of the anti-.beta..sub.1 monoclonal antibodies 
A.sub.II B.sub.2 (C. Damsky, UC, San Francisco), anti-CD29 (Upstate 
Biotechnology, Inc.), and 3S3 (J. Wilkins, U Manitoba). Lysates were 
pre-cleared and immune complexes collected with Protein A-Sepharose. For 
Western blotting, RIPA lysates or immune complexes were subjected to 7.5% 
or 10% SDS-PAGE, and proteins then electrophoretically transferred to 
polyvinylidene fluoride membranes (Immobilon-P, Millipore). Membranes were 
blocked in 5% non-fat milk/Tris-buffered saline Tween-20, and incubated 
with 0.5 .mu.g/ml affinity purified antibodies. Horseradish 
peroxidase-coupled goat anti-rabbit IgG was used in secondary incubations, 
followed by detection of reactive bands by enhanced chemiluminescence 
(ECL, Amersham). For blotting without use of secondary antibody (FIG. 3), 
affinity-purified 92-2 antibody was labeled with Biotin Hydrazide 
(Immunopure, Pierce Chemicals), according to the manufacturer's protocol, 
with visualization by peroxidase-conjugated streptavidin (Jackson 
ImmunoResearch Laboratories) and ECL. For re-probing, membranes were 
stripped according to manufacturer's instructions. 
EXAMPLE 4 
Overexpression of ILK Provides Growth Advantage 
The fibronectin-dependent regulation of ILK kinase activity was tested. 
Plating of rat intestinal epithelial cells, IEC-18 on fibronectin reduced 
ILK phosphorylation of MBP in immune complex kinase assays, relative to 
cells plated on plastic, or kept in suspension (FIG. 4a). This 
fibronectin-dependent reduction of ILK activity was abrogated in IEC-18 
cells expressing an activated H-ras allele, indicating that ras 
transformation disrupts ECM regulation of ILK activity in these cells. An 
expression vector containing the full-length ILK cDNA, pCMV-ILK, was 
stably transfected into IEC-18 cells. Twelve stable clones each, of 
pCMV-ILK and vector control transfectants, were selected and characterized 
for p59.sup.ILK expression levels. Two representative overexpressing 
subclones, ILK13-A1a3 and -A4a are illustrated (FIG. 4b). Overexpression 
of p59.sup.ILK disrupted the epithelial morphology of IEC-18 cells. ILK13 
clones were more refractile, and grew on LN, FN and VN with a stellate 
morphology, in marked contrast to the typical, `cobble-stone` morphology 
of the parental and ILK14 cells (FIG. 4c). We plated the ILK13-A1a3 and 
-A4a subclones, the control transfectants, ILK14-A2C3 and -A2C6, and 
IEC-18 cells, on varying concentrations of the integrin substrates, 
laminin (LN), fibronectin (FN) and vitronectin (VN). Adhesion of the ILK14 
and IEC-18 cells was equivalent, whereas that of the overexpressing 
subclones was significantly reduced, on all these substrates (FIG. 4d). 
Immunoprecipitation analysis indicated that cell surface integrin 
expression was unaffected. The effect of p59.sup.ILK overexpression on 
anchorage-independent growth was examined by assaying the colony forming 
ability of ILK transfectants in soft agarose. In marked contrast to IEC-18 
and transfectant controls, four independent p59.sup.ILK overexpressing 
subclones, ILK13-A4a, A1a3, A4d3 and A4C12, formed colonies in these 
assays (FIG. 4e). The proliferative rates of all of these clones on tissue 
culture plastic were equivalent to control rates. 
FIG. 4 shows the modulation of ILK kinase activity by ECM components. a, 
ILK phosphorylation of MBP was assayed in ILK immune complexes, from 
lysates of IEC-18 intestinal epithelial cells which were harvested from 
tissue culture plastic and either kept in suspension, or replated on 
fibronectin, for 1 hour. A H-ras-transformed variant of IEC-18, Ras37 
(transfected with Rasval12 in pRC/CMV vector), was assayed in parallel. 
The band shown is MBP. b, Expression levels of p59.sup.ILK in two 
representative clones of IEC-18 cells, transfected with an ILK expression 
construct (ILK13), two vector control clones (ILK14), and the parental 
IEC-18 cells are presented. The indicated amounts (.mu.g/lane) of whole 
cell RIPA lysates were run out on 10% SDS-PAGE gels, and p59.sup.ILK 
expression analyzed by Western blotting with affinity-purified 92-2 
antibody. c, Representative p59.sup.ILK overexpressing clone ILK13-A4a, 
vector control clone ILK14-A2C3, and parental IEC-18 cells were plated on 
the ECM substrates LN, FN and VN for 1 hour, then fixed, stained with 
toluidine blue and photographed (40.times.mag). d, Adhesion of the ILK 
overexpressing clones to LN, FN and VN was quantified. Key: IEC-18 
(black), ILK14-A2C6 (white), ILK13-A1a3 (dark grey), ILK13-A4a (light 
grey). Results are presented for 10 .mu.g/ml substrate, and are expressed 
as % adhesion (+/-s. d.) relative to IEC-18, for each substrate. The 
serial concentrations of ECM showed similar reductions in adhesion of the 
ILK13 subclones, and ILK14-A2C3 adhesion was identical to that of 
ILK14-A2C6, on all three substrates. Immunoprecipitation of 
surface-biotinylated IEC-18, ILK13, and ILK14 subclones, with the anti-FNR 
and anti-VNR sera, confirmed there was no change in expression of 
.alpha..sub.5 /.alpha..sub.3 .beta..sub.1 and .alpha..sub.v .beta..sub.3 
/.beta..sub.5 integrin subunits in the p59.sup.ILK overexpressors. Data 
are representative of two independent experiments. e, Four ILK13, 
p59.sup.ILK overexpressing clones were plated in soft agarose, and assayed 
for colony growth after three (experiment 1) and two (experiment 2) weeks. 
Parent and vector control transfectants were also assayed, and the ras 
val12 transformed clone, Ras-37, was used as a positive control. Bars 
represent the mean of duplicate determinations. Maximum colonies in IEC-18 
and ILK14 cells was 1/field. 
The rat intestinal epithelial cell line lEC-18, and a variant of this line 
transfected with an activated H-rasval12 allele, expressed from pRC/CMV, 
were grown on tissue culture plastic in 5% serum-containing medium, washed 
three times in minimum essential medium (MEM), and harvested with 5 mM 
EDTA. These were resuspended in 2.5 mg/ml BSA in MEM, and either kept in 
suspension, or plated on 10 .mu.g/ml fibronectin-coated plates, for 1 hour 
at 37.degree. C. NP-40 lysates (300 .mu.g) of these cells were 
immunoprecipitated with affinity-purified 91-3, and immune complex kinase 
assays (MBP substrate) performed, as described above. IEC-18 were 
transfected with the expression vector pRC/CMV, containing Plac5 in the 
forward orientation relative to the CMV promotor. Stable clones were 
selected in G418, and subcloned through two rounds of limiting dilution. 
In all, twelve each of ILK and vector control transfectant subclones were 
isolated. Protein concentrations were determined using the Bradford 
reagent (Bio-Rad). Two p59.sup.ILK overexpressors, ILK13-A1a3 and 
ILK13-A4a, and two vector transfectant controls, ILK14-A2C3 and -A2C6, 
were analyzed for effects of ILK overexpression on cell adhesion to ECM 
substrates. Adhesion was quantified according to published methods. For 
colony formation assays 3.times.10.sup.5 cells were plated in 35 mm wells, 
in 0.3% agarose, as described previously. Ras-37 were plated at 
2.times.10.sup.3 /well. Colonies were counted and scored per field (d=1 
cm) in duplicate wells, and defined as a minimum aggregate of 50 cells. 
These results demonstrate that p59.sup.ILK overexpression in the IEC 
epithelial cells provides a growth advantage, in the absence of 
proliferative signals normally provided by adhesion. 
The transduction of extracellular matrix signals through integrins 
influences intracellular (`outside-in`) and extracellular (`inside-out`) 
functions, both of which appear to require interaction of integrin 
cytoplasmic domains with cellular proteins. The association of ILK with 
.beta..sub.1 integrin subunits, and specific regulation of its kinase 
activity by adhesion to fibronectin, suggests that p59.sup.ILK is a 
mediator of integrin signaling. Thus the ankyrin repeat motif likely 
represents a protein interaction module specifying interactions of ILK 
with downstream, cytoplasmic or cytoskeletal proteins. Reduced ECM 
adhesion by the p59.sup.ILK overexpressing cells is consistent with our 
observation of adhesion-dependent inhibition of ILK activity, and suggests 
that p59.sup.ILK plays a role in inside-out integrin signaling. 
Furthermore the p59.sup.ILK -induced, anchorage-independent growth of 
epithelial cells indicates a role for ILK in mediating intracellular 
signal transduction by integrins. 
EXAMPLE 5 
The Effect of Anti-ILK on Cell Migration 
The role of ILK in cell motility has important implications for normal 
physiological processes such as inflammation and wound healing, as well as 
pathological conditions involving tumour invasiveness and metastatic 
tumour spread, or osteoporosis (bone is essentially an extracellular 
matrix secreted by osteoblast, or bone-forming cells, and this deposition 
can be modulated by integrin expression levels and function). Cell 
motility is a dynamic process, which is dependent on integrin-ECM 
interactions. The "on-off" switch function of protein kinases provides an 
ideal mechanism for the dynamic regulation of integrin affinity states for 
ECM substrates. The effect on cell migration of microinjecting highly 
specific anti-ILK antibodies (thereby inhibiting ILK function) into the 
cell's cytoplasm is assayed. These effects are assayed in endothelial 
cells plated on solid substrata, and are extended to include studies on 
cell migration through three-dimensional gels composed of ECM proteins. 
EXAMPLE 6 
Anti-Sense Oligonucleotides to Inhibit ILK Activity 
The sequence of ILK cDNA provides information for the design and generation 
of synthetic oligonucleotides for "anti-sense" inhibition of ILK activity. 
This term derives from the strategy of employing a reverse complement of 
the coding, or sense strand of a specific messenger RNA, known as an 
anti-sense oligonucleotide (AO). By binding to its complementary mRNA, the 
AO inhibits translation of that mRNA into protein, thereby preventing 
normal protein accumulation in the cell. ILK AO derived from the ILK mRNA 
sequence closest to the presumptive translational start site, as defined 
in FIG. 1, will be tested, as this is predicted to provide the most 
successful reagent. 
Regardless of the actual chemistry used to construct the AO, or 
modifications to an anti-ILK AO to improve its efficiency, the cDNA 
sequence of ILK provides the information for derivation of a specific AO. 
The cDNA sequence of ILK is also used to design oligonucleotide reagents, 
known as degenerate primers (due to the degeneracy of the genetic code), 
for use in polymerase chain reaction (PCR)-based screens for cDNAs 
structurally related to ILK. Similarly, the ILK cDNA is used to screen for 
related genes in a more conventional screen of genomic or cDNA libraries, 
by employing less stringent (i.e. milder) hybridization conditions during 
screening. In this way, distinct cDNA or DNA sequences significantly 
related to ILK (&gt;50% nucleotide identity) can be isolated, and a family of 
ILK-related kinases identified in a non-random fashion. 
EXAMPLE 7 
Mapping of ILK Chromosomal Locus to Assess Imprinted Copies of Gene 
High resolution mapping of the ILK chromosomal locus through fluorescent in 
situ hybridization (FISH) to metaphase (i.e. separated and identifiable) 
human chromosomes has placed the ILK gene on chromosome 11p15. FISH is 
known to those skilled in the art. High resolution mapping uses known 
marker genes in this region. Certain genes (e.g. insulin-like growth 
factor 2, IGF2) in the 11p15 region have been shown to be imprinted (i.e. 
preferentially expressed from either the maternally or paternally-derived 
chromosomes). This imprinting effectively provides a functional deletion 
or "knock-out" of one of the two inherited copies of a gene. Thus, 
mutation of the non-imprinted allele (copy) has a more profound outcome, 
since no compensatory activity is available from the imprinted allele. 
Also, 11p15 has been identified as a region subject to 
loss-of-heterozygosity, or LOH, in a subset of breast tumour patients. LOH 
results in the loss of one allele, for example by gene deletion, and is a 
mechanism underlying the contribution of a number of tumor suppressor 
genes to the development of various cancers (e.g. BRCA1 in breast, DCC in 
colon carcinoma, and RB1 in retinoblastoma). Thus ILK cDNA sequence is 
used to develop DNA reagents for the diagnosis and prognostic indications 
of a significant subset of breast cancers, and these reagents contribute 
to the molecular classification of such tumors. As mentioned above, the 
gene(s) on 11p15 contributing to some inherited cases of long QT syndrome 
are identified, and the candidacy of ILK as a causative gene for this 
cardiac condition, are evaluated by looking for alterations in ILK gene 
structure in families where 11p15 associations have been made. 
EXAMPLE 8 
Induction of in vivo Tumorigenesis by Overexpression of ILK 
Overexpression of ILK down-regulates E-cadherin which is an important 
epithelial cell adhesion molecule mediating cell-cell 
communcation/interaction. The loss of E-cadherin induced by overexpression 
of ILK in epithelial cells suggests that ILK may promote tumorigenicity in 
vivo. To test this, we injected cells expressing varying levels of ILK 
into athymic nude mice subcutaneously. Mice were inoculated subcutaneously 
with the cells expressing high (ILK13-A1a3 and A4a) or low (IEC-18 and 
ILK14-A2C3) levels of ILK (10.sup.7 cells/mouse in PBS). The mice were 
monitored for tumor formation at the site of inoculation after three 
weeks. Tumors arose within three weeks in 50% to 100% of the mice injected 
with the ILK13 cells (10.sup.7 cells/mouse) that overexpress ILK, whereas 
no tumors were detected in the mice that were injected with the same 
number of the IEC-18 or ILK14 cells expressing lower levels of ILK (Table 
1). Thus, overexpression of ILK in these epithelial cells promotes tumor 
formation in vivo. 
TABLE 1 
______________________________________ 
Tumorigenicity of ILK Overexpressing IEC-18 Cells 
Cell Line Number of Mice with Tumors at 3 weeks 
______________________________________ 
IEC-18 0/6 
ILK14-A2C3 0/6 
ILK13-A1a3 6/6 
ILK13-A4a 3/6 
______________________________________ 
EXAMPLE 9 
Increased Expression of ILK in Human Breast Carcinoma 
The expression of Integrin Linked Kinase in human breast carcinomas was 
determined by immunohistochemical staining of paraffin embedded sections 
from human breast cancer biopsies. Affinity purified anti-ILK polyclonal 
antibody was used followed by conjugated secondary antibody. The positive 
staining observed was completely abolished by absorption of the antibody 
to ILK-coupled sepharose beads. A total of 30 samples have been examined 
so far. In every case ILK expression levels are markedly elevated in tumor 
tissue compared to normal ducts and lobules. FIG. 5A shows a normal region 
showing well formed ducts with a single layer of epithelial cells. ILK 
staining is most prominent in epithelial cells. The stroma appears 
negative. FIG. 5B shows ductal carcinoma in situ (DCIS). Multiple cell 
layers are present with markedly elevated ILK staining in the tumor cells. 
Invasive carcinoma is depicted in FIGS. 5C and 5D. There is markedly 
elevated expression of ILK compared to the normal tissue shown in FIG. 5A. 
EXAMPLE 10 
Regulation of LEF-1 Expression and Complex Formation 
Overexpression of ILK results in a downregulation of E-cadherin expression, 
formation of a complex between .beta.-catenin and the HMG transcription 
factor, LEF-1, translocation of .beta.-catenin to the nucleus, and 
transcriptional activation by this LEF-1/.beta.-catenin complex. LEF-1 
protein expression is rapidly modulated by cell detachment from the 
extracellular matrix, and that LEF-1 protein levels are constitutively 
upregulated upon ILK overexpression. These effects are specific for ILK, 
since transformation by activated H-ras or v-src oncogenes do not result 
in the activation of LEF-1/.beta.-catenin. The results demonstrate that 
the oncogenic properties of ILK involve activation of the 
LEF-1/.beta.-catenin signaling pathway via elevation of LEF-1 expression. 
Overexpression of ILK in rat intestinal epithelial cells (IEC-18) induces a 
loss of epithelial morphology, characterized by a disruption of cell-cell 
adhesion and the acquisition of a fibroblastic morphology that includes 
enhanced fibronectin matrix assembly. This altered morphology is 
accompanied by the ability of the cells to progress through the cell cycle 
in an anchorage-independent manner and to form tumors in nude mice. To 
determine whether the loss of cell-cell adhesion was accompanied by an 
increased invasive phenotype, the invasiveness of IEC-18 parental cells 
and ILK-overexpressing (ILK-13) cells was tested in a collagen gel 
invasiveness assay. The data is shown in Table 2. 
The ILK-13 cells are much more invasive than the parental and control 
transfected (ILK-14) cells that have been transfected with an ILK 
anti-sense cDNA construct. Collagen-gel invasion by epithelial cells is 
normally associated with an epithelial to mesenchymal transformation 
characterized by the down regulation of E-cadherin expression. Notably, 
the expression of E-cadherin protein is completely lost in ILK 
overexpressing cells (ILK-13), but is maintained in control transfected 
cells, reduced in IEC-18 cells transfected with activated H-ras cDNA, and 
greatly reduced in v-src transformed cells. In contrast, the steady-state 
levels of the expression of the intracellular E-cadherin binding protein, 
.beta.-catenin, is unchanged by ILK overexpression and is similar in all 
IEC cell transfectants. 
The subcellular localization of .beta.-catenin was examined in these cells. 
In sharp contrast to the localization of .beta.-catenin at the plasma 
membrane and at cell-cell adhesion sites in the parental IEC-18 and 
control transfected cell clones (A2c3 and A2c6), .beta.-catenin is 
localized entirely in the nuclei of ILK overexpressing ILK-13 clones (A4a, 
A1a3). This ILK-induced nuclear localization of .beta.-catenin is 
dependent on an active kinase, since overexpression of a kinase-deficient 
ILK (E359K) did not induce nuclear translocation of .beta.-catenin which 
remains localized largely to the plasma membrane. Likewise, overexpression 
of kinase-deficient ILK also did not result in a loss of E-cadherin 
expression. The translocation of .beta.-catenin to the nucleus is a 
specific property of ILK, since in IEC-18 cells transfected with activated 
H-ras or v-src oncogenes, .beta.-catenin is not translocated to the 
nucleus, but is either localized to the plasma membrane or is expressed 
diffusely in the cytoplasm. Although these oncogenes also disrupt the 
epithelial morphology of IEC-18 cells and result in the downregulation of 
E-cadherin expression, the translocation of .beta.-catenin to the nucleus 
is a property unique to ILK expression, suggesting that loss of E-cadherin 
expression and .beta.-catenin nuclear translocation may be regulated 
differentially. Overexpression of ILK in mouse mammary epithelial cells 
also results in similar alterations in the phenotypic properties described 
above for the IEC-18 cells. 
Translocation of .beta.-catenin to the nucleus can be induced by the 
activation of the Wnt signaling pathway, which initially results in an 
elevation of free cytosolic .beta.-catenin due to decreased degradation. 
Alternatively, loss of expression or mutations in the tumor suppressor 
protein APC and certain mutations in the .beta.-catenin gene lead to 
cytosolic .beta.-catenin stabilization and nuclear translocation. The 
nuclear translocation of .beta.-catenin is associated with complex 
formation between .beta.-catenin and members of the HMG transcription 
factors, LEF-1/TCF which then activate (or silence) transcription of 
target genes. Since the steady state levels of .beta.-catenin were not 
changed by ILK overexpression "uncomplexed" .beta.-catenin levels were 
measured, as determined by binding to a cytoplasmic domain peptide of 
E-cadherin. "Uncomplexed " pools of .beta.-catenin in ILK overexpressing 
clones were found to be low and unaltered compared to IEC-18 cells or 
control ILK 14 clones. This indicates that most .beta.-catenin is likely 
complexed with nuclear components such as transcription factors and DNA. 
In contrast, free P-catenin pools in Ras and Src transformed cells were 
high consistent with decreased E-cadherin expression and indicating 
disruption of E-cadherin-.beta.-catenin interaction. However, the 
increased free pools of .beta.-catenin did not result in nuclear 
translocation of .beta.-catenin. 
The expression levels of LEF-1, a member of the family of HMG transcription 
factors that bind .beta.-catenin, were measured. The expression of LEF-1 
is dramatically higher in six independent ILK expressing ILK-13 cell 
clones as compared with six independent control transfected ILK-14 clones, 
as well as 2 activated H-ras transfected and v-src transfected IEC-18 
clones. E-cadherin expression is lost in all 6 ILK-13 cell lines. 
Transient induction of ILK expression using an ecdysone inducible ILK 
construct also resulted in an increase of LEF-1 expression. As expected, 
the increased levels of LEF-1 and the nuclear translocation of 
.beta.-catenin are associated with enhanced complex formation between 
LEF-1 and .beta.-catenin in the ILK overexpressing cells. 
LEF-1 is a transcription factor that is by itself, unable to stimulate 
transcription from multimerized binding sites, however in association with 
.beta.-catenin, LEF1/TCF proteins can augment promoter activity from 
multimerized binding sites. Transcriptional activation from a 
TCF/.beta.-catenin responsive promoter construct was examined in 
ILK-overexpressing cells and control kinase-deficient ILK expressing 
cells. High promoter activity was observed in ILK-overexpressing cells and 
the extent of transcriptional activation was reduced with promoter 
constructs containing mutations in the multimerized LEF-1/TCF binding 
sites. Moreover, nuclear extracts were analyzed from ILK-overexpressing 
cell clones and from cell clones transfected with an anti-sense or 
kinase-deficient ILK cDNA to identify proteins that bind the LEF/TCF 
binding site. The abundance of a nuclear factor in ILK-overexpressing 
cells that displays the same binding site specificity, immunoreactivity 
and electrophoretic mobility as LEF-1, was found to be markedly enhanced 
relative to the unrelated DNA-binding protein Oct-1. 
ILK binds to the cytoplasmic domain of .beta..sub.1 and .alpha..sub.3 
integrin subunits, and its kinase activity is downregulated upon cell 
adhesion to extracellular matrix (ECM) proteins. Overexpression of 
constitutively activated ILK overcomes this regulation of ILK activity by 
integrin occupation and results in decreased cell adhesion to ECM-protein. 
Cell adhesion to ECM suppresses LEF-1 expression, which is rapidly, but 
transiently, elevated upon cell detachment in ILK-14 and ILK13 cells. 
However in ILK overexpressing ILK-13 cells the elevation in LEF-1 levels 
are more robust and are maintained at high levels for as long as 16 hours 
in suspension. Furthermore, LEF-1 levels are also higher in adherent 
ILK-13 cells compared to ILK-14 cells. 
These data indicate that ILK overexpression overcomes the regulation of 
LEF-1 expression by adhesion-deadhesion, and that the maintenance of 
constitutively high levels of LEF-1 result in enhanced complex formation 
between LEF-1 and .beta.-catenin, translocation of .beta.-catenin to the 
nucleus, and transcriptional activation of responsive genes. Since 
TCF/.beta.-catenin has been shown to induce transcription of genes 
encoding homeobox proteins that regulate mesenchymal genes eg. Siamois in 
Drosophila, this pathway is likely to mediate the observed epithelial to 
mesenchymal transformation, as well as the oncogenic properties of ILK in 
these intestinal epithelial cells, since constitutive activation of 
TCF/.beta.-catenin is oncogenic in human colon carcinomas. The data 
presented here also suggest a connection between the expression of 
E-cadherin and the signaling properties of .beta.-catenin in mesenchymal 
induction in ILK transformed cells, in agreement with the work of others 
that E-cadherin can antagonize .beta.-catenin signaling, although the loss 
of E-cadherin expression does not always correlate with nuclear 
.beta.-catenin translocation e.g. in the v-src transformed cells. 
An additional pathway is demonstrated to that by activated Wnt-1 leading to 
increased LEF-1/.beta.-catenin complex formation and transcriptional 
activation. These data also corroborate previous work showing that 
overexpression of LEF-1 can work independently of Wnt to enhance 
LEF-1-.beta.-catenin complex induced transcription. Here it is shown that 
in contrast to the effects of Wnt-1, activated ILK can dramatically induce 
the formation and nuclear translocation of LEF-1/.beta.-catenin complexes 
without a corresponding increase in the free pool of .beta.-catenin. This 
ILK-regulated pathway may be modulated via cell adhesion to ECM, but can 
be constitutively activated by ILK overexpression. 
Methods 
Cells and cell culture. IEC-18 rat intestinal epithelial cells were stably 
transfected with a mammalian vector incorporating ILK to produce clones 
overexpressing wt ILK in the sense orientation (ILK-13) or antisense 
orientation (ILK-14), or to produce a kinase-deficient form of ILK 
(IEC-18GH31RH) described below. IEC-18 cells were also stably transfected 
to overexpress H-ras (Ras 33, Ras 37) (Buick et al. (1987) Exp. Cell. Res. 
170:300-309), and v-src (Src2, Src4) (Filmus et al. (1988) Mol. Cell. 
Biol. 8:4243-4249). Cells were grown in d-MEM containing 5% FCS, 2 mm 
L-glutamine, glucose (3.6 mg/ml), insulin (10 ug/ml), and G418 (40 ug/ml) 
was added to transfected cells to maintain selection pressure. 
Site directed mutagenesis of ILK kinase domain. Mutations were introduced 
into wt ILK-cDNA with the Promega Altered Sites II System (Promega, 
Madison Wis.). Mutant oligomers (with the altered nucleotide underlined) 
were used to change lysine at position 220 to an arginine (K220R, (SEQ ID 
NO:9) 5' CCTTCAGCACCCTCACGACAATGTCATTGCCC 3') and glutamic acid at 
position 359 to lysine (E359K, (SEQ ID NO:10) 5' 
CTGCAGAGCTTTGGGGGCTACCCAGGCAGGTG 3'). Mutant clones were confirmed by 
dideoxy sequencing and subcloned into pGEX4T-1 GST fusion vector 
(Pharmacia, Piscataway N.J.) to express GST-ILK in E. coli (BL21-DE3) and 
into pcDNA3 (Invitrogen, San Diego, Calif.) to stably transfect 
kinase-deficient ILK into the IEC-18 cell line (IEC-18GH31RH containing 
the E359K mutation). 
Inducible expression of ILK. Full length wt ILK cDNA (1.8 Kb) was subcloned 
into the Ecdysone-inducible expression vector pIND (Invitrogen, San Diego, 
Calif.) and 10 ug were transiently co-transfected with 10 ug of the 
complementary regulator vector pVgRXR into subconfluent cells growing in 6 
well plates with 20 ul of Lipofectin (Gibro-BRL, Gaithersburg, Md.). ILK 
expression was induced 6 hrs later with the addition of 1 uM muristerone 
A. 
Western blotting and immunoprecipitation. Cells were lysed for 10 minutes 
on ice in NP-40 lysis buffer (1% NP40, 50 mM Hepes, pH 7.4, 150 mM NaCl, 2 
mM EDTA, 2 mM PMSF, 1 mM Na-o-vanadate, 1 mM NaF, 10 ug/ml aprotinin, 10 
ug/ml leupeptin). Extracts were centrifuged with the resulting 
supernatants being the cell lysate used in assays. Lysates were 
electrophoresed through SDS-PAGE and transferred to Immobilon-P membranes 
(Millipore, Bedford, Md.). Antibodies used to probe Western blots were: 
rabbit anti-ILK, monoclonal anti-E-cadherin and monoclonal 
anti-.beta.-catenin (Transduction Labs, Lexington, Ky.), and rabbit 
anti-LEF-1 (Travis et al. (1991) Genes & Development 5:880-894). Bands 
were visualized with ECL chemiluminescent substrate (Amersham, 
Buckinghamshire, England). For immunoprecipitation, NP-40 lysates were 
rotated with primary antibody ON at 4.degree. C., then rotated with 
Protein G-Sepharose (Pharmacia, Uppsala, Sweden) for 2 hrs at RT. Beads 
were pelleted, boiled in electrophoresis sample buffer (non-reducing), 
centrifuged and supernatants were electrophoresed. Protein concentrations 
were determined by the Bradford assay (Bio-Rad, Hercules, Calif.). 
Invasion assay. Confluent cells were trypsinized and 7.5.times.10.sup.4 
cells in 1.5 ml of complete medium were seeded onto 1.5 ml of a three 
dimensional collagen gel in a 35 mm tissue culture dish (Montesano et al. 
(1985) Cell 42:469-477). Upon reaching confluence (3 days), the cultures 
were incubated for a further 4 days, then fixed in situ with 2.5% 
glutaraldehyde in 100 mM cacodylate buffer (pH 7.4), and photographed at 
different planes of focus. Invasion was quantitated by counting the number 
of cells which had migrated below the surface of the collagen gel. Five 
randomly selected fields measuring 1.0 mm.times.1.4 mm were photographed 
at a single level beneath the surface monolayer using a 10.times. phase 
contract objective. 
Indirect immunofluorescence. Cells were grown on cover slips, washed with 
PBS, fixed in 4% paraformaldehyde in PBS for 12 minutes, washed with PBS, 
permeabilized in 0.1% Triton X-100 in PBS for 10 minutes, blocked with 4% 
BSA in PBS for 30 minutes at RT, incubated with rabbit anti-.beta.-catenin 
(Hulsken et al. (1994) J. Cell Biol. 127:2061-2069) diluted 1:400 in 0.1% 
Triton X-100 for 60 minutes at 37.degree. C., washed with PBS, incubated 
with rhodamine conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, 
West Grove, Pa.) diluted 1:50 in 0.1% Triton X-100 for 30 minutes at 
37.degree. C., washed with PBS, then mounted onto a slide with Slow-Fade 
Antifade (Molecular Probes Inc., Eugene, Oreg.). Cells were viewed at 100 
fold magnification using a Zeiss Axiovert 135 fluorescence microscope. 
Reportergene assay. Cells were transiently transfected with 0.3 ug of a 
luciferase reporter gene construct containing a series of optimal or 
mutated LEF-1/TCF binding sites (Korinek et al. (1997) Science 
275:1784-1787), along with 0.05 ug of a CAT gene construct containing a 
ribosomal promoter (Hariharan et al. (1989) Genes & Development 
3:1789-1800) to control for transfection efficiency. Extracts were 
prepared and assayed 48 hours after transfection. 
Electrophoretic mobilty shift assay. Twenty .mu.g of nuclear extract were 
incubated with 1 fmole of .sup.32 P-labeled duplex oligonucleotide probe 
specific for LEF-1, in 20 .mu.l of binding buffer containing 200 ng 
poly[d(I-C)], 400 ng salmon sperm DNA, and electrophoresed through a 5% 
native polyacrylamide gel (Travis et al. (1991) Genes and Development 
5:880-894). For DNA competition, an 800-fold molar excess of 
oligonucleotide containing a specific LEF-1 binding site or a non-specific 
EBF-binding site (Hagman et al. (1991) EMBO J. 10:3409-3417) was included 
in the DNA-binding reaction. For antibody addition, 1 ul of polyclonal 
anti-LEF-1 antibody or 1 ul of monoclonal anti-.beta.-catenin antibody 
(Transduction Labs, Lexington, Ky.) were used. 
TABLE 2 
______________________________________ 
INVASION OF COLLAGEN GELS 
Cell Line Invading cells/field 
______________________________________ 
IEC-18 10 +/- 0.87 
ILK14/A2c 67.8 +/- 1.32 
ILK13/A1a 326.73 +/- 2.61 
ILK-13/A4a 83.6 +/- 4.68 
______________________________________ 
After seeding 7.5.times.10.sup.4 cells, the number of invading cells in 5 
photographic fields from 3 separate experiments (total of 15 fields/cell 
line) were counted. Results are given as the mean number of invading 
cells.+-.SEM. *p&lt;&lt;0.01 between ILK13/A1a3 compared to IEC-18 and ILK-14 
cells (Students unpaired t=test). 
EXAMPLE 11 
Regulation of Fibronectin Matrix Assembly, E-cadherin Expression and 
Tumorigenicity 
A common feature of many oncogenically transformed cells is that they lose 
the ability of assembling a fibronectin (Fn) matrix. However, exceptions 
to the rule of neoplastic cells lacking Fn matrix clearly exist. For 
example, Fn matrix assembly is dramatically enhanced in hairy cell 
leukemia cells. The specific phenotype (inhibition or stimulation of Fn 
matrix assembly) is probably determined by the origin of the neoplastic 
cells and the initial target of the oncogenic transformation. Because Fn 
matrix has a major impact on cell adhesion, migration, cell growth and 
cell differentiation, an understanding of the molecular mechanism by which 
cells control Fn matrix assembly may provide important information on 
tumorigenicity and may lead to new ways of controlling tumor growth. 
Binding of Fn by specific integrins is critical in initiating Fn matrix 
assembly. Fn fragments containing the RGD-containing integrin binding site 
or antibodies recognizing the integrin binding site inhibit Fn matrix 
assembly. In addition, antibodies to .alpha..sub.5 .beta..sub.1 integrin 
reduce the deposition of Fn into extracellular matrix by fibroblasts. In 
addition to .alpha..sub.5 .beta..sub.1 integrin, members of the 
.beta..sub.3 integrins (.alpha..sub.IIb .beta..sub.3 and .alpha..sub.v 
.beta..sub.3) also initiate Fn matrix assembly, although some of the other 
Fn binding integrins such as .alpha..sub.4 .beta..sub.1 or .alpha..sub.v 
.beta..sub.1 do not. The ability of cells to use multiple integrins to 
support Fn matrix assembly provides the cells with a versatile mechanism 
for control of Fn matrix assembly. It may also explain why certain cells, 
such as fibroblastic cells derived from .alpha..sub.5 integrin null mutant 
embryos, assemble a Fn matrix in the absence of .alpha..sub.5 
.beta..sub.1. The primary role of .alpha..sub.5 .beta..sub.1 in Fn matrix 
assembly appears to involve initiating the assembly, as Fn mutants lacking 
the .alpha..sub.5 .beta..sub.1 integrin binding site could not be 
assembled into Fn matrix unless in the presence of native Fn. 
Activation of specific Fn binding integrins, either by mutations at the 
integrin cytoplasmic domains or using activating antibodies, dramatically 
stimulates Fn matrix assembly. The ability of a cell to assemble a Fn 
matrix is not only controlled by the types of integrins it expresses but 
also regulated by the Fn binding activity of the integrins. The 
extracellular ligand binding affinity of integrins can be controlled from 
within the cells (inside-out signaling). 
Integrin-linked kinase (ILK) may be involved in regulating Fn matrix 
assembly. ILK binds to the cytoplasmic domains of both .beta..sub.1 and 
.beta..sub.3 integrins, and phosphorylates the .beta..sub.1 cytoplasmic 
domain in vitro. Overexpression of ILK in epithelial cells dramatically 
stimulated integrin-mediated Fn matrix assembly, down-regulated 
E-cadherin, and induced tumor formation in vivo. The data identify ILK as 
an important regulator of pericellular Fn matrix assembly, and suggest a 
critical role of this integrin-linked kinase in cell-cell interactions and 
tumorigenesis. 
Reagents 
All organic chemicals were of analytic grade and were obtained from Sigma 
Chemical Co. (St. Louis, Mo.) or Fisher Scientific Co. (Pittsburgh, Pa.) 
unless otherwise specified. Media for cell culture were from Gibco 
Laboratories (Grand Island, N.Y.). Fetal bovine serum was from HyClone 
Laboratories, Inc. (Logan, Utah). Polyclonal rabbit anti-.alpha..sub.5 
integrin cytoplasmic domain antibody AB47 was generated using a synthetic 
peptide representing the membrane distal region of the .alpha..sub.5 
integrin cytoplasmic domain ((SEQ ID NO:11) LPYGTAMEKAQLKPPATSDA). 
Polyclonal rabbit anti-Fn antibody MC54 was raised against purified plasma 
Fn and purified with a protein A-Sepharose affinity column (Wu et al. 
(1993) J. Biol. Chem. 268:21883-21888). Polyclonal rabbit anti-29 kDa 
fragment of Fn antibody was raised against the aminoterminal 29 kDa 
fragment of Fn and was further purified using Sepharose beads coupled with 
the 29 kDa fragment of Fn (Limper et al. (1991) J Biol. Chem. 
266:9697-9702). Anti-ILK polyclonal antibody 91-4 was prepared in rabbits 
as described previously (Hannigan et al. (1996) Nature 379:91-96). 
Monoclonal hamster anti-rat .alpha.5 integrin antibody (HM.alpha.5-1) and 
mouse anti-rat .beta..sub.3 integrin antibody (F11) were from PharMingen 
(San Diego, Calif.). Monoclonal mouse anti-vinculin antibody (hVIN-1) and 
purified rabbit IgG were purchased from Sigma (St. Louis, Mo.). The Fn 
fragments (110 kDa RGD containing integrin binding fragment, the 20 kDa 
and 70 kDa amino terminal fragments, and the 60 kDa gelatin binding were 
prepared as previously described (Quade and McDonald (1988) J. Biol. Chem. 
263:19602-19609). cDNA Vectors, Transfection and Cell Culture. Rat 
intestinal epithelial cells (IEC-18) were maintained in .alpha.-MEM medium 
(Gibco Laboratories, Grand Island, N.Y.) supplemented with 5% FBS (Atlanta 
Biologicals, Norcross, Ga.), 3.6 mg/ml glucose, 10 .mu.g/ml insulin and 2 
mM glutamine. The pRC/CMV and metallothionein promoter (MT) driven 
expression vectors containing sense and anti-sense full length ILK cDNA 
sequences were generated as described above. The expression vectors were 
transfected into IEC-18 cells using calcium phosphate and the transfected 
cells were selected with G418 as described. The expression of human ILK in 
IEC-18 cells transfected with the MT-ILK expression vectors (MT-ILK) was 
induced by growing the cells in .alpha.-MEM medium containing 125 .mu.M 
ZnSO.sub.4 and 2.5 .mu.M CdCl.sub.2 for 24 to 48 hours. The 
kinase-inactive ILK mutant (GH31R) was generated by a single point 
mutation (E.fwdarw.K) at amino acid residue 359 within the kinase 
subdomain VIII using the Promega Altered Site II in vitro Mutagenesis 
System. The mutated DNA was cloned into a pGEX expression system 
(Pharmacia), and expressed as a GST fusion protein. Kinase assays were 
carried out using the recombinant protein as described above and the 
results showed that the E.sup.359 .fwdarw.K point mutation completely 
inactivated the kinase activity. The cDNA encoding the kinase-inactive 
mutant was cloned into a pcDNA3 expression vector (Invitrogen), 
transfected into IEC-18 cells and stable transfectants were selected. 
Determination of ILK, E-cadherin and .beta..sub.1 integrin levels. The 
cellular levels of ILK and E-cadherin were determined by immunoblot using 
an affinity-purified polyclonal rabbit anti-ILK antibody 91-4, and an 
anti-E-cadherin antibody (Upstate Biotechnologies, Inc.). The cell surface 
expression of .alpha..sub.5 .beta..sub.1 integrins was estimated by 
immunoprecipitation of surface biotinylated cell lysates with a polyclonal 
rabbit anti-.alpha..sub.5 .beta..sub.1 antibody. 
Immunofluorescent Staining. Fn matrix assembly was analyzed by 
immunofluorescent staining of cell monolayers (Wu et al. (1995) Cell 
83:715-724). Cells were suspended in the .alpha.-MEM medium containing 5% 
FBS and other additives as specified in each experiment. Cells were plated 
in 12-well HTC.sup.R slides (Cel-Line, Inc., Newfield, N.J.; 50 
.mu.l/well) at a final density of 2.times.10.sup.5 cells/ml and cultured 
in a 37.degree. C. incubator under a 5% CO.sub.2 -95% air atmosphere. 
Cells were fixed with 3.7% paraformaldehyde, and staining with the 
polyclonal rabbit anti-Fn antibody MC54 (20 .mu.g/ml) and Cy3-conjugated 
goat anti-rabbit IgG antibodies (Jackson ImmunoResearch Lab, Inc, West 
Grove, Pa.; 2.5 .mu.g/ml). Stained cell monolayers were observed using a 
Nikon FXA epifluorescence microscope and representative fields were 
photographed using Kodak T-Max 400 or Ektachrome 1600 direct positive 
slide film. To obtain representative images, exposure times for different 
experimental conditions were fixed, using the positive, e.g., matrix 
forming cells, as the index exposure length. 
In double staining experiments, 3.7% paraformaldehyde fixed cells were 
permeablized with 0.1% Triton X-100 in TBS containing 1 mg/ml BSA. The 
cells were then incubated with primary antibodies from different species 
as specified in each experiment. After rinsing, the bound primary 
antibodies were detected with species-specific Cy3- and FITC-conjugated 
secondary antibodies. Stained cell monolayers were observed using a Nikon 
FXA epifluorescence microscope equipped with Cy3 and FITC filters. 
For inhibition studies, ILK13-A4a cells that overexpress ILK were plated in 
12-well HTC.sup.R slides in the .alpha.-MEM medium containing 5% FBS and 
other additives as specified (2 .mu.M anti-29 kDa Fn fragment antibody, 2 
.mu.M rabbit control IgG, or 4.2 .mu.M of one of the following Fn 
fragments: 110 kDa RGD containing integrin binding fragment of Fn, 70 kDa 
aminoterminal fragment of Fn or 60 kDa gelatin binding fragment of Fn). 
The cells were cultured for four hours, and then fixed and stained with 
the polyclonal rabbit anti-Fn antibody and the Cy3-conjugated goat 
anti-rabbit IgG antibodies as described above. 
Isolation and Biochemical Characterization of Extracellular Matrix Fn. To 
isolate and biochemically characterize extracellular matrix Fn, the cells 
were cultured in 100 mm tissue culture plates (Corning, Inc., Corning, 
N.Y.) in .alpha.-MEM medium supplemented with 5% FBS, 2 mM L-glutamine, 
3.6 mg/ml glucose, 10 .mu.g/ml insulin and other additives as specified in 
each experiment for two days. Then the cell monolayers were washed three 
times with PBS containing 1 mM AEBSF and harvested with a cell scraper. 
The extracellular matrix fraction was isolated by sequential extraction of 
the cells with (1) 3% Triton X-100 in PBS containing 1 mM AEBSF; (2) 100 
.mu.g/ml DNase I in 50 mM Tris, pH 7.4, 10 mM MnCl.sub.2, 1 M NaC, 1 mM 
AEBSF and (3) 2% deoxycholate in Tris, pH 8.8, 1 mM AEBSF (Wu et al., 
supra.) Fn in the deoxycholate insoluble extracellular matrix fraction was 
analyzed by immunoblot with polyclonal rabbit anti-Fn antibody MC54 and an 
ECL detection kit as previously described (Wu et al. (1995) J. Cell Sci. 
108:821-829). 
Colony formation in soft agar. ILK13-A1a3 cells that overexpress ILK 
(3.times.10.sup.5 /well), and Ras-37 cells that overexpress H-RasVal12 
(2.times.10.sup.3 /well) were plated in 35 mm wells, in 0.3% agarose and 
assayed for colony growth after three weeks as described above. Fn 
fragments were incorporated in the agar at the final concentrations 
indicated. 
Tumor formation in athymic nude mice. IEC-18, ILK14, or ILK13 cells were 
resuspended in PBS and inoculated subcutaneously into athymic nude mice 
(10.sup.7 /mouse). Six mice were inoculated per cell line. In situ tumor 
formation was assessed after 3 weeks. 
Tyrosine Phosphorylation of p125.sup.FAK in ILK cells. ILK13-Ala3 and 
ILK14-A2C3 cells growing in monolayer culture were harvested using 5 mM 
EDTA/PBS (Phosphate Buffered Saline, pH 7.6) and the cells were washed 
twice in PBS. Cells were resuspended in serum free medium and then 
transferred to plain tissue culture plates (Nunc), tissue culture plates 
precoated with 10 .mu.g/ml Fn (Gibco/BRL) or maintained in suspension. For 
the suspension control cells were kept in 50 ml rocker tube. After 1 hour 
incubation at 37.degree. C. in 5% CO.sub.2 cell monolayer (for the 
adherent controls) and cell pellet (for the suspension controls) were 
washed twice in ice-cold PBS and lysed in NP-40 lysis buffer (1% NP-40; 
150 mM NaCl; 50 mM Tris, pH 7.4; 1 mM EDTA, 1 mM PMSF, 0.2 U/ml aprotonin, 
2 .mu.g/ml leupeptin and 1 mM Sodium Vanadate). FAK was immunoprecipitated 
from 400-500 .mu.g of total cell extract using 4 .mu.g mouse monoclonal 
anti-p125.sup.FAK antibody and Protein A-Agarose conjugate (UBI). Immune 
complexes were washed three times in lysis buffer, boiled in SDS-PAGE 
sample buffer and run on a 7.5% gel. Resolved proteins were transferred to 
Immobilon-P (Millipore) and membrane blocked in 5% BSA (Sigma) in TBST 
(0.1% Tween-20 in Tris Buffered Saline, pH 7.4). Tyrosine-phosphorylated 
FAK was detected using the RC20H recombinant antibody (HRP-conjugate, 
Transduction) and ECL detection system (Amersham). 
Results 
Stimulation of Fn matrix assembly by ILK, To determine whether ILK plays a 
role in regulation of Fn matrix assembly, the ability of cells expressing 
different levels of ILK to assemble a Fn matrix was analyzed. IEC-18 rat 
intestinal epithelial cells assembled a small amount of Fn matrix 
consisting of mostly short fibrils. ILK13-A1a3 cells, which were isolated 
from the IEC-18 cells stably transfected with a pRC/CMV expression vector 
containing full length ILK coding sequence, express a much higher level of 
ILK than the parental IEC-18 cells. The ILK overexpressing ILK13-A1a3 
cells assembled an extensive Fn matrix resembling that formed by 
fibroblasts, whereas control transfectants (ILK14-A2C3), which express a 
similar level of ILK as the parental IEC-18 cells, assembled a small 
amount of Fn matrix that is indistinguishable from that of the IEC-18 
cells fibroblasts. To exclude the possibility that the observed effect 
depends on a specific clone, ten additional cell lines were analyzed that 
were independently isolated from the cells transfected with the 
pRC/CMV-ILK expression vector (ILK13-A4a, A1d11, A4c, A4c3 and A4i) or the 
control vector (ILK14-A2C6, A2a3, A2g3, A2g8 and A3a1) Fn matrix assembly 
was dramatically increased in all six ILK-overexpressing cell lines (Table 
3). On the other hand, all six control cell lines assembled a low level of 
Fn matrix resembling that of the parental IEC-18 cells. In marked contrast 
to overexpression of ILK, overexpression of an oncogenic H-Ras mutant in 
which the twelfth amino acid residue is mutated (H-RasVal12) in the IEC-18 
cells abolished the assembly of Fn fibrils. 
TABLE 3 
______________________________________ 
Fn matrix assembly by cells expressing different levels of ILK 
Extracellular 
Cell Line ILK Expression level Fn matrix level 
______________________________________ 
ILK13 (A1a3, A4a, A1d11, A4c 
High (wild type ILK) 
High 
ILK14 (A2C6, A2C3, A2a3, Low (wild type ILK) Low 
A2g3, A2g8 and A3a1), IEC-18, 
MT-ILK6 (E2) 
GH31RH High (kinase-inactive Low 
mutant) 
______________________________________ 
The ILK 13 cell lines were independently isolated from IEC18 rat 
intestinal epithelial cells that were stably transfected with a pRC/CMV 
expression vector containing full length ILK coding sequence and they 
express a much higher level of ILK than the parental IEC18 cells. The ILK 
14 cells were control transfectants (41). The MTILK1 (11B8) cells were 
isolated from IEC18 cells transfected with the sense ILK expression vecto 
(MTILK1). # The MTILK6 (E2) cells were isolated from IEC18 cells 
transfected with the antisense ILK expression vector (MTILK6). The GH31R 
cells were isolated from IEC18 cells transfected with a pCDNA3 expression 
vector encoding a ILK kinaseinactive mutant in which glutamic acid residu 
359 was replaced with a lysine residue. The relative ILK expression level 
were based on immunoblot analysis with antiILK antibodies. 
To further confirm a regulatory role of ILK in Fn matrix assembly, IEC-18 
cells were transfected with expression vectors containing full length ILK 
cDNA in the forward (sense) or the reverse (anti-sense) orientation that 
were under the control of metallothionein promoter (MT). The MT-ILK1 
(IIB8) cells, which were derived from the IEC-18 cells transfected with 
the sense ILK expression vector, expressed more ILK than the MT-ILK6 (E2) 
cells that were derived from the IEC-18 cells transfected with the 
anti-sense ILK expression vector. The difference in ILK expression was 
maximized when the cells were grown in the presence of Zn.sup.++ and 
Cd.sup.++. Consistent with a critical role of ILK in Fn matrix assembly, 
the ILK overexpressing MT-ILK1 (IIB8) cells exhibit a much high Fn matrix 
assembly than the MT-ILK6 (E2) cells that have a much lower level of ILK. 
Thus, overexpression of ILK, either driven by a CMV promoter or driven by 
a metallothionein promoter, stimulates Fn matrix assembly. 
Involvement of integrin-linked kinase activity in the cellular regulation 
of Fn matrix assembly. To test whether the kinase activity is involved in 
the stimulation of Fn matrix assembly by ILK, a kinase-inactive ILK mutant 
(GH31R) was overexpressed in the IEC-18 cells. Unlike cells overexpressing 
the wild type ILK, cells overexpressing the kinase-inactive ILK mutant did 
not assemble an increased amount of Fn into the extracellular matrix (FIG. 
1D). Thus, the kinase activity is critical in the cellular signal 
transduction leading to the up-regulation of Fn matrix assembly. 
Biochemical characterization of Fn matrix assembled by cells overexpressing 
ILK. The Fn matrix deposited by fibroblastic cells is characterized by 
insolubility in sodium deoxycholate. To determine whether Fn matrix 
induced by overexpression of ILK in the epithelial cells shares this 
characteristic, the cell layers were extracted with 2% sodium deoxycholate 
and the insoluble matrix fractions analyzed by immunoblotting. The cells 
overexpressing ILK (A1a3, A4a and IIB8) assembled much more Fn into the 
deoxycholate insoluble matrix than the cells that express relatively low 
level of ILK (A2C6, A2C3, and E2). By contrast, cells overexpressing 
H-RasVal12 failed to deposit detectable amount of Fn into the detergent 
insoluble matrix (H-Ras). These results are consistent with the 
immunofluorescent staining data. Taken together, they provide strong 
evidence supporting an important role of ILK in regulation of Fn matrix 
assembly. 
Participation of the RGD containing integrin-binding domain and the amino 
terminal domain of Fn in ILK stimulated Fn matrix assembly. 
Integrin-mediated Fn matrix assembly requires at least two discrete 
portions of Fn, the RGD containing integrin-binding domain and the 
aminoterminal domain. To determine whether these domains also participate 
in Fn matrix assembly induced by overexpression of ILK, the 110 kDa RGD 
containing fragment, the 70 kDa aminoterminal domain of Fn, and an 
antibody against the amino terminal domain of Fn (anti-29 kDa) were 
utilized. Both the antibody and the Fn fragments decreased the Fn fibril 
formation induced by ILK. The inhibition was specific, as neither 
irrelevant rabbit IgG nor a 60 kDa Fn Fragment lacking the amino terminus 
inhibited the Fn matrix assembly. Thus, both the RGD containing 
integrin-binding domain and the amino terminal domain of Fn are involved 
in Fn matrix assembly promoted by overexpression of ILK, suggesting a role 
of Fn-binding integrins in this process. 
Co-distribution of .alpha.5.beta.1 integrin and Fn matrix in cells 
overexpressing ILK. To begin to identify which Fn-binding integrin 
mediates Fn matrix assembly induced by overexpression of ILK, cells 
overexpressing ILK were stained with a hamster monoclonal anti-rat 
.alpha.5 integrin antibody and a rabbit polyclonal anti-Fn antibody. The 
double-staining experiments showed that .alpha..sub.5 .beta..sub.1 
integrin was co-localized with Fn fibrils in A1a3 cells that overexpress 
ILK. In contrast, staining of the cells with an anti-rat .beta..sub.3 
integrin antibody revealed no distinctive staining. These results suggest 
that .alpha..sub.5 .beta..sub.1 integrin, but not .beta..sub.3 integrins, 
participate in the Fn matrix assembly induced by overexpression of ILK. 
In contrast to cells that overexpress ILK, cells expressing a lower level 
of ILK (A2C6) have fewer clusters of .alpha..sub.5 .beta..sub.1 integrin 
that could be detected by immunofluorescent staining, although these cells 
express the same level of cell surface .alpha..sub.5 .beta.1 integrin as 
the cells overexpressing ILK. Moreover, in marked contrast to A1a3 cells 
that overexpress ILK, many of the structures containing .alpha..sub.5 
.beta..sub.1 integrin in the A2C6 cells lacked detectable Fn, indicating 
that overexpression of ILK enhances the binding of Fn to .alpha..sub.5 
.beta..sub.1 integrin. 
Effect of ILK overexpression on the formation of focal adhesion and matrix 
contacts. Cell adhesion to extracellular substrates is mediated by 
transmembrane complexes termed focal adhesions which contain integrin, 
vinculin and other cytoskeletal proteins. A connection between 
extracellular Fn and the intracellular actin cytoskeleton involving the 
integrin .beta. cytoplasmic domain is required for the assembly of Fn 
fibrils. A2C3 cells that express low levels of ILK formed abundant focal 
adhesions visualized by staining with an anti-vinculin antibody. However, 
only a small amount of .alpha..sub.5 .beta..sub.1 integrin and Fn were 
co-localized with the focal adhesions in A2C3 cells. 
Overexpression of ILK promoted co-localization of .alpha..sub.5 
.beta..sub.1 integrin and Fn with vinculin. Thus, while cells expressing a 
relatively low level of ILK are not defective in the assembly of focal 
adhesion, a higher level of ILK promotes the assembly of complexes 
containing vinculin, .alpha..sub.5 .beta..sub.1 integrin and Fn matrix. 
Overexpression of ILK down-regulates E-cadherin. E-cadherin is an 
important epithelial cell adhesion molecule mediating cell-cell 
interactions. Because overexpressing ILK in epithelial cells disrupted the 
characteristic "cobble-stone" epithelial morphology of the epithelial 
cells, the effect of ILK expression on the cellular level of E-cadherin 
was studied. The level of E-cadherin in cells expressing different amount 
of ILK was determined by immunoblot using an anti-E-cadherin antibody. The 
parental IEC-18 epithelial cells expressed abundant E-cadherin. 
Overexpression of H-RasVal12 in IEC-18 cells reduced the level of 
E-cadherin. Strikingly, E-cadherin was completely eliminated in ILK13-A1a3 
and A4a cells that overexpress ILK, whereas it was present at a normal 
level in ILK14-A2C3 and A2C6 cells that express a similar level of ILK to 
the parental IEC-18 cells (FIG. 8A). These results indicate an inverse 
correlation between the level of ILK and that of E-cadherin. 
In contrast to E-cadherin level, overexpression of ILK did not alter the 
ability of the cells to phosphorylate focal adhesion kinase 
(pp125.sup.FAK) in response to cell adhesion to Fn, indicating that 
tyrosine phosphorylation of pp125.sup.FAK does not transduce the signals 
leading to the alterations observed upon ILK overexpression, and in 
particular tyrosine phosphorylation of pp125.sup.FAK does not play a 
regulatory role in ILK induced Fn matrix assembly. 
Induction of in vivo tumorigenesis by overexpression of ILK. To assess a 
potential role of ILK in tumorigenesis, cells expressing varying levels of 
ILK were injected into athymic nude mice subcutaneously. Tumors arose 
within three weeks in 50% to 100% of the mice injected with the ILK13 
cells (10.sup.7 cells/mouse) that overexpress ILK, whereas no tumors were 
detected in the mice that were injected with the same number of the IEC-18 
or ILK14 cells expressing lower levels of ILK (Table 4). Thus, 
overexpression of ILK in these epithelial cells promotes tumor formation 
in vivo. 
TABLE 4 
______________________________________ 
Tumorigenicity of ILK overexpressing IEC-18 Cells 
Cell Line Number of Mice with Tumors at 3 weeks 
______________________________________ 
IEC-18 0/6 
ILK14-A2C3 0/6 
ILK13-A1a3 6/6 
ILK13-A4a 3/6 
______________________________________ 
Athymic nude mice were inoculated subcutaneously with the cells expressin 
high (ILK13A1a3 and A4a) or low (IEC18 and ILK14A2C3) levels of ILK 
(10.sup.7 cells/mouse in PBS). The mice were monitored for tumor formatio 
at the site of inoculation after three weeks. 
Inhibition of ILK induced cell growth in soft agar by amino terminal 
fragments of Fn that inhibit matrix assembly. One of the hallmarks of 
tumor forming cells is that their growth is less dependent on anchorage as 
measured by their ability to grow in soft agar culture. Similar to cells 
overexpressing H-Ras, cells overexpressing ILK were able to grow in soft 
agar. However, in marked contrast to the H-Ras overexpressing cells, ILK 
overexpressing cells assembled an abundant Fn matrix (Table 3). It was 
therefore tested whether the ability of the ILK overexpressing cells to 
grow in soft agar culture is related to the elevated level of Fn matrix 
assembly. The cells overexpressing ILK and the cells overexpressing H-Ras, 
respectively, were cultured in soft agar either in the presence or absence 
of the 70 kDa Fn amino terminal fragment, which inhibits the ILK induced 
Fn matrix assembly. The 70 kDa Fn fragment significantly inhibited the ILK 
induced "anchorage independent" growth in soft agar. Similar inhibition 
was observed with the 29 kDa fragment of Fn. In contrast, the H-Ras 
induced anchorage independent growth in soft agar was not inhibited by the 
70 kDa Fn fragmen. Moreover, the ILK induced cell growth in soft agar was 
not inhibited by the 60 kDa Fn Fragment which does not inhibit the Fn 
matrix assembly induced by ILK. These results suggest that the cell growth 
in soft agar induced by ILK, but not that induced by H-Ras, is at least 
partially mediated by a Fn matrix. 
Discussion 
The overexpression of ILK results in a loss of E-cadherin protein 
expression, offering a possible explanation for the loss of cell-cell 
contact in these cells. Indeed, losses of cell-cell adhesion have been 
implicated in tumorigenicity in vivo. ILK overexpressing cells are 
tumorigenic in nude mice in contrast to the parental IEC-18 intestinal 
epithelial cells and the control transfected clones. Thus, ILK can be 
considered to be a proto-oncogene. Another important finding is the 
apparent involvement of ILK in Fn matrix assembly. Overexpression of ILK 
in IEC-18 cells stimulated Fn matrix assembly. This is a property of 
transfected cell clones constitutively overexpressing ILK, and also of 
transfected clones in which ILK expression is induced using a 
metallothionein inducible promoter. Furthermore, Fn matrix assembly is 
impaired when an anti-sense ILK cDNA is induced resulting in decreased ILK 
expression. 
The ILK-stimulated Fn matrix assembly was inhibited by the amino-terminal 
domain of Fn, as well as the RGD-containing integrin binding domain of Fn, 
suggesting that RGD-binding integrins mediate ILK functions in Fn matrix 
assembly. Due to the unavailability of anti-integrin function blocking 
antibodies against rat integrins, it has not been possible to identify 
directly the specific integrin(s) involved in the enhanced Fn binding and 
matrix assembly. However, using immunofluorescence analysis, the 
.alpha..sub.5 .beta..sub.1 integrin, but not .alpha..sub.v .beta..sub.3, 
was co-localized with Fn fibrils in the ILK overexpressing cells, 
implicating .alpha..sub.5 .beta..sub.1 in the matrix assembly process. 
Furthermore, ILK overexpression promoted the co-localization of Fn with 
.alpha..sub.5 .beta..sub.1 and vinculin, whereas in the parental IEC-18 
cells and control transfected cells vinculin containing focal adhesion 
plaques were not co-localized with Fn. 
The kinase activity of ILK is clearly important in the stimulation of Fn 
matrix assembly, as overexpression of a kinase-inactive ILK mutant failed 
to enhance Fn matrix assembly. However, because ILK has potential binding 
sites for integrins and probably other intracellular signaling molecules, 
and because Fn matrix assembly can be regulated by post ligand occupancy 
events, it is possible that other activities of ILK may also play 
important roles in the stimulation of Fn matrix assembly. 
Although ILK overexpressing IEC-18 cells express same levels of integrins 
as the parental cells, the ILK overexpressing cells gain the ability to 
grow in an anchorage independent manner in soft agar, and are tumorigenic 
in nude mice, and they organize a prolific Fn matrix. The same IEC-18 
cells transfected with an activated form of H-ras, do not assemble a Fn 
matrix, but nevertheless are highly tumorigenic in nude mice. This 
represents a novel pathway of oncogenic transformation which is 
distinctive from H-Ras induced transformation and involves ILK and 
enhanced Fn matrix assembly. In fact, the ability to form a Fn matrix is 
important for the anchorage independent growth of transforming growth 
factor .beta. (TGF .beta.) treated fibroblasts. Fn matrix assembly also 
seems to be important for anchorage-independent growth in soft agar of the 
ILK overexpressing cells since inhibition of matrix assembly by the 29 kDa 
and 70 kDa amino terminal fragments of Fn, results in an inhibition in 
colony formation in soft agar. 
The expression of activated p21.sup.ras results in the disregulation of 
multiple signaling pathways and typically renders cells serum-independent, 
as well as anchorage independent for cell growth. On the other hand, the 
overexpression of ILK does not result in serum-independent cell growth, 
but induces anchorage-independent cell growth. These results indicate that 
ILK normally regulates adhesion-dependent signaling pathways and that the 
disregulation of ILK (e.g. by overexpression) induces 
anchorage-independent cell growth specifically. It is likely that ILK 
mediated signaling may be involved in the regulation of integrin 
inside-out signaling, as activated integrins are required for Fn matrix 
assembly. 
The ability to assemble an extensive Fn fibrillar matrix is a property of 
mesenchymal cells and it is intriguing that the stimulation of this 
activity by ILK overexpression in the epithelial cells is accompanied by a 
dramatic downregulation of cellular E-cadherin expression. Numerous 
previous studies have established that cellular E-cadherin level or 
activity is downregulated during epithelial-mesenchymal transition. 
Moreover, in a recent study, Zuk and Hay demonstrated that inhibition of 
.alpha..sub.5 .beta..sub.1 integrin, which is a substrate of ILK, 
significantly inhibited epithelial-mesenchymal transition of lens 
epithelium. It is now also widely accepted that many invasive carcinomas 
exhibit a loss of E-cadherin expression, and E-cadherin gene has been 
found to be a tumor/invasion-suppressor gene in human lobular breast 
cancer. The tumor suppressor gene fat in Drosophila is also homologous to 
cadherins. ILK may therefore be involved in coordinating cell-matrix 
adhesion and cell-cell adhesion in epithelial-mesenchymal transition, and 
overexpression of ILK may drive epithelial cells towards a mesenchymal 
phenotype and oncogenic transformation. 
The ILK stimulated Fn matrix assembly may allow enhanced interaction of Fn 
with .alpha..sub.5 .beta..sub.1. This integrin has recently been shown to 
be specific in supporting survival of cells on Fn, although no direct 
correlation was found between Fn matrix assembly and .alpha..sub.5 
.beta..sub.1 mediated cell survival. This latter conclusion was derived 
from the use of wild type .alpha..sub.5 .beta..sub.1 and .alpha..sub.5 
cytoplasmic deleted (.alpha..sub.5 .DELTA.C.beta..sub.1) mutants. It is 
likely that for cell survival, both receptor interaction with Fn, as well 
as proper intracellular interactions are required. ILK overexpression in 
IEC-18 cells induces cell survival in suspension cultures largely due to 
the up-regulation of expression of cyclin D.sub.1 and cyclin A proteins. 
EXAMPLE 12 
Expression of ILK in Human Colon Carcinoma Cells 
Tumor (T) or adjacent normal (N) tissue from patients biopsied for colon 
carcinoma were analyzed for the expression of ILK or LEF-1 by Western blot 
analysis. ILK activity was further determined by an in vitro kinase assay, 
as described in previous examples. The data are shown in Table 5. 
TABLE 5 
______________________________________ 
ILK Expression LEF-1 Expression 
ILK Activity 
Sample # 
N T N T N T 
______________________________________ 
369 + ++ - - + + 
371 ++ ++++ + ++++ + +++ 
373 ++ ++++ + ++++ + +++ 
438 - - - +/- + ++ 
443 + ++++ +/- ++++ 
444 +/- ++ +/- ++ ++ ++ 
445 +++ +++ + + ++ +++ 
450T7W ++++ ++++ ++ 
450TEW ++++ ++++ ++ 
______________________________________ 
These data demonstrate the strong expression of ILK in colon carcinomas, 
indicating an association with transformation. In accordance with the data 
presented in the previous example, LEF-1 expression is closely tied to ILK 
expression. 
EXAMPLE 13 
Phosphoinositide-3-OH Kinase-dependent Regulation of GSK-3 and PKB/AKT by 
ILK 
The amino acid sequence of ILK contains a sequence motif found in 
pleckstrin homology (PH) domains (Klarulund et al. (1997) Science 
275:1927-1930). This motif has been shown to be involved in the binding of 
phosphatidylinositol phosphates (Lemmon et al. (1996) Cell 85:621-624). 
Amino acids critical to the binding of such lipids to the PH domain are 
completely conserved in ILK. The phosphatidylinosital 3,4,5, triphosphate 
binding sites are the lysines at positions 162 and 209 (SEQ ID NO:2). The 
PH motifs are comprised of residues 158-165 and 208-212 (SEQ ID NO:2). 
There is a high degree of sequence identity within this motif between ILK 
and other PH-domain containing proteins such as cytohesin-1 (a .beta.2 
integrin cytoplasmic domain interacting protein) and GRP-1. It was 
determined that ILK activity is influenced by the presence of 
phosphatidylinosital3,4,5, triphosphate, and interacts with other kinase 
proteins in this pathway. 
Materials and Methods 
Stable-Transfected Cells. IEC-18 rat epithelial transformed cells are grown 
in Alpha-ME Media with 5% Fetal Calf Serum (GIBCO-BRL), insulin, glucose 
and L-glutamine. All cells are grown in the absence of antibiotics and 
anti-fungal agents. They are harvested and lysed at 80% confluency, with 
the Lysis Buffer used in the following Kinase Assays. The lysates are 
quantified with the Bradford Assay. 
Transient Transfection. On the day before transfection, the 293 Human 
Embryonic Kidney cells are split such that there will be approximately 1 
to 1.2 million cells (68% confluent) in a 100 mm (Falcon) dish at the time 
of transfection. The cells are fed with DME Media and 10% Donor Calf Serum 
(GIBCO-BRL). The cells are grown in the absence of antibiotics and 
anti-fungal agents. The use of poly-L-lysine is optional. 
Precipitate plasmids using the calcium/phosphate method with 40 .mu.g of 
DNA per dish (15 to 20 .mu.g of plasmids containing ILK construct; 7 to 10 
.mu.g of plasmids containing GSK-3B construct; use empty vectors when 
appropriate), and a 2.times.HEPES-buffered saline (HeBS) solution of ph 
7.05. Allow precipitates to transfect overnight in 3% carbon dioxide 
environment, in 7 ml of DME Media and 5% donor calf serum. The next 
morning, remove the precipitate and medium mixture. Then continue to 
propagate the cells with 10 ml of DME media and 10% donor calf serum until 
the time of harvest. If the cell become too confluent, they can be split. 
Harvest the cell lysates 48 to 60 hours after transfection. 
GSK-3B Kinase Assay. Lyse the cells directly from the dish and collect the 
cytoplasmic lysate [Lysis Buffer: 150 mM NaCl, 1% NP-40, 0.5% DOC, 50 mM 
ph 7.5 Hepes, 1 .mu.g/ml Leupeptin, 1 .mu.g/ml Aprotinin, 1 mm PMSF and 
0.1 mM Sodium orthovanadate]. Incubate overnight, 300 .mu.g of pre-cleared 
cell protein with 1 .mu.l of GSK-3B antibody (Alphonse antibody from James 
Woodgett) in a 500 .mu.l volume. Capture the immunocomplex by incubating 
25 to 30 .mu.l of protein A-sepharose beads with the lysate for 2 hours at 
4 degrees centigrade. Collect the beads and wash with cold Lysis Buffer 
and Kinase Last Wash Buffer, [10 mM Magnesium Chloride, 10 mM Manganese 
Chloride, 50 mM ph 7.0 Hepes, 0.1 mM Sodium Ortho-Vanadate and 1 mM DTT]. 
Remove all traces of the supernatant and add 25 .mu.l of Kinase Reaction 
Buffer [50 mM ph 7.0 Hepes, 10 mm Manganese Chloride, 10 mM Magnesium 
Chloride, 2 mM Sodium Fluoride, 1 mM Sodium orthovanadate, 1 .mu.l of 
Glycogen Syntase-1 peptide (from James Woodgett)/reaction and 5 
.mu.Ci/reaction of ATP(gamma 32 phosphate)] to the beads. Incubate the 
mixture for 25 minutes at 30.degree. C. and stop the reaction with the 
addition of 30 .mu.l of 2.times. reducing sample buffer. Incubate the 
mixture at 4.degree. for 10 minutes. Do not boil the samples. Run the 
samples on a Tricine Gel (Schlaeggen and von Jaggow 1987 Anal Biochem 
166:368-79) with 15 teeth, 1.5 mm Hoefer comb and apparatus overnight at a 
constant voltage of 110 Volts. Visualize the wet gel with a phosphorimager 
or via autoradiography. 
ILK Kinase Assay. This technique is similar to the GSK-3B Kinase assay. The 
only differences are the following. For the formation of the 
immunocomplex, 1.5-2 .mu.g of antibody is required per sample (200 to 300 
.mu.g of protein in a 500 to 600 .mu.l volume). The composition of the 
Kinase Reaction Buffer contains 5 .mu.g of myelin basic protein per 
reaction instead of the GSK-1 peptide. The reaction is stopped by the 
addition of 30 .mu.l of 2.times. non-reducing sample buffer, followed by 3 
min boiling of the samples. The samples are separated on a 15% 
SDS-polyacrylamide gel. The fixed and dried gel is visualized via 
autoradiography or phosphorimagery. 
Kinase Activation Assessment of Transient Transfected 293 HEK Cells. 48 
hours after transfection with the various constructs, 293 HEK cells are 
serum starved, because the cells must be quiescent prior to being 
activated by growth factors. The cultures are washed 3.times. with 
serum-free DMEM and incubated for 12 hrs in serum-free DMEM. 
For activating the cell, the serum-free media is removed and the cultures 
are incubated with DMEM (4 ml per 100 mm dish) supplemented with the 
appropriate concentration of growth factors (100 nM Insulin or 5 nM 
IGF-1), and in the presence or absence of a P13 Kinase inhibitor (50 .mu.M 
LY294002). The activation times vary. 
The activation is stopped by washing the cultures 3.times. with cold PBS, 
followed by lysing the cells on the dishes with NP40-DOC lysis buffer. 
Allow the lysis buffer to work for 30 minutes on ice, before harvesting. 
Spin the whole cell lysates at 15000 rpms for 15 min and collect the 
supernatant. Quantify the supernatant (cytoplasmic lysate) with the 
Bradford Assay. The lysates are now ready to be used for 
immunoprecipitation or mixed with 4.times. sample buffer for Western Blot 
Analysis. 
Assessment of ILK activation by insulin on IEC-18 cells. IEC 18 cells are 
rat colon epithelial cells that are cultured routinely in .alpha.-MEM 
medium supplemented with insulin (10 mg/liter), glucose 3.6 g/liter, and 
5% FCS. When IEC 18 cells are grown to 80% confluence, they are serum 
starved for 18 hours prior to activation by insulin. Before addition of 
insulin, media are removed and 4 ml of .alpha.-MEM+insulin 6 .mu.M is 
added to the 100 mm dishes. PI3 kinase inhibitors such as LY294002 at 50 
.mu.M or wortmannin at 200 nM are added optionally to block P13 kinase 
dependent ILK activation. At the designated times, dishes are washed 
3.times. with ice cold PBS and cells are lysed in 500 .mu.l lysis buffer: 
150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 50 mM Hepes pH 7.5, 10 
.mu.g/ml leupeptin, 1 mM PMSF, 2.5 .mu.l aprotinin/ml lysis buffer, NaF 5 
mM, Sodium vanadate 1 mM. After assessment of protein concentration by 
Bradford assay, 500 .mu.l samples containing 200 .mu.g of proteins are 
incubated for 2 hrs at 4.degree. C. with 20 .mu.l of Protein A-Sepharose 
to preadsorb the non specific kinases. The lysate is then incubated 
overnight with 2 .mu.g of rabbit anti ILK antiserum at 4.degree. C. under 
rotation. 
The immunocomplexes are then captured by incubating the lysate with 15 
.mu.l of Protein A Sepharose for 2 hrs at 4.degree. C. The beads are 
washed 2.times. with lysis buffer. The beads are washed 2.times. with last 
wash buffer: 10 mM MgCl.sub.2, 10 mM MnCl.sub.2, 50 mM Hepes pH 7.0, 0.1 
mM sodium orthovanadate, 2 mM NaF, 1 mM DTT. After aspirating completely 
the buffer, the beads are then mixed with 25 .mu.l of kinase reaction 
mixture: 22.5 .mu.l of kinase buffer (10 mM MgCl.sub.2, 10 mM MnCl.sub.2, 
50 mM Hepes pH 7.0, 1 mM sodium orthovanadate, 2mM NaF); 2 .mu.l of myelin 
basic protein at 2 mg/ml (UBI, #13-104), 5 .mu.Ci of .sup.32 P 
.gamma.-ATP. The kinase reaction is allowed to proceed for 25 min at 
30.degree. C. The reaction is stopped by addition of 30 .mu.l of 2.times. 
sample buffer and boiling for 3 min. The samples an then electrophoresed 
on a 12% SDS-PAGE gel. Phosphorylation level of MBP is assessed by 
phosphorimager analysis or exposure to an X ray film. 
Assessment of ILK kinase activity in 3T3 cells stably transfected with 
active or inactive P13 kinase. The cDNAs coding for the HA-tagged P110 
subunit of the PI3 kinase in pcDNA3 were used. 3T3 cells were grown in 
DMEM with 10% donor calf serum in exponential conditions. The 3T3 cells 
were harvested by trypsinization and washed once with HeBs buffer: 20 mM 
Hepes pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na.sub.2 HPO.sub.4, 6 mM 
glucose. 10.sup.7 cells were then resuspended in 0.8 ml of ice-cold HeBs 
containing 20 .mu.g of uncut DNA. Electroporation was performed with a 
Bio-Rad gene pulser set to 280 V, 960 .mu.F. After electroporation, cells 
were allowed to sit on ice for 10 min before being diluted into 24 ml 
DMEM, 10% DCS and plated on a 150 mm dish. After 2 day recovery, selection 
was initiated by the addition of G418 at the final concentration of 0.8 
mg/ml to the culture medium. After 2 weeks, the clones appeared and the 
transfectants were cloned by serial dilution and culture in 96 well 
microwell plates. Clones expressing the HA-tagged p110 subunit were 
expanded and used for ILK kinase assay in serum starved cells treated with 
or without with LY 294002. 
Transfection of 293 cells protocol. 293 cells have to be exponentially 
grown for optimal transfection. Typically they are passaged every 3 days 
by splitting them 1/10. Medium is DMEM medium supplemented with 10% donor 
calf serum. CaCl.sub.2 M solution: to 14.7 g of CaCl.sub.2 2H.sub.2 O, add 
50 ml of water to 50 ml. Filter sterilize through a 0.45 .mu.m 
nitrocellulose filter. Store aliquots at -20.degree. C. 2.times.HBS 
solution: to 16.4 g NaCl, add 11.9 g Hepes and 0.21 g Na.sub.2 HPO.sub.4 
and dissolve in 800 Ml H2O. Adjust the pH to 7.12 and add water to 1000 
ml. Filter sterilize through a 0.22 .mu.M filter and store at -20.degree. 
C. Plasmid solution: 15 .mu.g of ethanol precipitated plasmids are used 
per transfection. They are resuspended in sterile H.sub.2 O and mixed with 
62 .mu.l CaCl.sub.2 2M solution. H.sub.2 O is added to 500 .mu.l final. 
Plate 1.times.10.sup.6 293 cells per 100 mm dish in 10 ml medium 24 h prior 
to transfection. Mix 50 .mu.l of plasmid solution to 500 .mu.l of 
2.times.HBS solution dropwise at the same time as bubbling the combined 
mixture with a Pasteur pipette connected to a pipetman. Vortex the mixture 
for 1 min. and let it stand for 20 min. Add dropwise the 1 ml mixture to 
the cells and grow them in 3% CO.sub.2 atmosphere. After 16 hrs of 
culture, change the media and grow the cells in normal 5% CO.sub.2 
atmosphere. After 48-60 hrs, the cells are harvested for the assay. 
Assessment of AKT phosphorylation by ILK in 293 cells. 
Kinase assay. After cotransfection of 293 cells with HA-AKT and ILK, wild 
type or kinase dead, the cells are serum starved for 12 hours and 
submitted for activation by growth factors for designated times. Cells are 
then lysed with 500 .mu.l lysis buffer: 50 mM Tris-HCl pH 7.4, 0.5% NP40, 
1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM 5-glycerophosphate, 0.25 mM sodium 
vanadate, 1 .mu.M microcystin LR, PMSF 1 mM, aprotinin 2.5 .mu.l/ml, 
leupeptin 10 .mu.g/ml. Prepare a 1:1 slurry of protein G-anti HA mouse Mab 
beads as follows: Wash the proteinG-sepharose beads with solubilization 
buffer 3.times.. Add 2 .mu.g of anti HA antibody per assay point. Rotate 
at 4.degree. C. for 1 hr. Wash with solubilization buffer 3.times.. 
Resuspend to 1:1 with solubilization buffer and add 40 .mu.l to the 
lysates. Rotate the lysates with the beads for 1-2 hrs. Wash beads 3 
.times. with solubilization buffer containing 500 mM NaCl. Wash beads 
2.times. with kinase buffer: 20 mM HEPES pH 7.4 25 mM 
.beta.-glycerophosphate, 1 mM sodium vanadate, 1 mM DTT, 1 mM MgCl.sub.2, 
1 .mu.M microcystin LR, PMSF and leupeptin. 
Aspirate the beads completely. Add 20 .mu.l kinase buffer containing 60 
.mu.M Crosstide (From UBI catalog #12-331). Keep cold until ready for 
kinase assay. Add 10 .mu.l ATP solution (200 .mu.M cold ATP and 10 
.mu.Ci/sample .sup.32 P .gamma.-ATP in kinase buffer), vortex gently and 
place tubes in 30.degree. C. water bath. At 15 min, spot 20 .mu.l onto p81 
chromatography paper, let dry for about 2 min, and immerse into 1% 
phosphoric acid. Wash blots 6-10.times. with 1% phosphoric acid and count 
in scintillation counter. 
Western blot analysis of AKT (Ser473) phosphorylation state. After cell 
activation and lysis, the lysates are mixed with 4.times. sample buffer 
and heated to 95-100.degree. C. for 5 minutes and cooled on ice. 20 .mu.l 
of samples are run onto SDS-PAGE gels. Proteins are electotransfered on a 
PVDF membrane. Incubate membrane in 100 ml blocking buffer, i.e. TBS (Tris 
buffered saline) pH. 7.6 supplemented with 5% milk for 1-3 hrs. Incubate 
membrane and rabbit anti p-.sup.473 S AKT antiserum (New England Bio Labs 
Cat No #9270) at the 1:1000 dilution in 10 ml primary antibody dilution 
buffer with gentle agitation overnight at 4.degree. C. 
Primary antibody dilution buffer: 1.times.TBS, 0.1% Tween 20 with 5% BSA. 
Wash 3 times for 5 minutes each with 15 m TBST. Incubate membrane with 
horse radish peroxidase (HRP)-conjugated secondary antibody (1:20,000) 
with gentle agitation for 1 hr at room temperature. Wash membrane 3 times 
for 5 minutes each with 15 m TBST. Incubate membrane with ECL reagent 
(Amersham) for 1 min at room temperature. Drain membrane of excess 
developing solution, wrap in Saran wrap and expose to X-ray film. 
Assessment of regulation of ILK kinase by phosphoinositides. Ptdlns(3)P, 
Ptdins(3,4)P.sub.2 and Ptdlns(3,4,5)P.sub.3 were dried under nitrogen and 
resuspended at 0.1 mM in Hepes 10 mM, pH 7. 0 with phosphatidylserineand 
phosphatidylcholine, both at 1 mM. The lipid suspensions were vortexed and 
further sonicated for 20 min in order to generate unilamellar vesicles. 11 
.mu.l of ILK5-GST in kinase buffer were combined to 4 .mu.l of lipids and 
25 .mu.l of kinase reaction solution containing 2.5 .mu.l of MBP and 5 
.mu.Ci of .gamma..sup.32 P-ATP. The reaction proceeded for 30 or 2 hrs at 
30.degree. C. The reaction was stopped by adding an equal volume of 
2.times. sample buffer. The samples were run on a 12% non reducing 
SDS-PAGE gel. 
Results 
ILK activity is stimulated in vitro by phosphatidylinositol (3,4,5) 
trisphosphate (Ptdlns(3,4,5)P3) but not by phosphatidylinositol(3,4) 
bisphosphate (Ptdlns(3,4)P2), or phosphatidylinositol(3) monophosphate 
(Ptdlns(3)P). 
Since Ptdlns(3,4,5,)P3 is specifically generated upon receptor-mediated 
stimulation of PI(3)Kinase activity, it was determined whether ILK 
activity is stimulated in a PI(3)K dependent manner. PI(3)K is activated 
in response to a very wide range of extracellular stimuli, which include 
growth factors and cytokines, as well as by cell adhesion to ECM. The 
Ptdlns(3,4,5)P3 product of PI(3)K is a second messenger that acts on 
pathways that control cell proliferation, cell survival, and metabolic 
changes often through the activation of P70 ribosomal S6 Kinase 
(p70.sup.S6k) and protein kinase B (PKB), also known as AKT. PKB/AKT is a 
protooncogene and has been shown to be activated in a PI(3)K-dependent 
manner in response to growth factors, cytokines and cell-ECM interactions. 
To determine whether ILK is activated in a PI(3)K-dependent manner, 
quiescent, serum-starved, IEC-18 intestinal epithelial cells were treated 
with insulin, which is known to activate PI(3)K. ILK activity is rapidly 
stimulated by insulin and this activation is inhibited by prior treatment 
of the cells with Wortmannin (200 nM), a specific inhibitor of PI(3)K. 
Another inhibitor, Ly294002, also inhibits this activation. ILK activity 
is rapidly stimulated upon plating cells on fibronectin. This activation 
is also PI(3)Kinase-dependent, since it is inhibited by LY294002. These 
data demonstrate that ILK activity is stimulated by growth factors, such 
as insulin, and also by cell-ECM interactions, in a PI(3)K dependent 
manner, most probably resulting from the direct interaction of PI(3)K 
generated Ptdlns(3,4,5)P3 with ILK. 
To further demonstrate the role of PI(3)K in ILK activation, NIH3T3 cells 
were stably transfected with either constitutively activated P110 subunit 
of PI(3)K, or a kinase-dead mutant of PI(3)K, and ILK activity was 
determined in the transfected clones. ILK activity is several-fold higher 
in cells expressing constitutively active P110 subunit of PI(3)K, compared 
to control cells, or those expressing kinase-dead PI(3)K. Furthermore, the 
stimulated ILK activity in these cells is inhibited by prior incubation 
with Ly294002. 
Since ILK overexpression in epithelial cells results in the translocation 
of .beta.-catenin to the nucleus, it was determined whether the activity 
of GSK-3, a kinase that normally phosphorylates .beta.-catenin, is 
regulated by ILK. GSK-3 activity is inhibited when cells encounter Wnt, a 
matrix associated protein involved in cell fate determination. The 
inactivation of GSK-3 results in the inhibition of phosphorylation of 
.beta.-catenin and its subsequent stabilization and nuclear accumulation. 
ILK may also contribute to the nuclear localization of .beta.-catenin by 
inhibiting GSK-3 activity. 
Although GSK-3 is expressed in all IEC-18 cell transfectants, its activity 
is dramatically inhibited in the ILK overexpressing ILK-13 cells, but not 
in IEC-18 cells stably expressing a kinase-dead ILK. As expected, ILK 
activity is about 5-fold higher in ILK-13 cells compared to the control 
cells. To determine whether this inhibition of GSK activity is due to ILK, 
transient transfection assays were carried out in 293 human embryonal 
kidney epithelial cells. Co-transfection of HA-tagged-GSK-3 together with 
wild type ILK results in profound inhibition of GSK-3 activity, 
demonstrating that kinase active ILK can inhibit GSK-3 activity. 
Co-transfection with kinase-dead ILK did not result in GSK-3 inhibition, 
but reproducibly resulted in increased GSK-3 activity over basal levels. 
These results suggest that the kinase-dead ILK may be acting in a 
dominant-negative manner by suppressing the function of transfected and 
endogenous ILK. 
Since GSK-3 activity can also be regulated by PKB/AKT in a PI(3)K-dependent 
manner, and since it has previously been shown by others that integrin 
engagement stimulates PI(3)K activity leading to the activation of 
PKB/AKT, it was determined whether ILK might be upstream of PKB and may 
regulate its phosphorylation and activation. Co-transfection in 293 cells 
of HA-tagged PKB with wild-type ILK results in specific phosphorylation of 
PKB on serine-473, with the concomitant activation of its activity. 
Furthermore, co-transfection with kinase-dead ILK results in a distinct 
inhibition of serine-473 phosphorylation, demonstrating again that this 
form of ILK may be competing with endogenous ILK and thus behaving in a 
dominant-negative manner in the regulation of phosphorylationand 
activation of PKB. The identification of the protein kinases involved in 
the PI(3)K-mediated activation of PKB has been the subject of intense 
study, and has been extensively reviewed recently (Downward (1997) Science 
279:673-674). Ptdlns(3,4,5)P3 can bind to the PH domain of PKB resulting 
in its targeting to the plasma membrane and exposure of threonine-308. A 
constitutively active kinase, PDK-1, then phosphorylates PKB on 
threonine-308. However, this phosphorylation alone is not sufficient to 
fully activate PKB, which also needs to be phosphorylated on serine-473 by 
an as yet unidentified kinase (PDK-2), in a Ptdlns(3,4,5)P3-dependent 
manner. The present data shows that ILK, which is activated by 
Ptdlns(3,4,5)P3, can phosphorylate PKB on serine-473, resulting in its 
full activation, thus demonstrating that ILK is directly upstream of PKB 
in the transduction of PI(3)K-dependent signals to PKB. 
In summary, the activity of ILK can be stimulated by Ptdlns(3,4,5)P3 in a 
PI(3)K-dependent manner and that it can then phosphorylate PKB on 
serine-473, resulting in its activation. ILK also inactivates GSK-3 
activity. This inhibition may be indirect, occurring via PKB/AKT, as this 
kinase can phosphorylate GSK-3 on serine-9, but it is possible that ILK 
can also directly phosphorylate GSK-3 and inactivate it, independently of 
PKB. It will be interesting to determine whether, like ILK, PKB activation 
also results in the nuclear translocation of .beta.-catenin and activation 
of Lef-1/.beta.-catenin transcriptional activity, or whether the pathways 
bifurcate at this point, resulting in ILK activating the .beta.-catenin 
pathway, whereas PKB may target other pathways such as P70S6Kinase and 
control of protein translation or the inactivation of BAD, a pro-apoptotic 
BcL-2 family member. 
All publications and patent applications cited in this specification are 
herein incorporated by reference as if each individual publication or 
patent application were specifically and individually indicated to be 
incorporated by reference. 
The present invention has been described in terms of particular embodiments 
found or proposed by the present inventor to comprise preferred modes for 
the practice of the invention. It will be appreciated by those of skill in 
the art that, in light of the present disclosure, numerous modifications 
and changes can be made in the particular embodiments exemplified without 
departing from the intended scope of the invention. For example, due to 
codon redundancy, changes can be made in the underlying DNA sequence 
without affecting the protein sequence. Moreover, due to biological 
functional equivalency considerations, changes can be made in protein 
structure without affecting the biological action in kind or amount. All 
such modifications are intended to be included within the scope of the 
appended claims. 
References 
1. Damsky C. H., and Werb Z. Curr. Opin. Cell Biol. 4, 772-781 (1992). 
2. Hynes R. O. Cell 69, 11-25 (1992). 
3. Clark E. A. and Brugge J. S. Science 268, 233-239 (1995). 
4. Fields S. and Song O. Nature 340, 245-246 (1989). 
5. Lux S. E., John K. M. and Bennett V. Nature 344, 36-42 (1990). 
6. Inoue J.-I., et al. Proc. Natl. Acad. Sci. U.S.A. 89, 4333-4337 (1992). 
7. Lukas J., et al. Nature 375, 503-506 (1993). 
8. Schaller M. D., et al. Proc. Natl. Acad. Sci. U.S.A. 89, 5192-5196 
(1992). 
9. Hanks, S. K., Calalb M. B., Harper M. C. and Patel S. K. Proc. Natl. 
Acad. Sci. U.S.A. 89, 8481-8491 (1992). 
10. Dedhar S., Saulnier R., Nagle R. and Overall C. M. Clin. Exp. 
Metastasis 11, 391-400 (1993). 
11. Filmus J., et al., Oncogene 9, 3627-3633 (1994). 
12. O'Toole T. E., et al. J. Cell Biol. 124, 1047-1059 (1994). 
13. Chen Y.-P., et al., J. Biol. Chem. 269, 18307-18310 (1994). 
14. Kapron-Bras C., Fitz-Gibbon L., Jeevaratnam P., Wilkins J. and Dedhar 
S. J. Biol. Chem. 268, 20701-20704 (1993). 
15. Chen Q., Kinch M. S., Lin T. H., Burridge K. and Juliano R. L. J. Biol. 
Chem. 269, 26602-26605 (1994). 
16. Schlaepfer D. D., Hanks S. K., Hunter T. and van der Geer P. Nature 
372, 786-791 (1994). 
17. Kozak M. Cell 44, 283-292 (1986). 
18. Altschul S. F., Gish W., Miller W., Myers E. W., and Lipman D. J. 
(1990) Basic alignment search tool. J. Mol. Biol. 215, 403-410. 
19. Hanks S. K., Quinn A. M. and Hunter T. Science 241, 42-52 (1988). 
20. Zervos A. S., Gyuris J. and Brent R. Cell 72, 223-232 (1993). 
21. Argraves W. S., et al. J Cell Biol. 105, 1183-1190 (1987). 
22. Gietz D., St. Jean A., Woods R. A. and Schiestl R. H. Nucl. Acids Res. 
20, 1425 (1992). 
23. Sambrook J., Fritsch E. F. and Maniatis T. Molecular Cloning: A 
laboratory manual, 2nd ed. (Cold Spring Harbor Laboratory Press, New York, 
1989). 
24. Otey C. A., Pavalko F. M. and Burridge K. J. Cell Biol. 111, 721-729 
(1990). 
25. Cooper J. A., Sefton B. M. and Hunter T. Methods Enzymol. 99, 387-402 
(1983). 
26. Stephens L. C., Sonne J. E., Fitzgerald M. L. and Damsky C. H. J. Cell 
Biol. 123, 1607-1620 (1993). 
27. Harlow E. and Lane D. Antibodies: A Laboratory Manual. (Cold Spring 
Harbor Laboratory Press, New York, 1988). 
28. Leung-Hagesteijn C. Y., Milankov K., Michalak M., Wilkins J. and Dedhar 
S. J. Cell Sci. 107, 589-600 (1994). 
29. Buick, R. N., Filmus J. and Quaroni, A. Exp. Cell Res. 170, 300-309 
(1987). 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 11 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1789 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - GAATTCATCT GTCGACTGCT ACCACGGGAG TTCCCCGGAG AAGGATCCTG CA - 
#GCCCGAGT 60 
- - CCCGAGGATA AAGCTTGGGG TTCATCCTCC TTCCCTGGAT CACTCCACAG TC - 
#CTCAGGCT 120 
- - TCCCCAATCC AGGGGACTCG GCGCCGGGAC GCTGCTATGG ACGACATTTT CA - 
#CTCAGTGC 180 
- - CGGGAGGGCA ACGCAGTCGC CGTTCGCCTG TGGCTGGACA ACACGGAGAA CG - 
#ACCTCAAC 240 
- - CAGGGGGACG ATCATGGCTT CTCCCCCTTG CACTGGGCCT GCCGAGAGGG CC - 
#GCTCTGCT 300 
- - GTGGTTGAGA TGTTGATCAT GCGGGGGGCA CGGATCAATG TAATGAACCG TG - 
#GGGATGAC 360 
- - ACCCCCCTGC ATCTGGCAGC CAGTCATGGA CACCGTGATA TTGTACAGAA GC - 
#TATTGCAG 420 
- - TACAAGGCAG ACATCAATGC AGTGAATGAA CACGGGAATG TGCCCCTGCA CT - 
#ATGCCTGT 480 
- - TTTTGGGGCC AAGATCAAGT GGCAGAGGAC CTGGTGGCAA ATGGGGCCCT TG - 
#TCAGCATC 540 
- - TGTAACAAGT ATGGAGAGAT GCCTGTGGAC AAAGCCAAGG CACCCCTGAG AG - 
#AGCTTCTC 600 
- - CGAGAGCGGG CAGAGAAGAT GGGCCAGAAT CTCAACCGTA TTCCATACAA GG - 
#ACACATTC 660 
- - TGGAAGGGGA CCACCCGCAC TCGGCCCCGA AATGGAACCC TGAACAAACA CT - 
#CTGGCATT 720 
- - GACTTCAAAC AGCTTAACTT CCTGACGAAG CTCAACGAGA ATCACTCTGG AG - 
#AGCTATGG 780 
- - AAGGGCCGCT GGCAGGGCAA TGACATTGTC GTGAAGGTGC TGAAGGTTCG AG - 
#ACTGGAGT 840 
- - ACAAGGAAGA GCAGGGACTT CAATGAAGAG TGTCCCCGGC TCAGGATTTT CT - 
#CGCATCCA 900 
- - AATGTGCTCC CAGTGCTAGG TGCCTGCCAG TCTCCACCTG CTCCTCATCC TA - 
#CTCTCATC 960 
- - ACACACTGGA TGCCGTATGG ATCCCTCTAC AATGTACTAC ATGAAGGCAC CA - 
#ATTTCGTC 1020 
- - GTGGACCAGA GCCAGGCTGT GAAGTTTGCT TTGGACATGG CAAGGGGCAT GG - 
#CCTTCCTA 1080 
- - CACACACTAG AGCCCCTCAT CCCACGACAT GCACTCAATA GCCGTAGTGT AA - 
#TGATTGAT 1140 
- - GAGGACATGA CTGCCCGAAT TAGCATGGCT GATGTCAAGT TCTCTTTCCA AT - 
#GTCCTGGT 1200 
- - CGCATGTATG CACCTGCCTG GGTAGCCCCC GAAGCTCTGC AGAAGAAGCC TG - 
#AAGACACA 1260 
- - AACAGACGCT CAGCAGACAT GTGGAGTTTT GCAGTGCTTC TGTGGGAACT GG - 
#TGACACGG 1320 
- - GAGGTACCCT TTGCTGACCT CTCCAATATG GAGATTGGAA TGAAGGTGGC AT - 
#TGGAAGGC 1380 
- - CTTCGGCCTA CCATCCCACC AGGTATTTCC CCTCATGTGT GTAAGCTCAT GA - 
#AGATCTGC 1440 
- - ATGAATGAAG ACCCTGCAAA GCGACCCAAA TTTGACATGA TTGTGCCTAT CC - 
#TTGAGAAG 1500 
- - ATGCAGGACA AGTAGGACTG GAAGGTCCTT GCCTGAACTC CAGAGGTGTC GG - 
#GACATGGT 1560 
- - TGGGGGAATG CACCTCCCCA AAGCAGCAGG CCTCTGGTTG CCTCCCCCGC CT - 
#CCAGTCAT 1620 
- - GGTACTACCC CAGCCTGGGG TCCATCCCCT TCCCCCATCC CTACCACTGT GC - 
#GCAAGAGG 1680 
- - GGCGGGCTCA GAGCTTTGTC ACTTGCCACA TGGTGTCTCC CAACATGGGA GG - 
#GATCAGCC 1740 
- - CCGCCTGTCA CAATAAAGTT TATTATGAAA AAAAAAAAAA AAAAAAAAA - # 
1789 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 452 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Asp Asp Ile Phe Thr Gln Cys Arg Glu Gl - #y Asn Ala Val Ala Val 
1 5 - # 10 - # 15 
- - Arg Leu Trp Leu Asp Asn Thr Glu Asn Asp Le - #u Asn Gln Gly Asp Asp 
20 - # 25 - # 30 
- - His Gly Phe Ser Pro Leu His Trp Ala Cys Ar - #g Glu Gly Arg Ser Ala 
35 - # 40 - # 45 
- - Val Val Glu Met Leu Ile Met Arg Gly Ala Ar - #g Ile Asn Val Met Asn 
50 - # 55 - # 60 
- - Arg Gly Asp Asp Thr Pro Leu His Leu Ala Al - #a Ser His Gly His Arg 
65 - #70 - #75 - #80 
- - Asp Ile Val Gln Lys Leu Leu Gln Tyr Lys Al - #a Asp Ile Asn Ala Val 
85 - # 90 - # 95 
- - Asn Glu His Gly Asn Val Pro Leu His Tyr Al - #a Cys Phe Trp Gly Gln 
100 - # 105 - # 110 
- - Asp Gln Val Ala Glu Asp Leu Val Ala Asn Gl - #y Ala Leu Val Ser Ile 
115 - # 120 - # 125 
- - Cys Asn Lys Tyr Gly Glu Met Pro Val Asp Ly - #s Ala Lys Ala Pro Leu 
130 - # 135 - # 140 
- - Arg Glu Leu Leu Arg Glu Arg Ala Glu Lys Me - #t Gly Gln Asn Leu Asn 
145 1 - #50 1 - #55 1 - 
#60 
- - Arg Ile Pro Tyr Lys Asp Thr Phe Trp Lys Gl - #y Thr Thr Arg Thr 
Arg 
165 - # 170 - # 175 
- - Pro Arg Asn Gly Thr Leu Asn Lys His Ser Gl - #y Ile Asp Phe Lys Gln 
180 - # 185 - # 190 
- - Leu Asn Phe Leu Thr Lys Leu Asn Glu Asn Hi - #s Ser Gly Glu Leu Trp 
195 - # 200 - # 205 
- - Lys Gly Arg Trp Gln Gly Asn Asp Ile Val Va - #l Lys Val Leu Lys Val 
210 - # 215 - # 220 
- - Arg Asp Trp Ser Thr Arg Lys Ser Arg Asp Ph - #e Asn Glu Glu Cys Pro 
225 2 - #30 2 - #35 2 - 
#40 
- - Arg Leu Arg Ile Phe Ser His Pro Asn Val Le - #u Pro Val Leu Gly 
Ala 
245 - # 250 - # 255 
- - Cys Gln Ser Pro Pro Ala Pro His Pro Thr Le - #u Ile Thr His Trp Met 
260 - # 265 - # 270 
- - Pro Tyr Gly Ser Leu Tyr Asn Val Leu His Gl - #u Gly Thr Asn Phe Val 
275 - # 280 - # 285 
- - Val Asp Gln Ser Gln Ala Val Lys Phe Ala Le - #u Asp Met Ala Arg Gly 
290 - # 295 - # 300 
- - Met Ala Phe Leu His Thr Leu Glu Pro Leu Il - #e Pro Arg His Ala Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - Asn Ser Arg Ser Val Met Ile Asp Glu Asp Me - #t Thr Ala Arg Ile 
Ser 
325 - # 330 - # 335 
- - Met Ala Asp Val Lys Phe Ser Phe Gln Cys Pr - #o Gly Arg Met Tyr Ala 
340 - # 345 - # 350 
- - Pro Ala Trp Val Ala Pro Glu Ala Leu Gln Ly - #s Lys Pro Glu Asp Thr 
355 - # 360 - # 365 
- - Asn Arg Arg Ser Ala Asp Met Trp Ser Phe Al - #a Val Leu Leu Trp Glu 
370 - # 375 - # 380 
- - Leu Val Thr Arg Glu Val Pro Phe Ala Asp Le - #u Ser Asn Met Glu Ile 
385 3 - #90 3 - #95 4 - 
#00 
- - Gly Met Lys Val Ala Leu Glu Gly Leu Arg Pr - #o Thr Ile Pro Pro 
Gly 
405 - # 410 - # 415 
- - Ile Ser Pro His Val Cys Lys Leu Met Lys Il - #e Cys Met Asn Glu Asp 
420 - # 425 - # 430 
- - Pro Ala Lys Arg Pro Lys Phe Asp Met Ile Va - #l Pro Ile Leu Glu Lys 
435 - # 440 - # 445 
- - Met Gln Asp Lys 
450 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 258 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - Asn Met Lys Glu Leu Lys Leu Leu Gln Thr Il - #e Gly Lys Gly Glu Phe 
1 5 - # 10 - # 15 
- - Gly Asp Val Met Leu Gly Asp Tyr Arg Gly As - #n Lys Val Ala Val Lys 
20 - # 25 - # 30 
- - Cys Ile Lys Asn Asp Ala Thr Ala Gln Ala Ph - #e Leu Ala Glu Ala Ser 
35 - # 40 - # 45 
- - Val Met Thr Gln Leu Arg His Ser Asn Leu Va - #l Gln Leu Leu Gly Val 
50 - # 55 - # 60 
- - Ile Val Glu Glu Lys Gly Gly Leu Tyr Ile Va - #l Thr Glu Tyr Met Ala 
65 - #70 - #75 - #80 
- - Lys Gly Ser Leu Val Asp Tyr Leu Arg Ser Ar - #g Gly Arg Ser Val Leu 
85 - # 90 - # 95 
- - Gly Gly Asp Cys Leu Leu Lys Phe Ser Leu As - #p Val Cys Glu Ala Met 
100 - # 105 - # 110 
- - Glu Tyr Leu Glu Gly Asn Asn Phe Val His Ar - #g Asp Leu Ala Ala Arg 
115 - # 120 - # 125 
- - Asn Val Leu Val Ser Glu Asp Asn Val Ala Ly - #s Val Ser Asp Phe Gly 
130 - # 135 - # 140 
- - Leu Thr Lys Glu Ala Ser Ser Thr Gln Asp Th - #r Gly Lys Leu Pro Val 
145 1 - #50 1 - #55 1 - 
#60 
- - Lys Trp Thr Ala Pro Glu Ala Leu Arg Glu Ly - #s Lys Phe Ser Thr 
Lys 
165 - # 170 - # 175 
- - Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Tr - #p Glu Ile Tyr Ser Phe 
180 - # 185 - # 190 
- - Gly Arg Val Pro Tyr Pro Arg Ile Pro Leu Ly - #s Asp Val Val Pro Arg 
195 - # 200 - # 205 
- - Val Glu Lys Gly Tyr Lys Met Asp Ala Pro As - #p Gly Cys Pro Pro Ala 
210 - # 215 - # 220 
- - Val Tyr Glu Val Met Lys Asn Cys Trp His Le - #u Asp Ala Ala Met Arg 
225 2 - #30 2 - #35 2 - 
#40 
- - Pro Ser Phe Leu Gln Leu Arg Glu Gln Leu Gl - #u His Ile Lys Thr 
His 
245 - # 250 - # 255 
- - Glu Leu 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 256 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Ile Pro Arg Glu Ser Leu Arg Leu Glu Val Ly - #s Leu Gly Gln Gly Cys 
1 5 - # 10 - # 15 
- - Phe Gly Glu Val Trp Met Gly Thr Trp Asn Gl - #y Thr Thr Lys Val Ala 
20 - # 25 - # 30 
- - Ile Lys Thr Leu Lys Pro Gly Thr Met Met Pr - #o Glu Ala Phe Leu Gln 
35 - # 40 - # 45 
- - Glu Ala Gln Ile Met Lys Lys Leu Arg His As - #p Lys Leu Val Pro Leu 
50 - # 55 - # 60 
- - Tyr Ala Val Val Ser Glu Glu Pro Ile Tyr Il - #e Val Thr Glu Phe Met 
65 - #70 - #75 - #80 
- - Thr Lys Gly Ser Leu Leu Asp Phe Leu Lys Gl - #u Gly Glu Gly Lys Phe 
85 - # 90 - # 95 
- - Leu Lys Leu Pro Gln Leu Val Asp Met Ala Al - #a Gln Ile Ala Asp Gly 
100 - # 105 - # 110 
- - Met Ala Tyr Ile Glu Arg Met Asn Tyr Ile Hi - #s Arg Asp Leu Arg Ala 
115 - # 120 - # 125 
- - Ala Asn Ile Leu Val Gly Asp Asn Leu Val Cy - #s Lys Ile Ala Asp Phe 
130 - # 135 - # 140 
- - Gly Leu Ala Arg Leu Ile Glu Asp Asn Glu Ty - #r Thr Ala Arg Gln Gly 
145 1 - #50 1 - #55 1 - 
#60 
- - Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Gl - #u Ala Ala Leu Tyr 
Gly 
165 - # 170 - # 175 
- - Arg Phe Thr Ile Lys Ser Asp Val Trp Ser Ph - #e Gly Ile Leu Leu Thr 
180 - # 185 - # 190 
- - Glu Leu Val Thr Lys Gly Arg Val Pro Tyr Pr - #o Gly Met Val Asn Arg 
195 - # 200 - # 205 
- - Glu Val Leu Glu Gln Val Glu Arg Gly Tyr Ar - #g Met Pro Cys Pro Gln 
210 - # 215 - # 220 
- - Gly Cys Pro Glu Ser Leu His Glu Leu Met Ly - #s Leu Cys Trp Lys Lys 
225 2 - #30 2 - #35 2 - 
#40 
- - Asp Pro Asp Glu Arg Pro Thr Phe Glu Tyr Il - #e Gln Ser Phe Leu 
Glu 
245 - # 250 - # 255 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 263 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - Ile Pro Trp Cys Asp Leu Asn Ile Lys Glu Ly - #s Ile Gly Ala Gly Ser 
1 5 - # 10 - # 15 
- - Phe Gly Thr Val His Arg Ala Glu Trp His Gl - #y Ser Asp Val Ala Val 
20 - # 25 - # 30 
- - Lys Ile Leu Met Glu Gln Asp Phe His Ala Gl - #u Arg Val Asn Glu Phe 
35 - # 40 - # 45 
- - Leu Arg Glu Val Ala Ile Met Lys Arg Leu Ar - #g His Pro Asn Ile Val 
50 - # 55 - # 60 
- - Leu Phe Met Gly Ala Val Thr Gln Pro Pro As - #n Leu Ser Ile Val Thr 
65 - #70 - #75 - #80 
- - Glu Tyr Leu Ser Arg Gly Ser Leu Tyr Arg Le - #u Leu His Lys Ser Gly 
85 - # 90 - # 95 
- - Ala Arg Glu Gln Leu Asp Glu Arg Arg Arg Le - #u Ser Met Ala Tyr Asp 
100 - # 105 - # 110 
- - Val Ala Lys Gly Met Asn Tyr Leu His Asn Ar - #g Asn Pro Pro Ile Val 
115 - # 120 - # 125 
- - His Arg Asp Leu Lys Ser Pro Asn Leu Leu Va - #l Asp Lys Lys Tyr Thr 
130 - # 135 - # 140 
- - Val Lys Val Cys Asp Phe Gly Leu Ser Arg Le - #u Lys Ala Ser Thr Phe 
145 1 - #50 1 - #55 1 - 
#60 
- - Leu Ser Ser Lys Ser Ala Ala Gly Thr Pro Gl - #u Trp Met Ala Pro 
Glu 
165 - # 170 - # 175 
- - Val Leu Arg Asp Glu Pro Ser Asn Glu Lys Se - #r Asp Val Tyr Ser Phe 
180 - # 185 - # 190 
- - Gly Val Ile Leu Trp Glu Leu Ala Thr Leu Gl - #n Gln Pro Trp Gly Asn 
195 - # 200 - # 205 
- - Leu Asn Pro Ala Gln Val Val Ala Ala Val Gl - #y Phe Lys Cys Lys Arg 
210 - # 215 - # 220 
- - Leu Glu Ile Pro Arg Asn Leu Asn Pro Gln Va - #l Ala Ala Ile Ile Glu 
225 2 - #30 2 - #35 2 - 
#40 
- - Gly Cys Trp Thr Asn Glu Pro Trp Lys Arg Pr - #o Ser Phe Ala Thr 
Ile 
245 - # 250 - # 255 
- - Met Asp Leu Leu Arg Pro Leu 
260 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 271 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - Ile Pro Asp Gly Gln Ile Thr Val Gly Gln Ar - #g Ile Gly Ser Gly Ser 
1 5 - # 10 - # 15 
- - Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gl - #y Asp Val Ala Val Lys 
20 - # 25 - # 30 
- - Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gl - #n Leu Gln Ala Phe Lys 
35 - # 40 - # 45 
- - Asn Glu Val Gly Val Leu Arg Lys Thr Arg Hi - #s Val Asn Ile Leu Leu 
50 - # 55 - # 60 
- - Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Al - #a Ile Val Thr Gln Trp 
65 - #70 - #75 - #80 
- - Cys Glu Gly Ser Ser Leu Tyr His His Leu Hi - #s Ile Ile Glu Thr Lys 
85 - # 90 - # 95 
- - Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Ar - #g Gln Thr Ala Gln Gly 
100 - # 105 - # 110 
- - Met Asp Tyr Leu His Ala Lys Ser Ile Ile Hi - #s Arg Asp Leu Lys Ser 
115 - # 120 - # 125 
- - Asn Asn Ile Phe Leu His Glu Asp Leu Thr Va - #l Lys Ile Gly Asp Phe 
130 - # 135 - # 140 
- - Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gl - #y Ser His Gln Phe Glu 
145 1 - #50 1 - #55 1 - 
#60 
- - Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pr - #o Glu Val Ile Arg 
Met 
165 - # 170 - # 175 
- - Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser As - #p Val Tyr Ala Phe Gly 
180 - # 185 - # 190 
- - Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Le - #u Pro Tyr Ser Asn Ile 
195 - # 200 - # 205 
- - Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gl - #y Arg Gly Tyr Leu Ser 
210 - # 215 - # 220 
- - Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pr - #o Lys Ala Met Lys Arg 
225 2 - #30 2 - #35 2 - 
#40 
- - Leu Met Ala Glu Cys Leu Lys Lys Lys Arg As - #p Glu Arg Pro Leu 
Phe 
245 - # 250 - # 255 
- - Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Al - #a Arg Ser Leu Pro 
260 - # 265 - # 270 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - GGCCGAATTC GCTGGAATTG TTCTTATTGG C - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - GGCCGGATCC TCATTTTCCC TCATACTTCG G - # - # 
31 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - CCTTCAGCAC CCTCACGACA ATGTCATTGC CC - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: Other 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - CTGCAGAGCT TTGGGGGCAT CCCAGGCAGG TG - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - Leu Pro Tyr Gly Thr Ala Met Glu Lys Ala Gl - #n Leu Lys Pro Pro Ala 
1 5 - # 10 - # 15 
- - Thr Ser Asp Ala 
20 
__________________________________________________________________________