RECOMBINANT YEAST CELL AND ASSAY USING SAME

Abstract: Yeast cell having the ability to express the kinase domain of N-Pak. The expressed kinase domain restores the mating function of a ste20 mutant and such cells are therefore useful in an assay to screen for inhibitors of N-Pak. Such inhibitors may be useful in treatment and/or prevention of Alzheimer's Disease.

Description: RECOMBINANT YEAST CELL AND ASSAY USING SAME Field of the invention: The present invention concerns expression vectors containing a yeast promoter and nucleic acid encoding the kinase domain of mammalian N-Pak, recombinant yeast host cells expressing the N-Pak kinase domain , and assays to detect inhibitors of N-Pak activity. Background of the Invention: Rac and Cdc42 are members of the Rho family of small GTP binding proteins. These GTPases are involved signal transduction processes affecting morphogenesis and assembly of cytoskeletal structures involved in cell motility and metastasis. Recently, a family of senne/threonine protein kmases characterized by their binding to Cdc42 and Rac have been described Based upon this characteristic, they have been named p21 -activated kmases (PAK) These constitute a new subfamily of kmases, characterized by high homology to the Saccharomyces cerevisiae Ste20 kinase A mammalian PAK (p65 PAK ) was identified by its specific binding to GTP-bound forms of Cdc42 and Rac1 ( Manser et al, 1994) Previously, a gene for a second member of this family was isolated from a rat brain cDNA library based on its ability to interact specifically with the C-terminal portion of the Alzheimer β amyloid precursor protein (a fragment comprising the C- terminal 100 ammo acids of β-APP) The gene encoding this protein appears to be specifically expressed in neuronal tissue The sequence which was found to be closely related to that of p65 PAK and to the yeast STE20 gene product (Ramer and Davis, 1993), was designated N-Pak (nervous system specific p_21 activated kinase) The sequence of this N-Pak cDNA clone and corresponding ammo acid sequence are disclosed in WO 94/0581 1 , U.S. patent application serial numbers 08/559.397, 08/144,555, and 07/938,184, the disclosures of which are incorporated herein by reference in their entirety. In the last few years, numerous additional PAKs, characterized by the presence of a sequence conserved C-terminal kinase domain and upstream p21 -binding and activation domains, have been reported. Thus, these kmases comprise a ubiquitous new family of senne/threonine kinases, referred to as the PAK/Ste20 family Three mammalian isoforms have been described to date. In rodents, the 68-kDa α-PAK (Manser et al, 1994) and the 65 kDa β-PAK (Manser et al, 1995; Bagrodia et al, 1995a) appear to be enriched in brain, while the 62 kDa γ-isoform (Teo et al, 1995) is widely expressed in all tissues. Human homologues of the rat α-isoform, hPAK1 (Brown et al, 1996) and the γ-isoforms hPAK2 (Knaus et al, 1995) and hPAK65 (Martin et al, 1995) have been described. In addition to the numerous vertebrate species now shown to have PAK kinases, homologues have been described in insects such as Drosophila (Harden, et al. 1996) and several fungal species. Genes encoding Ste20 homologues have been isolated from the budding yeast S.cerevisiae (Ramer and Davis 1993; Cvrckova.et al, 1995), the fission yeast Schizosaccharomyces pombe (Ottilie et al, 1995; Marcus et al 1995) and the dimorphic human opportunistic pathogenic yeast Candida albicans (Leberer et al, 1996; Kohler and Fink, 1996) In budding yeast, the Ste20 protein is a component of a MAPK signal transduction pathway leading to mating (Ramer and Davis, 1993). This pathway is activated by hormone stimulation of a trimeric G protein coupled mating receptor. Ste20 serves as a link between Gβγ and downstream mitogen-activated protein kinases of this well-studied signalling cascade and is therefore essential for activation of mating specific genes (Leberer et al, 1992; Wu et al, 1995). Recent evidence suggests an additional role for Cdc42 and Ste20 or Ste20 homologues in yeast morphogenesis (Cvrckova et al, 1995; Mosch, H-Uet al, 1996). Likewise, in fission yeast, disruption of the pakl gene causes aberrant actin localization and reduces mating (Marcus et al. 1995; Ottilie et al, 1995). Recent evidence suggests a role for mammalian Paks in signalling to p38 and JNK MAP kinases ( Zhang et al, 1995; Bagrodia et al, 1995b; Brown et al, 1996). In addition, human PAK1 and PAK2 have been shown to be regulated by G-protein coupled receptors (Knaus et al, 1995). Therefore, there appears to be a common role for the PAK family members in signalling pathways which utilize Cdc42 and Rac1 . Given the homology of mammalian PAK family proteins to the yeast Ste20 protein, the ability of the mammalian protein to substitute for the yeast protein has been examined. Zhao et al, (1995) found that the rat p65PAK was unable to complement a S.cerevisiae ste20 mutant. They postulated that the mammalian enzyme was unable to phosphorylate the appropriate downstream target. Subsequently, the mouse gene m-PAK-3 was tested in a similar manner and found to give extremely weak suppression of the mating defect; mating occurred at a level that was 3-4% of a wild type strain (Bagrodia et al, 1995a). Brown et al (1996) reported that full length hPAK can restore mating of a ste20-null mutant to nearly normal wild type levels. This led these authors to suggest that the differences in ammo acids (8 out of 545) between rat and human might be sufficient to account for this difference, and that one or more of these differences might define residues important for protein-protein interaction. The establishment of this class of serine/threonine kinases as important intermediates in cell signal transduction has generated much interest in their activity and role in a variety of cellular processes such as differentiation, cytoskeletal assembly, motility and growth, particularly in light of the finding that the brain specific N-Pak protein binds specifically to the cytoplas ic domain of β-APP Heretofore, the expression of rat N-PAK in yeast has been unknown. We have discovered that N-PAK truncated to exclude the N-termmal region containing the Cdc42 binding domain can functionally substitute for the yeast Ste20 protein. REFERENCES: Bagrodia, S., S.J.Taylor, C.L. Creasy, J. Chernoff and R. A. Cerione (1995a) Identification of a mouse p21 Cdc42/Rac activated kinase. J. Biol. Chem 270:22731 -22737. Bagrodia, S.. B.Derijard, R.J. Davis and R.J. Cerione (1995b) Cdc42 and PAK-mediated signalling leads to JNK and p38 mitogen-activated protein kinase activation. J.Biol. Chem 270:27995-27998. Brown, J.L., L. Stowers, M. Baer, J.Trejo, S.Cough nand and J. Chant. (1996) Human Ste20 homologue hPAKI links GTPases to the JNK MAP kinase pathway. Current Biology 6:598-605. Cvrckova, F., CIDe Virgilio, E. Manser, J.R. Pringle and K. Nasmyth (1995) Ste20-like protein kinases are required for normal localization of cell growth and for cytokinese in budding yeast. Genes and Devel. 9:1817-1830. Harden, N., J.Lee, H Y Loh, Y-M.Ong, I. Tan, T. Leung, E. Manser and L.ϋm. (1996) A Drosophila homologue of the Rac- and Cdc42-actιvated senne/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures Molec Cell. Biol. 1 6: 1896-1908. Knaus, U.G., S. Morris, H-J Dong, J.Chernoff and G.M. Bokoch (1995) Regulation of human leukocyte p21 -activated kinases through G protein- coupled receptors. Science 269.221 -223. Kohler, J R. and G R Fink (1996) Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signalling components have defects in hyphal development Proc. Natl. Acad. Sci. USA 93.13223-13228 Leberer, E., D. Dignard, D Harcus, D Y Thomas and M Whiteway (1992) The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protem βγ subunits to downstream signalling components EMBO.J 1 1 4815-4824 Leberer, E, D Harcus, I D Broadbent, K L. Clark, D. Dignard, K Ziegelbauer, A. Schmidt, N P. Gow, A P Brown and D Y Thomas (1996) Signal transduction through homologues of the Ste20 and Ste7 protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc. Natl. Acad. Sci USA 93.13217-13222. Manser, E., T Leung, H Salihudd , Z-S.Zhao and L.Lim ( 1 994) A brain senne/threonine protein kinase activated by Cdc42 and Rac1 Nature 367.40-46 Manser, M., C.Chong, Z-S. Zhao, T. Leung, G. Michaels, C. Hall and L.Lim. (1995) Molecular cloning of a new member of the p21 -Cdc42/Rac-actιvated kinase (PAK) family J Biol Chem 270 25070-25078 Marcus, S., A Polvenno, E Chang, D Robbins, M H. Cobb and M Wigler (1995) Shk1 , a homologue of the Saccharomyces cerevisiae Ste20 and mammalian P65PAK protein kinases, is a component of a Ras/Cdc42 signalling molecule in the fission yeast Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA 92:6180-6184. Martin, G.A., G. Bollag, F McCormick and A Abo. (1995) A novel seπne kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20 EMBO J. 14 1970-1978. Mosch, H-U., R.L. Roberts and G.R Fink (1996) Ras2 signals via the Cdc42/Ste20/mιtogen activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc Natl. Acad. Sci. USA 93:5352- 5356. Ottihe, S., P J. Miller, D. O. Johnson. C.L. Creasy, M A. Sells. S. Bagroda, S. Forsburg and J. Chemoff. ( 1995) Fission yeast pakY encodes a protein kinase that interacts with Cdc42p and is involved in the control of cell polarity and mating. EMBO J. 14 5980-5919 Ramer, S W and R W Davis (1993) A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae Proc Natl Acad. Sci, USA 90:425-456 Teo, M., E. Manser and L.Lim (1995) Identification and molecular cloning of a p21 cdc42/rac- activated seπne threonme kinase that is rapidly activated by thrombin in platelets. J. Biol. Chem 270. 26690-26697 Wu,C , M Whiteway, D Y Thomas and E. Leberer (1995) Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal related kinase kinase (MEK)kιnase kinase from Saccharomyces cerevisiae. J Biol. Chem. 270:15984-15992. Zhang, S., J. Han, M.A. Sells, J Chemoff, U G. Knaus, R.J. Ulevitch and G.M. Bokoch. (1995) Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1 J Biol. Chem 270:23934- 23926. Zhao, Z-S., T Leung, E. Manser and L.Lim (1995) Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24 Molec Cell Biol 15.5246-5257 Summary of the Invention The mammalian neuronal specific senne/threonine kinase N-Pak has high homology to the yeast Saccharomyces cerevisiae STE20 gene product. Ste20 acts as a signalling molecule in the yeast mating signal transduction pathway, where it comprises the first kinase (MAPKKKK) in a phosphorylation cascade. We have discovered that the kinase domain of the mammalian N-Pak protein can suppress the mating defect of a yeast mutant functionally deficient for the STE 20 gene Restoration of signal transduction was demonstrated by the ability to mate at high frequency as well as by activation of a reporter gene (e g ^ lac-Z) regulated by a yeast promoter (e g ,the promoter region of a phermone inducible yeast gene) Thus, the present invention is directed to a recombmant yeast host cell comprising recombmant nucleic acid encoding the kinase domain of mammalian N-Pak or homolog thereof, wherein, when expressed said kinase domain or homolog thereof has senne/threonine kinase activity but no Cdc42 binding activity The present invention is also directed to an expression vector comprising a yeast promoter and nucleic acid encoding the kinase domain of mammalian N-Pak or homolog thereof, wherein, when expressed said kinase domain or homolog thereof has senne/threonine kinase activity but no Cdc42 binding activity Further, the present invention is directed to a method for identifying a compound that interferes with the activity of mammalian N-Pak comprising: (a) incubating a recombmant yeast cell that expresses the binding domain of mammalian N-Pak or functional homolog thereof with a test compound; (b) determining whether the mating signal transduction pathway is inhibited relative to a control without the test compound. Brief Description of the Drawing: Figure 1 Schematic representation of plasmid pJG215 Detailed Description of the Invention All DNA sequences are represented herein by formulas whose left to right orientation is in the conventional direction of 5' to 3' Nucleotide base abbreviations used herein are conventional in the art, i.e., T is thymme, A is adenme, C is cytosme and G is guanme It is also contemplated that the present invention encompasses modified sequences As used in the present application, the term "modified", when referring to a nucleotide sequence, means a nucleotide sequence which differs from the wild-type sequence found in nature The DNA sequences of the present invention can be obtained using various methods well-known to those of ordinary skill in the art The expression vectors can be partly or wholly synthesized chemically and/or partly or wholly prepared through genetic engineering techniques Fragments can be sequentially ligated (via appropriate terminal restriction sites or complementary terminal sequences) so as to form the correct linear sequence of nucleotides. The sequences of the present invention can also be modified (i.e., mutated) to prepare various mutations These mutations may permit higher levels of production of the expressed peptide. easier purification of the peptide, or provide additional restriction endonuclease recognition sites. All such modified DNA and polypeptide molecules are included within the scope of the present invention Expression vectors Expression vehicles of the invention for production of the kinase domain of mammalian N-Pak include plasmids or other vectors. In general, such vectors contain control sequences that allow expression in various types of hosts Suitable expression vectors containing the desired coding and control sequences may be constructed using standard recombmant DNA techniques known in the art, many of which are described in Sambrook et al. (1989), Molecular Cloning A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. An expression vector as contemplated by the present invention is at least capable of directing the replication of the vector in both bacteria and yeast and expression of the DNA sequence of interest in yeast One class of vectors utilizes yeast DNA elements that provide autonomously replicating origins such as the yeast 2u element or ARS1 sequence which yield extrachromosomal plasmids A second class of vectors relies upon the integration of the desired gene sequences into the host ceil chromosome. The vectors must also incorporate a bacterial origin of replication Suitable bacterial origins of replication include, for example the Co/E1 , pSC101 and M13 origins of replication Expression vectors useful of the present invention typically contain a promoter located 5' to (i e , upstream of) the DNA sequence to be expressed, and a transcription termination sequence located 3' to (i.e., downstream of) the sequence to be expressed Suitable promoters include, for example, the yeast ADH1 promoter The promoter sequence may also be inducible, to allow modulation of expression (e g , by the presence or absence of nutrients or other mducers in the growth medium) Examples include the yeast GAL 1 CUP1 and MET25 promoters Suitable termination sequences include, for example the yeast CYC1 termination and polyadenylation sequences The expression vectors may also include other regulatory sequences for optimal expression of the desired product Such sequences include secretory leader sequences, which provide for secretion of the expressed product or direct membrane localization, and restriction enzyme recognition sequences which provide sites for cleavage by restriction endonucleases All of these materials are known in the art and some are commercially available A suitable expression vector may also include marking sequences, which allow phenotypic detection and/or selection of transformed yeast or bacterial cells Such a marker may provide prototrophy to an auxotrophic host (e g , ammo acid biosynthetic genes), biocide resistance or supersensitivity (e g , antiobiotic resistance) or a phenotypically detectable signal (e g , fluorescence) The selectable marker gene can be either directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by transformation Examples of yeast selectable markers include Basidium pullulans AUR1-C gene, the S cerevisiae URA3 or LEU2 genes and the like Examples of bacterial selectable markers include the ampicillin resistance gene A preferred vector is pYESII, containing the Co/E1 and 2μ origins of replication the yeast URA3 and bacterial amp R genes, and the yeast GAL 1 promoter sequence (Invitrogen) In a further alternative, the constructs may be introduced into a cell by transformation in conjunction with a gene allowing for selection where the construct will become integrated into the host genome or persist episomally. Usually, the construct will be part of a vector having homologous sequences for integration or a replication system recognized by the host cell. Host cells The present invention additionally concerns hosts for the expression of the N-PAK gene constructs. Suitable host cells are yeasts. Examples include Saccharomyces species (e.g.,S. cerevisiae), Candida species (e.g., C. albicans), and Schizosaccharomyces species (e.g., S. pombe) in which endogenous activities do not interfere with the function of the protein under study. Especially preferred is the strain S.cerevisiae ATCC 74424 (referred to herein as SGY 1707 or YJG 200-16C). The term "ATCC" as used herein refers to the accession number of the American Type Culture Collection, 12301 Parklawn Drive Rockville Md 20852, the depository for the organism referred to Saccharomyces cerevisiae ATCC 74424 was deposited with the ATCC on August 20, 1997, under the provisions of the Budapest Treaty. Expression vectors may be introduced into host cells by various methods known in the art. For example, transformation of host cells with expression vectors can be carried out by the alkali cation method. However, other methods for introducing expression vectors into host cells, for example, electroporation and spheroplast transformation can also be employed. Host cells containing an expression vector may be identified by any of a number of methods known in the art The protein desired to be expressed in the present invention comprises at least the kinase domain of mammalian N-Pak and does not have the Cdc42 binding domain Types of N-Pak useful herein include rat, human, muπne, and the like The expression vectors and host cells of the invention may also be engineered to express larger sequences of N-Pak protein which can result in expression of other binding sites such as those that bind to the C-terminal portion (C100) of β-APP These other binding sites may be desirable for study of the interaction between N-Pak and proteins to which it binds. It is also contemplated that the present invention include functional homologs of the kinase domain of mammalian N-Pak. In this context, "functional homologs" are these peptides that exhibit senne/threonine kinase activity capable of complementing the yeast ste20 deletion. It is preferred that a functional homolog useful herein have at least about 50% homology (i.e., 50% positional similarity or identity) to the kinase domain of rat N-Pak. It has been discovered that expression of the N-Pak kinase domain in yeast in the absence of the Cdc42 binding domain allows for functional substitution for the yeast Ste20 protein in systems in which such substution was heretofore not seen (see Zhao et al, 1995). Therefore, truncation to the N-Pak gene to exclude the Cdc42 binding domain is important to the present invention. The DNA useful herein encoding the kinase domain of N-Pak optionally includes the codons upstream of the kinase domain up to the Cdc42 binding domain. That is, the DNA useful in the present invention optionally encodes the ammo acids flanking the N- termmal portion of the kinase domain but does not extend to the Cdc42 binding domain. Thus, in the case of rat N-Pak, the DNA useful herein encodes a peptide that starts anywhere from ammo acid number 138 to am o acid number 254 and stops after the kinase domain, i.e., typically at ammo acid number 544 (all reference to rat N-Pak ammo acid position numbers and nucleotide position numbers refer to the rat cDNA and ammo acid sequence of N-Pak as disclosed in WO 94/0581 1 ). Likewise, in the case of human N-Pak, the DNA useful herein encodes a peptide that starts anywhere from ammo acid number 133 to ammo acid number 269 and stops after the the kinase domain, i.e., typically at ammo acid number 545 (all reference to human N-Pak am o acid position numbers and nucleotide position numbers refer to the human cDNA and ammo acid sequence of h- Pak1 as disclosed in Brown et al., (1996), Curr. Biol., 6(5):598-605). Thus, a preferred embodiment is when the expression vector of the invention comprises a fragment of the rat N-Pak gene comprising a fragment of the rat N-Pak gene having a sequence starting at nucleotide 394 through nucleotide 820 and ending at nucleotιde1635. Similarly, another preferred embodiment is when the expression vector of the invention comprises a fragment of the human N-Pak gene having a sequence starting at nucleotide 517 through nucleotide 928 and ending at nucleotιde1691 . The DNA sequences of expression vectors, plasmids or DNA molecules of the present invention may be determined by various methods known in the art. For example, the dideoxy chain termination method as described in Sanger et al., Proc. Natl. Acad. Sci USA 74, 5463-5467 (1977), or the Maxam-Gilbert method as described in Proc. Natl Acad. Sci. USA 74 560-564 (1977) may be employed It should, of course, be understood that not all expression vectors and DNA regulatory sequences will function equally well to express DNA sequences. Neither will all host cells function equally well with the same expression system. However, one of ordinary skill in the art may make a selection among expression vectors, DNA regulatory sequences, and host cells using the guidance provided herein without undue experimentation and without departing from the scope of the present invention. The present invention further concerns an assay method for identifying a compound that interferes with the activity of mammalian N-Pak comprising: (a) incubating a test compound with a recombmant yeast cell that does not express Ste20 but expresses the kinase domain of N-Pak or functional homolog thereof, and, (b) determining whether the mating signal transduction pathway of the yeast cell is inhibited relative to a control without the test compound. In the assay of the invention it is preferred that the recombmant yeast cell does not express the Cdc42 binding domain of N-Pak. The assay of the invention can be performed by introducing a recombmant yeast strain of the invention into a growth medium under conditions resulting in constitutive expression of the N-Pak kinase domain in the presence of a test compound and monitoring the ability of the yeast to mate, using a quantitave mating assay. It is preferred to use a strain that is engineered to express a reporter in addition to the N-Pak kinase domain and then monitor expression of the reporter, thereby obviating the need for a mating phermone or actual mating In addition, it is also necessary that that the endogenous yeast Ste20 gene be deleted from or otherwise inactivated in the host cell. The reporters useful herein can be, for example, enzymes such as β-galactosidase, luciferase, green fluorescent protein, and the like. The assay method of the invention can be used to detect inhibitors of N-Pak kinase activity. Since N-Pak has been shown to bind to the C100 fragment of β-APP in vitro, the assay of the invention can useful to screen for compounds that inhibit the interaction between N-Pak and β-APP C100, provided that the sequence of N-Pak coding for the C100 binding domain is also expressed. The componds so identified are therefore useful in treatment and/or prevention of Alzheimer's Disease. The reporter construct can be altered, such that any desired reporter gene could be employed. This would be placed under the control of a promoter for a gene normally induced by mating, le, a gene positively regulated by the transcriptional activator Ste12. For example, instead of a heterologous gene for a non- essential protein, a yeast gene encoding a biosynthetic enzyme could be used. In a strain deleted for the chromosomal copy of this gene, growth would be dependent upon expression of the reporter gene A reverse system, whereby expression of the reporter is detremental to the cell, can also be constructed and is contemplated to be within the scope of the present invention. One example would employ the URA3 gene as a reporter. The URA3 gene would be fused to a mating responsive promoter such as FL/S1 . N-Pak induced signalling through the pathway would allow a ura3, ste20 host strain to grow in the absence of uracil. Inhibition of signalling would result in reduced growth. In the presence of the uracil analogue 5 fluoro-orotic acid (5FOA), Ura + cells are inhibited, while ura cells continue to grow. In this case, signalling through the pathway causes growth inhibition, while inhibition of signalling, by turning off expression of the URA3 reporter, will result in growth on 5-FOA media. Growth of the host cells may be achieved by one of ordinary skill in the art by the use of an appropriate medium. Appropriate media for growing host cells include those which provide nutrients necessary for the growth of the cells. A typical medium for growth includes necessary carbon sources, nitrogen sources, and trace elements. Inducers may also be added. The term "mducer", as used herein, includes any compound enhancing formation of the desired protein or peptide. Carbon sources may include sugars such as glucose, galactose, raffmose, and the like; organic acids such as sodium acetate, and the like; and alcohols such as ethanol. A preferred medium comprises Yeast Nitrogen Base (Difco) containing 2% glucose or 2% galactose and all necessary ammo acids. The pH of the medium is preferably adjusted to about 5 to 6, more preferably about 5 The process of the present invention is performed under conditions suitable for expression of the desired peptide. The pH of the medium is preferably maintained between 4.0 and 6.0, most preferably between 4.5 and 5.5, during the growth of host cells. A suitable temperature range for the process of the invention is from about 28°C to about 32°C Pressure is not known to be critical to practice of the invention and for convenience about atmospheric pressure is typically employed The process of the invention is preferably carried out under aerobic conditions. The yeast assay of the invention can be used for high throughput screening for agents that inhibit the activity of N-Pak. Inhibition would most probably be due to direct effects on the kinase activity, however, interference with signalling by other mechanisms, such as by altering the conformation of N-Pak such that it has reduced ability to interact with upstream activators or downstream target proteins might also be detected. The present system can also be useful for studying structure-function relationships of the N-Pak protein. It also can be useful for study of the role of the regions 5' of the kinase domain in regulation of activity In addition, it could be used to define specific regions that interact with other proteins such as β-APP. The method has utility for identifying compounds that specifically inhibit protein-protein interactions necessary for N-Pak activity Thus, the compounds mdentified by practice of the method of the invention can be useful to treat or prevent degenerative human diseases The following examples are further illustrative of the present invention. These examples are not intended to limit the scope of the present invention, and provide further understanding of the invention EXAM PLES MATERIALS AND METHODS Strains and strain construction The strains of S cerevisiae employed are YSR10 ( MATa, can 1- 100, ade2- 1, hιs3- 1 1, 15, Ieu2-3, 1 12, trp 1- 1 ,ura3- 1), YSR30 {MATa ,ste20 1 .LEU2, can l- 100, ade2-1, hιs3- 1 1, 15, Ieu2-3, 1 12, trp 1- 1,ura3- 1) kindly supplied by Dr. S. Ramer (Ramer and Davis, 1993) , SY2263 {MATa, ste 1 1 :ura3, FUS1/HIS3@hιs3, FUS1/lacZ@mf2 1, ura3, trpl, Ieu2, adel) and SY2068 {MATa, ste7::LEU2, , FUS1/HIS3@hιs3, FUS1/lacZ@mf2 1, ura3, trp l, Ieu2, adel), kindly supplied by Dr. George Sprague. Strain YJG 200-16C, a STE20 deπvitive containing the integrated FUS 1/lacZ reporter {MATa, ste20 1::LEU2, FUS1/lacZ@mf2 1 ade, hιs3-1 1, 115, Ieu2-3, 112, trpl, ura3 ) was derived by crossing strain YSR30 with SY2068. Plasmids were maintained and amplified in Eschenchia coil strain HB101 . All yeast strains were cultivated according to standard methods (Rose et al, 1990 ), using appropriately supplemented Yeast Nitrogen Base (YNB w/o ammo acids, Difco) and either glucose or galactose at 2%. Yeast were transformed with plasmid DNA by the alkali cation method, using a commercially prepared kit (Bio 101 ). Genetic procedures for crossing and tetrad dissection were as described (Rose et al 1990 ). To obtain a host strain containing, in addition to the STE20 mutation, an integrated FL/S1/lacZ reporter, the STE20 strain YSR30 was crossed with SY2068, a STE7 strain containing a FUS1 /lacZ reporter integrated into the chromosome. In order to obtain mating between the two sterile strains, the parent strains were each transformed with a plasmid containing a gene which complemented the respective sterile mutation, either pFL {GAL 1p/STE20) or pJG204 {GAL lp/STE7) The strains were pregrown on raffinose selective minimal medium, induced with galactose for four hours, then co-plated on rich medium. Diploids were obtained by micromanipulation and propagated on rich medium to allow plasmid loss to occur. Following sporulation, tetrads were dissected for analysis. Fourteen complete tetrads were scored for auxotrophic markers and mating type. As both the STE7 and STE20 gene disruptions were made with LEU2, it was not possible to establish which spore progeny contained the STE7 vs STE20 mutation by this analysis. When tested for lacZ by filter assay for β- galactosidase activity, several segregants showed a weak but scorable positive, suggesting that the FUSl/\acZ construct was present in these segregants. PCR analysis of intact yeast cells , done essentially as described by Sathe et al (1991 ), was used to score the S7 " E20 and S7Ε7 genotypes and confirm the presence of the FUSMlacZ reporter. To determine the mating type of selected sterile strains, each was first transformed with a plasmid containing the STE20 or STE7 gene, then tested for mating with the Mata and Mat tester strains. A single candiate STE20 , STE7, FUS1/\acZ segregant, YJG200-16B, was identified. Plasmids and plasmid construction: Plasmid pFLC-1 , containing the full length STE20 gene and pSTE20ΔN, containing an N-terminally deleted allele of STE20 { Ramer and Davis, 1993) were kindly supplied by Dr. Sandy Ramer. The STE20ΔN allele encodes a protein which lacks the first 494 ammo acids of the protein. The Cdc42 binding domain is absent but the kinase is fully functional and acts in a dominant fashion, giving a mating response in the absence of mating hormone. Each is a 2μm, URA3 based shuttle vector in which the STE20 gene is regulated by the GAL 1 promoter. Plasmid pYES, a 2 μm , URA3 cloning vector (Clonetech) was employed to subclone the N-Pak gene. All plasmid manipulations were done basically by the techniques described in Sambrook et al (1989). Plasmids were transformed into and amplified in E.coli strain HB101 . The GAL 1 promoter vector, pLL16AΔlac, was constructed by replacing the URA3 gene in the low copy GAL 1/\acZ containing plasmid pBM746 with TRP1, deleting the lacZ gene and inserting a polyiinker between the BamHI and Sail sites originally flanking the lacZ gene. The complete coding sequence of STE7 (Teague et al, 1986) and the kinase domain of STE11 (ammo acids 342-717) (Rhodes et al. 1990) were generated as BamHI- Sail fragments by PCR and subcloned behind the GAL 1 promoter in pLL16A lac. The plasmids are pJG212 (STE7 7ΔN) and pJG204 {STE7) respectively. As with STE20, an N-termmal deletion of STE1 1 gives a constitutively activated kinase. The functionality of each of these PCR-generated clones was demonstrated by the ability to restore mating competence to the STE1 1 or STE7 mutant. The N-terminally deleted fragment of N-Pak was isolated as a Hincll-BamHI fragment (nt 397-1700) and subcloned behind the GAL 1 promoter in pYES. (For pJG215-K, pYES was cut with Asp718 made blunt with Klenow and then BamHI cut; in pJG215-L, pYES was cut with Hindlll, filled in, then BamHI cut). Each of these constructs is expected to produce a protein initiating at the first ATG downstream of the fusion junction. Thus the translated product will start at the methinione residue which is constitutes ammo acid 138 in the full length protein. The promoter/N-PAK junction of each of the plasmids was sequenced to verify the fusion. Note, the protein produced does not contain the Cdc42 binding consensus sequence. Enzyme assays: The ability to produce β-galactosidase was initially determined using the filter assay method (Breeden and Nasmyth 1985). Single colonies were streaked onto Whatman 50 filter paper placed on selective minimal agar medium containing either glucose or galactose as carbon source. After a 48-72 hour incubation, the filters were immersed in liquid nitrogen for 30 seconds, air dried and assayed. For this, the filters were placed in petri dishes on top of 3MM paper which was presoaked in 3.5 ml of reaction mixture per 100 mm dish and incubated at 30 oC for 4 to 24 hours. The reaction mixture contains X-gal (5-bromo-4-chloro-3-indolyl-β-D- galactoside) at 1 mg/mi in Z buffer (Miller, 1972) For quantitative determination, enzyme assays were performed on cell extracts, using the fluorometπc substrate methyl-umbelliferone galactoside Quantitative mating assays Quantatative mating reactions were performed as described by Sprague (Meth Enzymol 194, 1991 ) with some modification Strain DC17 {MATa, his) was used as the mating test strain. Cultures were pregrown overnight on selective minimal medium containing 2% raffmose and 0 2% glucose. Galactose to a final concentration of 2% was added and the cultures incubated for an additional four hours to induce gene expression. The cells were resuspended to give 4 x10 8 cells/ml of DC17 and 8 x 10 7 cells/ml of the MATa strain to be tested A 25 μl aliquots of each MATa strain was plated on 5 ml of rich medium contained in a well of a 25 mm diameter well microtiter dish After the fluid was completely absorbed into the agar, 25 μl of the MATα tester strain was added This resulted in a 5-fold excess of tester strain Controls using each strain alone were included The mating mixtures were incubated for four hours at 30°C, then harvested in 0 5 ml of sterile water, serially diluted and aliquots plated on minimal agar Only those cells which had formed diploids can grow on minimal medium, as each haploid strain contains different auxotrophic mutations After incubation at 30°C for three days, the number of colonies growing on the plates was determined. The frequency of mating is expressed as the total number of diploids formed in the mated sample Preliminary experiments showed that it was essential to use rich medium and that all fluid placed on the agar must be totally absorbed to obtain mating As galactose appeared to have a toxic effect on some of the transformed strains, a short galactose induction period was employed. RESULTS. The results are in the following tables Table 1 : Quantitative mating analysis of transformed STE20 strains. The various transformed haploid strains were tested for mating efficiency as described in Materials and Methods TABLE 2 Filter assay for β-galactosidase Plasmid Gene Non-induced The number of pluses indicates the relative intensity of the resulting blue color. Table 3: Activation of the mating signal transduction pathway by N-Pak β- alactosidase levels in lucose repressed and alactose induced cultures All plasmids are in strain YJG200-16C (STE20 ). Activity is expressed as units per 25 μl aliquot of culture extract (2.5x10 7 cells). One unit of enzyme is defined as the amount needed to hydrolyze one picomole of substrate per minute under the standard reaction conditions.

Claims: WHAT IS CLAIMED IS: 1 . A recombmant yeast host cell comprising recombinant nucleic acid encoding the kinase domain of mammalian N-Pak or homolog thereof, wherein, when expressed said recombinant nucleic acid has senne/threonine kinase activity but not Cdc42 binding activity. 2. the recombinant yeast cell of Claim 1 wherein said recombinant nucleic acid is DNA. 3. The recombinant yeast cell of Claim 1 wherein said recombinant nucleic acid encodes the kinase domain of mouse, rat, or human N-Pak. 4. The recombinant yeast cell of Claim 1 wherein said recombinant nucleic acid encodes a human N-Pak peptide starting at ammo acid 133 to 269 and stopping at the end of the kinase domain of human N-Pak. 5. The recombinant yeast cell of Claim 1 wherein said recombinant nucleic acid encodes a rat N-Pak peptide starting at ammo acid 138 to 254 and stopping at the end of the kinase domain of rat N-Pak. 6. The recombinant yeast cell of Claim 1 wherein said homolog has at least about 80% homology to the kinase domain of of rat N-Pak. 7. The recombinant yeast cell of Claim 1 wherein said nucieic acid comprises a yeast promoter. 8. The recombinant yeast cell of Claim 7 wherein said promoter is the GAL 1 promoter. 9. The recombinant yeast cell of Claim 1 wherein said nucleic acid further encodes a reporter. 10. The recombinant yeast cell of Claim 9 wherein said reporter is a FUSl/lacZ construct. 1 1 . The recombinant yeast cell of Claim 1 lacking a functional STE20 gene. 12. The recombinant yeast cell of Claim 1 which is a strain of Saccharomyces, Candida, or Schizosaccharomyces. 13. Saccharomyces cereviciae ATCC , 14. An expression vector comprising a yeast promoter and nucleic acid encoding the kinase domain of mammalian N-Pak or homolog thereof, wherein, when expressed said nucleic acid has senne/threonine kinase activity. 15. The expression vector of Claim 14 wherein said nucleic acid is DNA. 16. The expression vector of Claim 14 wherein said nucleic acid encodes the kinase domain of mouse, rat, or human N-Pak. 17. The expression vector of Claim 14 wherein said nucleic acid encodes a human N-Pak peptide starting at ammo acid 133 to 269 and stopping at the end of the kinase domain of human N-Pak. 18. The expression vector of Claim 14 wherein said nucleic acid encodes a rat N-Pak peptide starting at ammo acid 138 to 254 and stopping at the end of the kinase domain of rat N-Pak. 19. The expression vector of Claim 14 wherein said homolog has at least about 50% homology to the kinase domain of of rat N-Pak. 20. The expression vector of Claim 14 wherein said promoter is the GAL 1 promoter. 21 . The expression vector of Claim 14 wherein said nucleic acid further encodes a reporter. 22. The expression vector of Claim 21 wherein said reporter is a FUSl/lacZ construct. 23. The expression vector of Claim 14 wherein said nucleic acid encoding the kinase domain of mammalian N-Pak or homolog thereof is a fragment of the rat N-Pak gene consisting essentially of a sequence starting at nucleotide 394 through nucleotide 820 and ending at nucleotide1635. 24. The expression vector of Claim 14 wherein said nucleic acid encoding the kinase domain of mammalian N-Pak or homolog thereof is a fragment of the human N-Pak gene consisting essentially of a sequence starting at nucleotide 517 through nucleotide 928 and ending at nucleotide1691 . 25. The expression vector of Claim 14 which is a plasmid. 26. Plasmids pJG215-K9, pJG215-K21 , and pJG215-L8. 27. A method for identifying a compound that interferes with the activity of mammalian N-Pak comprising: (a) incubating a test compound with a recombinant yeast cell that does not express Ste20, and expresses the kinase domain of N- Pak or functional homolog thereof, and; (b) determining whether the mating signal transduction pathway of the yeast cell is inhibited relative to a control without the test compound. 28. The method of Claim 27 wherein the recombinant yeast cell does not express the Cdc42 binding domain of N-Pak. 29. The method of Claim 27 wherein said recombinant yeast cell expresses a reporter and inhibition of the mating signal transduction pathway is determined by detecting decreased expression to said reporter. 30. The method of Claim 29 wherein said reporter is β- galactosidase.

Inventors: GORMAN JESSICA A, MANLY SUSAN, GORMAN, JESSICA, A., MANLY, SUSAN
Applicants: SQUIBB BRISTOL MYERS CO, BRISTOL-MYERS SQUIBB COMPANY
Application year: 1998
Main classification: C12N1
IPC codes: C12N1, C12N9, C12N15, C12Q1