KINASE WEE1 FUSION PROTEIN COMPOSITIONS, NUCLEOTIDE SEQUENCES, EXPRESSION SYSTEMS, AND METHODS OF USE

Abstract: The invention describes recombinant fusion proteins containing Wee1 protein sequences involved in checkpoint regulation of cell cycle, nucleic acid molecules that encode the fusion protein, and methods using the proteins for screening for compounds which modulate Wee1 or Cds1 function.

Description: KINASE EE1 FUSION PROTEIN COMPOSITIONS, NUCLEOTIDE SEQUENCES, EXPRESSION SYSTEMS, AND METHODS OF USE Technical Field The invention relates to methods and compositions for screening for compounds that inhibit or modulate phosphorylation of Weel fusion proteins by kinase Cdsl. Background Cell cycle checkpoints ensure that chromosomal DNA is replicated and repaired prior to nuclear division (Hartwell et al, Science, 246:629, 1989; L. Hartwell, Cell, 71:543, 1992; and S.J. Elledge, Science. 274:1664, 1996). In mammalian cells, loss of checkpoint control results in rearrangements, amplification, and loss of chromosomes, events that are causally associated with cancer. In yeasts, checkpoint controls are vital for survival when DNA is damaged or replication is inhibited. Cdc2, the cyclin-dependent kinase that initiates mitosis, is the ultimate target of the DNA replication and repair checkpoints and is regulated by checkpoint kinases that couple mitosis to the completion of DNA replication and repair. The repair checkpoint kinase Chkl regulates Cdc25, a phosphatase which activates Cdc2. In the fission yeast Schizosaccharomyces pombe, Cdc2 is inhibited by phosphorylation on tyrosine-15. This phosphorylation is catalyzed by the kinases Weel and Mikl and reversed by the phosphatase Cdc25. Inhibitory phosphorylation of Cdc2 is crucial for replication and repair checkpoints in fission yeast and human cells (Enoch et al, Cell, 60:665, 1990; Lundgren et al, Cell, 64:1111, 1991; Jin et al, J. Cell Biol.. 134:963 (1996); Blasina et al, Mol . Biol . Cell, 8:1013, 1997; and Rhind et al, Genes Dev. , 11:504, 1997). Chkl apparently enforces the DNA repair checkpoint by -2- phosphorylating and inhibiting Cdc25 (Rhind et al, Genes Dev. , 11:504, 1997; Furnari et al, Science, 277:1495, 1997; Sanchez et al, Science. 277:1497 (1997); and Peng et al, Science, 277:1501, 1997). Chkl is only required for the repair checkpoint, not the replication checkpoint evoked by hydroxyurea (HU) (Walworth et al, Nature, 363:368, 1993; and al-Khodairy et al, Mol . Biol. Cell. 5:147, 1994). Thus, the kinase Weel participates in the regulation of cell cycle checkpoints during mitosis by catalyzing phosphorylation of the cyclin-dependent kinase that initiates mitosis, Cdc2. The role of Weel in these regulatory processes for controlling cell cycle are not well characterized. The kinase Cdsl has also been implicated in the checkpoint process, although its role is not understood (Marukami et al, Nature, 374: 817, 1995). There is a need for systems to characterize the role of Weel and Cdsl in regulation of cell cycle, and for screening for compounds which inhibit or modulate the effectors of these processes, particularly inhibitors of Weel or Cdsl function . Brief Summary of the Invention It has now been discovered that Weel is phosphorylated by Cdsl, and that Cdsl has an important role in the regulation of the replication checkpoint for mitosis. It has also been discovered that Weel can be used in various forms, including truncated proteins based on Weel, in compositions and methods for evaluating the components of the Weel-dependent processes related to replication checkpoint, particularly Cdsl. The invention therefore describes various nucleic acids which encode a truncated Weel protein, including expression vectors containing the nucleic acids for expressing the Weel protein. Also described are various forms of truncated Weel protein, particularly fusion proteins containing a Weel amino acid residue sequence which defines the functional site for phosphorylation by Cdsl. The invention describes methods for identifying compounds which modulate Weel function, or which modulate the ability of Cdsl to phosphorylate Weel. Other uses will be apparent to one skilled in the art in light of the present disclosures. Description of the Drawings Figures 1A-1D illustrate the association of an HU-regulated kinase with the NH 2 -terminus of Weel as described in Example 2. Figure 1A is a photograph of a SDS-polyacryla ide gel analysis of GST:Weel 152 and degradation products produced in bacteria and purified with GSH-Sepharose . The major degradation products are GST fused to -70 a ino-acids of Weel (GST:Weel 70 ) and unfused GST. Proteins were stained with Coomassie Blue. Figure IB is a photograph of an analysis of proteins from cells that were treated with 12 M HU for a 1 hr time course at 30 °C . Cell lysates were incubated with GST: eel proteins bound to GSH-Sepharose. The GSH-Sepharose was washed and assayed for associated kinase activity. The positions of GST:Weel 152 and GST:Weel 70 following 12% SDS-polyacrylamide gel electrophoresis are shown. Figure 1C is a photograph of an analysis of proteins which illustrates that mutational inactivation of cdc22, encoding the large subunit of ribonucleotide reductase, enhances phosphorylation of GST:Weel 70 . Cells carrying the temperature sensitive cdc22-M45 allele or wild-type cells were grown at 25 °C . The cultures were split and maintained - 4 - at 25 °C or incubated at 35.5 °C for 90 min. GST: Weel phosphorylation assays were performed as described above. Figure ID is a composite graph and photograph which illustrates activation of the kinase that phosphorylates GST:Weel 70 in extracts from cdc25-22 cells released into HU from a G2 arrest. A culture of cdc25-22 cells grown at 25 °C was shifted to 35.5 °C for 4 hr. The culture was split and treated with 12 mM HU or mock-treated for 45 min at 35.5 °C . Both cultures were returned to 25 °C and samples taken every 10 min. Septation index marks the completion of mitosis. Lysates from each time point were assayed for GST : Weel 70 kinase . GST: Weel 70 phosphorylation occurred in the HU-treated samples after exit from M, whereas phosphorylation of GST: Weel 70 remained low in samples from the mock-treated culture. In the mock-treated culture, activity of the kinase that phosphorylates GST: eel 152 increased as cells underwent mitosis. Figures 2A-2D illustrate that Cdsl is the kinase that phosphorylates GST: Weel 70 as described in Example 2. Figure 2A is a photograph of an analysis of proteins which illustrates that phosphorylation of GST: Weel 70 is dependent on Radl and Rad3. Wild-type, radl and rad3 strains cultures were split and treated for 1 hr with HU, or mock-treated, and then assayed for GST: Weel 70 phosphorylation as described in Figure 1. Figure 2B is a photograph of an analysis of proteins which illustrates that GST: Weel 70 phosphorylation is dependent on Cdsl but not Chkl. Wild-type, chkl and cdsl cultures were split and treated for 1 hr with HU or mock-treated. Phosphorylation of GST:Weel was assayed. Figure 2C is a photograph of an analysis of proteins which illustrates that Cdsl interacts with GST: Weel but not GST. Lysates from HU-treated cells expressing epitope tagged Cdsl (Cdsl HAHIS ) expressed from the cdsl genomic locus were incubated with GST or GST: Weel proteins bound to GSH-Sepharose. After extensive washing, samples were resolved by 10% SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with antibodies to HA and GST. Figure 2D is a photograph of an analysis of proteins which illustrates that Weel interacts with GST:Cdsl but not GST:Chkl in vivo . Strains carrying integrated nmtl : GST : chkl + or nmtl : GST : cdsV constructs were grown in minimal medium (EMM2) without thiamine at 30 °C for 16 hr to induce expression of GST fusion proteins. Cells were lysed and GST fusion proteins purified with GSH-Sepharose. After extensive washing, samples were resolved by 10% SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with antibodies to Weel, Cdc25 and GST. A -110 kDa protein corresponding to Weel was detected in the GST:Cdsl sample, whereas a -80 kDa protein corresponding to Cdc25 was detected in the GST: Chkl sample. The -110 kDa protein was not detected in samples prepared from A weel cells. GST:Cdsl and GST:Chkl migrate at positions corresponding to -82 and -80 kDa, respectively. Figures 3A-3D illustrate that HU stimulates Cdsl kinase activity, binding of Cdsl to GST: Weel 70 in lysates and accumulation of Mikl as described in Example 2. Figure 3A is a photograph of an analysis of proteins which illustrates that HU stimulates Cdsl binding to GST: eel 70 . A culture of cells expressing Cdsl HAHIS from the cdsl genomic locus was split and treated for 3.5 hr with HU or mock-treated. The cultures were split again and samples processed for Ni 2+ -NTA purification of Cdsl HAHIS in denaturing conditions or purification of proteins that bind GST:Weel proteins in nondenaturing conditions. After extensive washing, samples were resolved by 10% SDS-polyacrylamide gel electrophoresis -6- and subjected to immunoblot analysis. Figure 3B is a photograph of an analysis of proteins which illustrates that HU activates Cdsl kinase activity. In a separate experiment Cdsl HAHIS was purified in native conditions and assayed for autophosphorylation activity (upper panel) . Immunoblot analysis confirmed that similar amounts of Cdsl HAHIS were recovered in the HU and mock-treated samples (lower panel) . Figure 3C is a photograph of an analysis of proteins which illustrates that Cdsl and Rad3-dependent increase in Mikl abundance in HU-treated cells. Wild-type, cdsl and rad3 cells in which genomic mikl encoded Mikl HAHIS protein were incubated for 4 hrs in YES at 25 °C with 12 mM HU or mock-treated. Mikl HAHIS protein was purified by Ni 2+ -NTA purification in denaturing conditions and detected by immunoblotting with antibodies to HA. Figure 3D is a photograph of cell cultures which illustrates that Mikl is important for survival of HU treatment. Serial dilutions of wild-type and mikl cells were plated in YES medium supplemented with 0 or 6 mM HU and incubated for 2 days at 30 °C . Figure 4 illustrates that Cell cycle arrest caused by expression of large amounts of GST: Cdsl as described in Example 2. Cells having an integrated nmtl : GST: cdsV construct were incubated in nmtl-repressing medium (promoter "off") or inducing medium (promoter "on") for 19 hrs at 30 °C . GST:Cdsl expression induced cell cycle arrest in wild-type, rad3 and chkl strains. Flow cytometry analysis confirmed that these cells were arrested with a 2C DNA content. Cells expressing Cdc2Y15F, a form of Cdc2 in which tyrosine at position 15 is replaced with phenylalanine, were insensitive to GST:Cdsl. Figures 5A-5B are graphs which illustrate that - 7 - replication checkpoint is abolished in cdsl chkl cells as described in Example 2. Figure 5A shows that synchronous cultures were obtained by centrifugal elutriation. Progression through mitosis in the presence of 12 mM HU was monitored by scoring binucleate or septated cells. The cdsl chkl double mutant and rad3 cells underwent mitosis in the presence of HU, whereas division was arrested in control strains (wild-type, cdsl and chkl single mutants) . Figure 5B shows that the cdsl chkl double mutant and rad3 cells are equally hypersensitive to killing by 12 mM HU. Wild-type and chkl cells were not sensitive to HU, whereas cdsl cells were moderately sensitive. Detailed Description of the Invention The invention describes compositions and systems for screening for compounds which effect the role of Weel in cell cycle checkpoints. In particular, the invention describes a recombinant truncated protein containing Weel which can be used in combination with other components of the cell cycle, particularly Cdsl, Chkl, Cdc2, Cdc25 and the like checkpoint proteins, in methods to identify and characterize regulatory pathways and to identify compounds which modulate and/or inhibit components in the pathways, particularly Cdsl. The invention describes particular fusion protein constructs, particularly GST:Weel fusion proteins, which are useful in the assays. The invention also describes an isolated nucleotide sequence which encodes truncated kinase Weel or a fusion protein containing truncated kinase Weel. Also described are expression vectors for expression of a fusion protein of this invention. In addition, the invention describes an assay for screening for modulators of check point regulation using - 8 - Weel fusion proteins. A particular assay screens for compounds which modulate activity of kinase Weel or kinase Cdsl. A. Compositions The invention describes compositions of truncated Weel proteins, isolated nucleic acids which encode the Weel proteins, expression vectors containing the nucleic acids useful for expressing a Weel protein of this invention. The Weel proteins are useful for evaluation Weel protein function, particularly for identifying modulators of Weel or Cdsl activity. 1. Truncated Weel Protein In one embodiment, the invention describes a truncated Weel protein which is useful for evaluating Weel function and for screening for modulators of Weel or its effector molecule, Cdsl. A truncated Weel protein of this invention is a polypeptide having an amino acid residue sequence derived from a native Weel protein, and which comprises the region of Weel which is a functional substrate of Cdsl. Preferably, the truncated Weel protein has a sequence which substantially corresponds to the native Weel amino acid residue sequence, and more preferably has the same amino acid residue sequence as is found in native Weel protein . By "substantially corresponds" means that there can be substitutions and/or deletions in the native Weel protein amino acid residue sequence so long as the biological activity of the Weel protein as a functional substrate of Cdsl is not substantially effected. That is, the truncated Weel protein with a modified sequence performs in the assays as described in the Examples for purposes of measuring Weel - 9- activity . It is understood that the length of a Weel protein can vary so long as the region for performing as a functional Cdsl phosphorylation substrate is present, as described herein. A preferred truncated Weel protein comprises an amino acid residue sequence which corresponds to amino acid residues 11-70 of native Weel, preferably residues 11-72 or 10-70 or 10-72. Particularly preferred is a protein that comprises residues 11-72. In a related embodiment, a truncated Weel protein comprises residues 11-152 or residues 10-152. Other combinations can also be easily be described and constructed by the methods described herein, and therefore, the invention should not be construed as so limited. Weel is a large protein and the complete amino acid residue sequence and corresponding cDNA nucleotide sequence are well known and available on the Genbank database as accession number M16508. By the term "truncated" is meant that the Weel protein is not the full length, wild-type Weel protein; instead a truncated protein contains amino acid residue deletions at either the amino or carboxy terminus of the wild-type sequence as described herein. Particularly preferred deletions include proteins in which residues 1-9, residues 1-10, residues 73-152 or residues 71-152 have been deleted. Particularly preferred truncated Weel proteins are described in the Examples, and have an amino acid residue sequence shown in SEQ ID NO 1 for the GST : Weel (11-72) protein construct and shown in SEQ ID NO 3 for the GST:Weel (11-152) protein construct. It is preferred that a subject truncated Weel protein be provided in the form of a fusion protein. That is, a protein which comprises a truncated Weel protein of the - 10- present invention operatively linked to a second protein where the operative linkage is in the form of a peptide bond typically found between amino acids of a polypeptide . Fusion proteins are well known in the art and are used because of the advantages provided by the presence of a second protein function provided by that second protein. These functions can include tags to facilitate isolation or identification of the fusion protein, to produce a bifunctional protein, and the like functions. In view of the state of the art relating to fusion proteins, the nature of the second protein is not considered to be the essential feature of the invention, and therefore is not to be construed as limiting. A preferred fusion protein includes glutathione-S-transferase (GST) . Exemplary fusion proteins, nucleotides, expression vectors and method for preparing the fusion proteins, nucleotides and vectors are described in the Examples. 2. Nucleic Acids and Expression Vectors In another embodiment, the invention contemplates an isolated nucleic acid molecule which encodes a truncated Weel protein of this invention, and expression vectors capable of expressing the protein off of the nucleic acid. A typical nucleic acid comprises a nucleotide sequence which, according to the genetic code, encodes a Weel protein of this invention, and therefore, the actual nucleotide sequence can vary depending upon the actual amino acid residue sequence and upon codon usage, as is well known. A preferred nucleic acid encodes a protein comprising amino acid residues 11-70, and preferably comprises residues 10-70, residues 11-72, residues 11-152 or residues 10-152. A preferred nucleic acid encodes a fusion protein as described herein, preferably a GST:Weel fusion protein, and - 11 - particularly a GST : eel ( 11-72 ) or GST : eel ( 11-152 ) protein. Although not intended to be so limited, a preferred nucleic acid has a nucleotide sequence which encodes a Weel protein of this invention as shown in SEQ ID NO 2 or 4, or which encodes a GST: Weel fusion protein as shown in SEQ ID NO 2 or 4. Also contemplated are expression vectors for expression of a truncated Weel protein of the present invention. An expression vector comprises a nucleic acid of the present invention operatively linked to the recombinant nucleic acid elements necessary for transcription and translation of the nucleic acid to express the encoded Weel. Such transcription and translation expression control elements are well known in the art, and art not to be construed as limiting. These expression control elements can further include transcription enhancers, transcription promoters, translation initiators and terminators, and the like elements . Preferred expression vectors are described in the Examples, or are commercially available from a variety of sources. Particularly preferred are the pGEX expression vectors commercially available. The complete nucleotide sequence of two preferred vectors are shown in SEQ ID NO 2 and 4, and correspond to pGEX-GST :Weel (11-72 ) and pGEX- GST:Weel (11-152) , respectively. B . Methods for Identifying Weel Modulators The invention also contemplates a variety of methods for evaluating Weel function, and particularly for identifying modulators of Weel or modulators of Cdsl, which phosphorylates Weel. As shown by the examples, the analysis of the various components of the regulation of the cell cycle, particularly the replication checkpoint, can be -12- carried out in a variety of formats, including by the use of isolated proteins, cell lysate containing one or more of the components, genetic strains of cells to provide different components of the checkpoint, all in combinations with the truncated Weel protein of this invention. A preferred method involves the evaluation of the interaction between Cdsl and Weel, where it is seen that Weel is a phosphorylation substrate for Cdsl. The site in Weel for phosphorylation is in the amino terminal portion of the Weel protein designated as the functional Cdsl phosphorylation substrate as described herein. Thus, in one embodiment, the invention describes a method for identifying a compound which modulates Cdsl kinase activity comprising the steps of: a) combining: i) a test compound; ii) a truncated Weel protein of this invention, or a homolog thereof; and iii) a composition comprising Cdsl protein; b) maintaining the combination of step (a) under conditions sufficient for Cdsl to phosphorylate said truncated Weel protein; and c) determining the amount of phosphorylated Weel protein whereby the amount of phosphorylated Weel indicates whether said test compound modulates the activity of Cdsl. In preferred embodiments, a truncated Weel protein comprises amino acid residues 11-70 of the native Weel protein as described herein, and preferably comprises amino acid residues 11-72, or 10-72 or 11-152 or 10-152 of native Weel protein. In this method, the test compound can be any substance believed to have an effect on the ability of Cdsl to phosphorylate Weel, and can include hydroxyurea (HU) , -13- components of the replication checkpoint pathway such as Cdc22, Cdc25, Chkl, Mikl, and the like components, genetic variants or mutants of these components of the checkpoint pathway, and any other molecule or test substance believed to be a drug candidate for modulating cell cycle checkpoints by inhibiting the phosphorylation interaction between Cdsl and Weel. The test reaction admixture further contains a truncated Weel protein according to the present invention, and can be provided in a variety of forms as described herein. The admixture further contains a composition comprising Cdsl protein which is capable of phosphorylating a truncated Weel protein of this invention in an amount sufficient to produce detectable phosphorylation under reaction conditions. The source of Cdsl protein in a composition containing Cdsl protein can vary widely, and therefore the invention is not to be construed as so limited. For example, the protein can be in the form of an isolated protein provided in vitro to a reaction admixture. The Cdsl protein can be isolated by a variety of known methods including biochemical extraction or immuno-adsorption from cell lysates, expression of recombinant protein using cloned Cdsl cDNA, and the like methods known in the checkpoint kinase arts. Cdsl protein can also be provided by preparing a lysate of a cell containing Cdsl protein as is described herein. Many different genetic strains of cells, such as yeast (e.g., S. pombe) stains, are available which express wild type (wt) or genetically variant Cdsl proteins suitable for use in the methods of the present invention, which strains and the preparation and handling of cell lysates are described in more detail in the Examples. In addition, as is shown herein, Cdsl binds to Weel, and therefore can be -14- isolated by use of the Sepharose bead isolation of GST: Weel protein following admixture with cell lysates containing Cdsl, as described in the Examples. The conditions for maintaining the combined admixture to allow Cdsl catalyzed phosphorylation of Weel are generally well known in the art, can vary widely depending upon the reaction format, and therefore are not to be considered as limiting. Examples of reaction conditions are described in the Examples and in the art, and include a variety of reaction components to support the biochemistry of phosphorylation of Weel by Cdsl, including buffers, phosphate donors and the like. Methods for determining amounts of a phosphorylation reaction can also vary widely, and depend in part on the nature of the reaction chemistry. Typically, the phosphate donor has a labeled phosphate leaving group, such as gamma- P 32 , and the like labeled substrate. Detection can involve isolating the Weel protein and measuring the amount of label present on the Weel protein, such as is described in the Examples using SDS-gel electrophoresis and autography to measure the amount of radioactivity associated with the Weel protein . Relative amounts of phosphorylation can be determined using comparisons to control reactions with known amounts of Cdsl protein, Weel protein and control reaction conditions, as is well known. In a related embodiment, the invention contemplates a method for identifying a compound which modulates kinase Weel activity comprising the steps of: a) combining: i) a test compound; ii) a truncated Weel protein of this invention, or a homolog thereof; and - 15 - iii) a reagent for Weel; b) maintaining the combination of step (a) under conditions suitable for Weel to react with said reagent; and c) determining the amount Weel reacts with said reagent whereby the amount of reaction indicates whether said test compound modulates the activity of Weel. In preferred embodiments, a truncated Weel protein comprises amino acid residues 11-70 of the native Weel protein as described herein, and preferably comprises amino acid residues 11-72, or 10-72 or 11-152 or 10-152 of native Weel protein. Many aspects of the above method are similar to the method described herein above for identifying modulators of Cdsl phosphorylation of Weel, and therefore will not be discussed again. In the method, a reagent for Weel refers to any compound or protein which interacts with Weel in a detectable reaction, and can include a cofactor for the reaction, a substrate for Weel kinase, or an enzyme which itself uses Weel as a substrate, any of which are modified in the reaction such that the amount of reaction can be measured. Thus, for example, the reagent can be a phosphate donor substrate which reacts in the reaction admixture, such as a Cdsl kinase substrate that donates a labeled phosphate to Weel in the presence of Cdsl (e.g., [gamma-P 32 ] ATP) . In this case, the determining comprises measuring phosphorylation of Weel protein. Alternatively, the reagent can be a Weel substrate such as Cdc22 which is phosphorylated by Weel as described herein, and the determining comprises measuring phosphorylation of Cdc22 similar to the procedures described herein. Further exemplary method formats are described in the -16- Examples . Examples The following examples illustrate, but do not limit, the present invention. 1. Preparation of GST:Weel Fusion Proteins To study the role of Weel on Chkl, a model system was designed using fusion proteins based on Weel. The rationale was developed because it was known that Chkl is only required for the repair checkpoint, not the replication checkpoint evoked by hydroxyurea (HU) (Walworth et al, Nature, 363:368, 1993; and al-Khodairy et al, Mol . Biol. Cell , 5:147, 1994) . However, an HU-induced arrest of cell cycle is rapidly abrogated after inactivation of temperature sensitive Weel protein in a mikl background, showing that the replication checkpoint requires inhibitory phosphorylation of Cdc2. Weel and Mikl are not individually required for an HU arrest, indicating that Weel and Mikl may be coordinately regulated by the replication checkpoint. To test this hypothesis, a construct was made to evaluate whether Weel was phosphorylated by an HU-activated kinase, with attention focused on the NH 2 -terminal regulatory domain (Aligue et al, J. Biol. Chem. , 272:13320, 1997). To that end, a fusion of glutathione-S-transferase (GST) protein to amino-acids 10-152 of Weel was designed and constructed, and is designated GST: Weel 152 (also referred to herein as GST : eel (11-152) . First, a PCR fragment was made encoding amino acids 10 to 152 of Weel using the known nucleotide sequence and amino acid residue sequence of the native Weel protein, incorporating a 5' Ndel site and 3 1 Notl site/stop codon into the fragment. This fragment was then cloned into a - 17 - modified pGEX vector and transformed into the E. coli BL21 (DE3) expression host [Shiozaki et al, Meth . Enz . , 283:506 (1997)]. The expression vector construct is referred to herein as pGEX-GST : Weel (11-152 ) , and has a complete nucleotide sequence shown in SEQ ID NO 4. Similarly, a fusion of GST protein to amino-acids 11-72 of Weel was designed and constructed, and is designated GST:Weel 70 (also referred to herein as GST : Weel (11-72 ) . The expression vector construct is referred to herein as pGEX-GST : Weel ( 11-72 ) , and has a complete nucleotide sequence shown in SEQ ID NO 2. Expression of a GST: Weel fusion protein by the pGEX expression vector in the BL21 host was induced by the addition of IPTG (1 mM) to exponentially growing cells at OD600 = 0.6 in LB media plus 50 μg/ml ampicillin at 37 C. After 2 hours at 37 C, cells were collected by centrifugation and stored at -70 C in aliquots (200 μl pellet volume) . Cells were lysed in 600 μl lysis buffer [50 mM Tris (pH8), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% NP-40, 5 μg/ml leupeptin/aprotinin/pepstatin, 1 mM PMSF] by sonication, centrifuged at 13,000 x g for 10 min at 4 C. GSH-Sepharose (50 μl bead volume) was added to the supernatant and incubated at 4 C for 30 min. The beads were then washed 3 times with 1 ml lysis buffer to yield expressed, isolated recombinant GST:Weel fusion protein. The protein preparations yielded full-length GST: Weel 152 and GST:Weel 70 (Figure 1A) , the latter being a truncation product containing -70 amino acids of the amino terminal portion of Weel protein. 2. Evaluation of Weel Function in Cell-Cycle The GST: Weel 152 and GST:Weel 70 proteins prepared in Example 1 were used to study yeast cellular functions. To - 18 - that end, the isolated proteins were bound to glutathione (GSH) -Sepharose and mixed with cell lysates from the fission yeast Schizosaccharomyces pombe (S. pombe) harvested at intervals during incubation with hydroxyurea (HU) . The cell lysates were obtained from a variety of strains of S. pombe cells having the following genotypes: PR109, wild-type; NB2117, cdsl : :ura4+; NB2118, cdsl:2HA6His:ura4+; NR1826, rad3::ura4+; OM591, cdc22-M45; NR1593, radl: :ura4+; NR1592, chkl::ura4+; BF1758, nmtl :GST: chkl :leul+; BF1916, nmtl : GST : cdsl : leul+; OM2173, mikl:2HA6His:ura4+; OM2183, mikl : 2HA6His :ura4+ cdsl : :ura4+; PR712, mikl: :ura4+; and BF2115, cdsl::ura4+ chkl::ura4+. All strains were leul-32 ura4-D18. All nmtl promoter constructs were integrated at the leul-32 locus. Growth media and general methods for S. pombe have been described [Moreno et al, Meth. Enzymol .. 194:795 (1991)]. Unless otherwise indicated, yeast cultures were grown in YES medium at 30 C. YES consists of glucose, yeast extract and amino-acid supplements. Hydroxyurea was used at a concentration of 12 mM. Purification of GST fusion proteins and hexahis tagged proteins expressed in S. pombe, immunoblotting and kinase assays were performed as described [Shiozaki et al, Nature, 378:739 (1995)]. The proteins bound to GSH-Sepharose were washed, incubated with [gamma- 32 P] ATP and analyzed by SDS-polyacrylamide gel electrophoresis (Figure IB) . GST: Weel 152 was phosphorylated in the 0 min sample, whereas GST: Weel 70 phosphorylation was negligible. Phosphorylation of both proteins increased during incubation in HU . Unfused GST was not phosphorylated. Thus GST:Weel 70 associates with and is phosphorylated by an HU-regulated kinase, whereas GST: eel 152 is phosphorylated by this kinase and another that is active prior to HU treatment. -19- Hydroxyurea prevents DNA replication by inhibiting ribonucleotide reductase and thereby reducing deoxyribonucleotide pools, but HU may also have other effects. However, mutational inactivation of cdc22, which encodes the large subunit of ribonucleotide reductase (Fernandez-Sarabia et al, Mol . Gen. Genet., 238:241, 1993), caused phosphorylation of GST:Weel 70 in cell lysates (Figure IC) , indicating that the HU effect is due to inhibition of DNA replication. GST: Weel 70 phosphorylation was assayed in lysates from temperature sensitive cdc25-22 cells that were arrested in G2, incubated in HU for 45 min, and then released from G2 arrest in medium containing HU . Hydroxyurea failed to induce phosphorylation of GST: Weel 70 in lysates prepared from cdc25-22 cells arrested in G2 (Figure ID) . Upon release from G2 , GST: Weel 70 phosphorylation and cell septation increased coincidentally . Septation marks the end of mitosis (M) and the beginning of DNA replication (S), thus GST: eel 70 phosphorylation increased as cells encountered the HU arrest. No increase in GST: Weel 70 phosphorylation was detected in a mock HU-treated culture (Figure ID) . These observations indicate that the kinase that phosphorylates GST: Weel 70 is activated when cells attempt DNA replication with insufficient deoxyribonucleotides . Phosphorylation of GST:Weel 70 was abolished in lysates made from radl and rad3 checkpoint mutants (Figure 2A) , suggesting that the kinase which phosphorylates GST: Weel 70 may be involved in the replication checkpoint. Experiments were undertaken to identify this kinase. Chkl was excluded by experiments using lysates made from chkl cells (Figure 2B) . The involvement of Cdsl, a protein kinase that is important for survival of HU treatment (Murakami et al, -20- Nature . , 374:817, 1995), was indicated by the observation that GST: eel 70 phosphorylation was abolished in a lysate prepared from HU-treated cdsl cells (Figure 2B) . An experiment was performed to determine whether Cdsl co-precipitated with GST:Weel 70 . Lysates from HU-treated cells in which genomic cdsl encoded a protein with a COOH-terminal HAHIS tag (consisting of two HA epitopes followed by hexahistidine) were incubated with GST:Weel or GST proteins, washed and analyzed by immunoblotting. Cdsl HAHIS precipitated with GST:Weel but not unfused GST (Figure 2C) . Rad3 did not associate with GST: Weel. Cdsl purified from fission yeast phosphorylated GST: Weel 70 in vitro, producing a two-dimensional tryptic phosphopeptide map that was identical to a map made from GST: Weel 70 phosphorylated by its associated kinase. These data indicate that Cdsl is the HU-regulated kinase which associates with and phosphorylates GST:Weel 70 . Association of Cdsl and Weel was also examined in vivo. GST: Cdsl was expressed from the thiamine-repressible nmtl promoter. To tag genomic cdsl+ with a sequence encoding two copies of the HA epitope and hexahistidine, a Pstl-Notl fragment having nucleotides 205-1465 of the cdsl+ open reading frame was made by PCR. This was introduced into pRIP42-SpclHA6H after digestion with Pstl and Notl enzymes [Shiozaki et al, Meth. Enz . , 283:506 (1997)]. The construct was linearized with Nhel and transformed into PR109. Integration and tagging was confirmed by Southern DNA hybridization and immunoblotting, with function of CdslHAHIS confirmed by lack of HU sensitivity. The nmtl : GST : cdsl+ construct was prepared as described for the nmtl : GST : chkl+ construct (Furnari et al, Science. 277:1495, 1997). -21- As a control, a strain that expressed GST:Chkl was used. The GST fusion proteins were purified with GSH-Sepharose and analyzed by immunoblotting. Cdc25 (but not Weel) associated with GST: Chkl, whereas Weel (but not Cdc25) associated with GST:Cdsl (Figure 2D) . HU may increase the abundance of Cdsl, or it may make Cdsl competent to associate with Weel, or perhaps the replication checkpoint enhances the kinase activity of Cdsl. Cells expressing Cdsl HAHIS were treated with HU or mock-treated, lysates were made and precipitated with GST:Weel proteins. Association of Cdsl HAHIS with GST:Weel was detected in lysates from cells treated with HU, whereas no Cdsl HAHIS associated with GST: Weel in mock-treated samples (Figure 3A) . Ni 2+ -NTA precipitation of Cdsl HAHIS via the hexahistidine sequence showed that Cdsl HAHIS was equally abundant in HU-treated and mock-treated cell lysates. Therefore, HU-treatment enhanced association of Cdsl with GST: Weel. The in vivo interaction between GST: Cdsl and Weel shown in Figure 2D did not require HU treatment, presumably because GST:Cdsl was over expressed. Cdsl HAHIS was also purified from HU or mock-treated cells and assayed in an autophosphorylation assay. Phosphorylation of Cdsl was increased in samples from cells treated HU (Figure 3B) . Similar results were obtained using GST: Weel 70 as a substrate. Thus HU apparently both activates Cdsl as a kinase and stimulates binding to Weel. Phosphorylation of Weel by Cdsl may play a role in the DNA replication checkpoint. However, weel mutants arrest division in response to HU (Enoch et al, Cell , 60:665, 1990), thus another protein that controls Cdc2 Tyr 15 phosphorylation must also be regulated by the replication checkpoint. The kinase Mikl phosphorylates Cdc2 on Tyr 15 (Lundgren et al, Cell, 64:1111, 1991), therefore the -22- abundance of Mikl was monitored in HU-treated cells using a strain expressing Mikl HAHIS from the genomic locus. Immunoblot analysis showed that the amount of Mikl HAHIS increased in cells treated with HU (Figure 3C) . The abundance of mikl mRNA was unchanged in HU-treated cells, thus HU must influence Mikl synthesis or turnover. The HU-induced increase of Mikl HAHIS was largely abolished in cdsl and rad3 cells (Figure 3C) , indicating that this response was regulated by the replication checkpoint. Mikl is a dose-dependent inhibitor of mitosis (Lundgren et al, Cell , 64:1111, 1991), thus the increase of Mikl abundance in HU-treated cells should help to enforce the checkpoint. Indeed, mikl mutants exhibited enhanced sensitivity to HU (Figure 3D) . Expression of a large amount of GST: Cdsl from the nmtl promoter caused a cell cycle arrest, as indicated by highly elongated cells (Figure 4) . The arrest occurred in rad3 and chkl mutants, indicating that Cdsl functions downstream of Rad3 and independently of Chkl. Expression of GST: Cdsl had no effect in cells that express Cdc2Y15F, a form of Cdc2 that cannot be phosphorylated by Weel or Mikl. These findings support the conclusion that Cdsl transmits a checkpoint signal. Cells lacking Cdsl arrest division in response to HU, indicating that the Cdsl signal is supplemented by other checkpoint proteins (Murakami et al, Nature, 374:817, 1995). The replication checkpoint was examined in synchronous cultures of cdsl chkl double mutant cells, cdsl , chkl , rad3 single mutants or wild-type cells. Cells were first grown in YES (yeast extract and glucose) medium to an OD600 of 1.0 and then treated with HU (12 mM) for 90 min at 30 C. Synchronous cells were then obtained by centrifugal elutriation with a Beckman JE-5.0 elutriation rotor. These cells were diluted to an OD600 of 0.3 in YES containing 12 mM HU and grown at 30 C for a further 4 hr. Cells were scored for progression through mitosis by microscopic observation. HU sensitivity studies were carried out with cells grown to an OD600 of 0.3 in YES medium and then diluted to 13,000 cells/ml in YES media containing HU (12 mM) , followed by growth at 30 C for a further 8 hr . Samples (75 μl) were taken at regular intervals, plated onto YES plates and grown for 4 days at 30 C to determine survival. HU arrested division in wild-type cells, cdsl and chkl single mutants (Figure 5A) . In contrast, cdsl chkl double mutant cells underwent division with kinetics that were similar to rad3 cells. Thus the checkpoint is abolished in cdsl chkl cells. Indeed, the cdsl chkl double mutant cells were acutely sensitive to killing by HU (Figure 5B) . These findings are consistent with a recent study (Lindsay et al, Genes Dev. , 12:382, 1998) and support a model in which Cdsl has a direct role in enforcing the replication checkpoint. These findings indicate that Cdsl and Chkl are dual effectors of the replication checkpoint. Chkl regulates Cdc25, whereas Cdsl appears to phosphorylate Weel and is required to increase the abundance of Mikl. The effect of phosphorylation of Weel by Cdsl is unknown and remains to be confirmed with in vivo studies, but it is striking that the replication checkpoint both increases the kinase activity of Cdsl and stimulates binding to Weel in cell extracts. The large HU-induced increase in the abundance of Mikl helps to explain why cdc25 weel double mutant cells arrest division in response to HU and reaffirms the importance of Cdc2 Tyr 15 phosphorylation in the replication checkpoint (Enoch et al, Genes Dev. , 6:2035, 1993). Cdsl shares significant sequence identity with the kinase Rad53p, a protein involved in the - 24 - DNA replication checkpoint in Saccharomyces cerevisiae (S.J. Elledge, Science, 274:1664, 1996). Effectors of the replication checkpoint evoked by hydroxyurea (HU) were previously unknown. Treatment of fission yeast with HU stimulated the kinase Cdsl which phosphorylates the kinase Weel, an inhibitor of Cdc2. The protein kinase Cdsl was also required for a large HU-induced increase in the amount of Mikl, a second inhibitor of Cdc2. HU-induced arrest of cell division was abolished in cdsl chkl cells. Thus Cdsl and Chkl jointly enforce the replication checkpoint. The foregoing specification, including the specific embodiments and examples, is intended to be illustrative of the present invention and is not to be taken as limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention.

Claims: -25- What Is Claimed Is: 1. An isolated nucleotide sequence that encodes a truncated Weel protein comprising amino acid residues 11-72 of the native Weel protein, and homologs thereof. 2. The nucleotide sequence of claim 1 wherein said protein comprises amino acid residues 10-72 of the native Weel protein. 3. The nucleotide sequence of claim 1 wherein said protein comprises amino acid residues 11-152 of the native Weel protein. 4. The nucleotide sequence of claim 1 wherein said protein is a fusion protein comprising said Weel protein amino acid residues operatively linked to glutathione-S- transferase (GST) . 5. The nucleotide sequence of claim 4 wherein said fusion protein is GST : eel ( 11-72 ) and has an amino acid residue sequence shown in SEQ ID NO 1. 6. The nucleotide sequence of claim 4 wherein said fusion protein is GST : eel ( 11-152 ) and has an amino acid residue sequence shown in SEQ ID NO 3. 7. The nucleotide sequence of claim 4 wherein said fusion protein is GST : Weel (11-72 ) and said nucleotide sequence has a sequence shown in SEQ ID NO 2. 8. The nucleotide sequence of claim 4 wherein said fusion protein is GST : Weel (11-152 ) and said nucleotide sequence has a sequence shown in SEQ ID NO 4. 9. An expression vector comprising a nucleotide sequence that encodes a truncated Weel protein comprising amino acid residues 11-72 of the native Weel protein, wherein said vector is capable of expressing said truncated Weel protein encoded by said nucleotide sequence. -26- 10. The expression vector of claim 9 wherein said protein comprises amino acid residues 10-72 of the native Weel protein. 11. The expression vector of claim 9 wherein said protein comprises amino acid residues 11-152 of the native Weel protein. 12. The expression vector of claim 9 wherein said protein is a fusion protein comprising said Weel protein amino acid residues operatively linked to glutathione-S- transferase (GST) . 13. The expression vector of claim 12 wherein said fusion protein is GST : Weel (11-72 ) and has an amino acid residue sequence shown in SEQ ID NO 1. 14. The expression vector of claim 12 wherein said fusion protein is GST : Weel ( 11-152 ) and has an amino acid residue sequence shown in SEQ ID NO 3. 15. The expression vector of claim 12 wherein said fusion protein is GST : Weel ( 11-72) and said vector comprises a nucleotide sequence shown in SEQ ID NO 2. 16. The expression vector of claim 12 wherein said fusion protein is GST : Weel ( 11-152 ) and said vector comprises a nucleotide sequence shown in SEQ ID NO 4. 17. A recombinant truncated Weel protein comprising amino acid residues 11-72 of the native Weel protein. 18. The recombinant truncated Weel protein of claim 17 wherein said protein comprises amino acid residues 10-72 of the native Weel protein. 19. The recombinant truncated Weel protein of claim 17 wherein said protein comprises amino acid residues 11-152 of the native Weel protein. 20. The recombinant truncated Weel protein of claim 17 wherein said protein is a fusion protein comprising said Weel protein amino acid residues operatively linked to glutathione-S-transferase (GST) . 21. The recombinant truncated Weel protein of claim 20 wherein said fusion protein is GST : Weel ( 11-72 ) and has an amino acid residue sequence shown in SEQ ID NO 1. 22. The recombinant truncated Weel protein of claim 20 wherein said fusion protein is GST : Weel ( 11-152 ) and has an amino acid residue sequence shown in SEQ ID NO 3. 23. A method for identifying a compound which modulates Cdsl kinase activity comprising the steps of: a) combining: i) a test compound; ii) a truncated Weel protein comprising amino acid residues 11-72 of the native Weel protein, or a homolog thereof; and iii) a composition comprising Cdsl protein; b) maintaining the combination of step (a) under conditions sufficient for Cdsl to phosphorylate said truncated Weel protein; and c) determining the amount of phosphorylated Weel protein whereby the amount of phosphorylated Weel indicates whether said test compound modulates the activity of Cdsl. 24. The method of claim 23 wherein said test compound is hydroxyurea. 25. The method of claim 23 wherein said test compound is a protein selected from the group consisting of Cdc22, Cdc25, Chkl, Mikl and genetic variants thereof. 26. The method of claim 23 wherein conditions comprise a phosphate-labelled Cdsl kinase substrate. 27. The method of claim 23 wherein said composition comprising Cdsl protein is a cell lysate . -28 - 28. The method of claim 23 wherein said composition comprising Cdsl protein comprises isolated Cdsl protein. 29. The method of claim 23 wherein said protein comprises amino acid residues 10-72 of the native Weel protein. 30. The method of claim 23 wherein said protein comprises amino acid residues 11-152 of the native Weel protein . 31. The method of claim 23 wherein said truncated Weel protein is a fusion protein. 32. The method of claim 31 wherein said fusion protein comprises said truncated Weel protein operatively linked to glutathione-S-transferase (GST) . 33. The method of claim 32 wherein said fusion protein is GST : eel ( 11-72 ) and has an amino acid residue sequence shown in SEQ ID NO 1. 34. The method of claim 32 wherein said fusion protein is GST :Weel (11-152) and has an amino acid residue sequence shown in SEQ ID NO 3. 35. A method for identifying a compound which modulates kinase Weel activity comprising the steps of: a) combining: i) a test compound; ii) a truncated Weel protein comprising amino acid residues 11-72 of the native Weel protein, or a homolog thereof; and iii) a reagent for Weel; b) maintaining the combination of step (a) under conditions suitable for Weel to react with said reagent; and c) determining the amount Weel reacts with said reagent whereby the amount of reaction indicates whether said test compound modulates the activity of Weel. -29- 36. The method of claim 35 wherein said determining comprises measuring phosphorylation of said Weel protein. 37. The method of claim 35 wherein said determining comprises measuring phosphorylation of said reagent. 38. The method of claim 35 wherein said test compound is hydroxyurea. 39. The method of claim 35 wherein said test compound is a protein selected from the group consisting of Cdc22, Cdc25, Cdsl, Chkl, Mikl and genetic variants thereof. 40. The method of claim 35 wherein said reagent is Cdc2 protein. 41. The method of claim 35 wherein said reagent is a labelled kinase substrate. 42. The method of claim 35 wherein said reagent is a Weel substrate. 43. The method of claim 35 wherein said protein comprises amino acid residues 10-72 of the native Weel protein. 44. The method of claim 35 wherein said protein comprises amino acid residues 11-152 of the native Weel protein . 45. The method of claim 35 wherein said truncated Weel protein is a fusion protein. 46. The method of claim 45 wherein said fusion protein comprises said truncated Weel protein operatively linked to glutathione-S-transferase (GST) . 47. The method of claim 46 wherein said fusion protein is GST :Weel (11-72) and has an amino acid residue sequence shown in SEQ ID NO 1. 48. The method of claim 46 wherein said fusion protein is GST :Weel (11-152) and has an amino acid residue sequence shown in SEQ ID NO 3.

Inventors: BODDY MICHAEL N, FUNARI BETH, RUSSELL PAUL, BODDY, MICHAEL, N., FUNARI, BETH, RUSSELL, PAUL
Applicants: BODDY MICHAEL N, FUNARI BETH, RUSSELL PAUL, SCRIPPS RESEARCH INST, BODDY, MICHAEL, N., FUNARI, BETH, RUSSELL, PAUL, THE SCRIPPS RESEARCH INSTITUTE
Application year: 1999
Main classification: C12N15
IPC codes: C07K14, C07K19, C12N9, C12N15, C12Q1