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• W2074088249 abstract "Ste20p from Saccharomyces cerevisiaeis a member of the Ste20/p21-activated protein kinase family of protein kinases. The Ste20p kinase is post-translationally modified by phosphorylation in a cell cycle-dependent manner, as judged by the appearance of phosphatase-sensitive species with reduced mobility on SDS-polyacrylamide gel electrophoresis. This modification is maximal during S phase, and correlates with the accumulation of Ste20p fused to green fluorescent protein at the site of bud emergence. Overexpression of Cln2p, but not Clb2p or Clb5p, causes a quantitative shift of Ste20p to the reduced mobility form, and this shift is dependent on Cdc28p activity. The post-translational mobility shift can be generated in vitro by incubation of Ste20p with immunoprecipitated Cln2p kinase complexes, but not by immunoprecipitated Clb2p or Clb5p kinase complexes. Ste20p is therefore a substrate for the Cdc28p kinase, and undergoes a Cln2p-Cdc28p mediated mobility shift as cells initiate budding and DNA replication. In cells that express only the Cln2p G1 cyclin, minor overexpression of Ste20p causes aberrant morphology, suggesting a proper coordination of Ste20p and Cln-Cdc28p activity may be required for the control of cell shape. Ste20p from Saccharomyces cerevisiaeis a member of the Ste20/p21-activated protein kinase family of protein kinases. The Ste20p kinase is post-translationally modified by phosphorylation in a cell cycle-dependent manner, as judged by the appearance of phosphatase-sensitive species with reduced mobility on SDS-polyacrylamide gel electrophoresis. This modification is maximal during S phase, and correlates with the accumulation of Ste20p fused to green fluorescent protein at the site of bud emergence. Overexpression of Cln2p, but not Clb2p or Clb5p, causes a quantitative shift of Ste20p to the reduced mobility form, and this shift is dependent on Cdc28p activity. The post-translational mobility shift can be generated in vitro by incubation of Ste20p with immunoprecipitated Cln2p kinase complexes, but not by immunoprecipitated Clb2p or Clb5p kinase complexes. Ste20p is therefore a substrate for the Cdc28p kinase, and undergoes a Cln2p-Cdc28p mediated mobility shift as cells initiate budding and DNA replication. In cells that express only the Cln2p G1 cyclin, minor overexpression of Ste20p causes aberrant morphology, suggesting a proper coordination of Ste20p and Cln-Cdc28p activity may be required for the control of cell shape. p21-activated protein kinase cyclin-dependent kinase polyacrylamide gel electrophoresis green fluorescent protein glutathione S-transferase hemagglutinin temperature-sensitive myelin basic protein. The Ste20p protein kinase, the founding member of the Ste20/PAK1 family of protein kinases, plays important roles in a number of cellular processes in the yeast Saccharomyces cerevisiae (1Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 59-66Crossref PubMed Scopus (189) Google Scholar). Ste20p was initially identified because of its role in yeast mating (2Leberer E. Dignard D. Harcus D. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4815-4824Crossref PubMed Scopus (347) Google Scholar) Subsequently, the Ste20p kinase was found to be required for pseudohyphal growth in diploid S. cerevisiae cells (3Liu H. Styles C. Fink G.R. Science. 1993; 262: 1741-1744Crossref PubMed Scopus (428) Google Scholar) and to be needed for invasive growth in nutrient limited haploid cells (4Roberts R.L. Fink G.R. Genes Dev. 1994; 8: 2974-2985Crossref PubMed Scopus (526) Google Scholar). In addition, the kinase has a yet poorly defined essential function it shares with the related kinase Cla4p; although cells deleted for eitherSTE20 or CLA4 are viable, the double mutants are inviable (5Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (310) Google Scholar). One of the potential areas of overlap between the Ste20p and Cla4p kinases is in the regulation of myosin-I function; both kinases can phosphorylate and activate the myosin-I isoform encoded byMYO3 (6Wu C. Lytvyn V. Thomas D.Y. Leberer E. J. Biol. Chem. 1997; 272: 30623-30626Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). MYO3 itself has an overlapping essential function with the related myosin-I MYO5 (7Goodson H.V. Anderson B.L. Warrick H.M. Pon L.A. Spudich J.A. J. Cell Biol. 1996; 133: 1277-1291Crossref PubMed Scopus (186) Google Scholar). Because of the multiple roles of the Ste20 kinase, it is important that regulatory mechanisms exist to target the kinase activity to specific substrates. Overexpression of an N-terminally deleted kinase lacking regulatory domains is lethal (8Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar). One potential targeting mechanism is cellular localization; the Ste20 protein visualized by green fluorescent protein (GFP) tagging is localized to the regions of polarized growth (9Leberer E. Wu C. Leeuw T. Fourest-Lieuvin A. Segall J.E. Thomas D.Y. EMBO J. 1997; 16: 83-97Crossref PubMed Scopus (166) Google Scholar, 10Peter M. Neiman A.M. Park H.O. van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (191) Google Scholar). The functional significance of this localization is underscored by the observation that mutations which perturb the localization, but not other aspects of Ste20 function, cause inviability in cla4-defective cells (9Leberer E. Wu C. Leeuw T. Fourest-Lieuvin A. Segall J.E. Thomas D.Y. EMBO J. 1997; 16: 83-97Crossref PubMed Scopus (166) Google Scholar, 10Peter M. Neiman A.M. Park H.O. van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (191) Google Scholar). This localization of Ste20p appears to depend on association with Cdc42p, as deletion of the Cdc42p binding domain of Ste20p is lethal in combination with Cla4p inactivation (9Leberer E. Wu C. Leeuw T. Fourest-Lieuvin A. Segall J.E. Thomas D.Y. EMBO J. 1997; 16: 83-97Crossref PubMed Scopus (166) Google Scholar, 10Peter M. Neiman A.M. Park H.O. van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (191) Google Scholar). Differential associations of Ste20p with other proteins may also influence its function. The Ste5 protein, which serves as a specificity determinant for components of the pheromone response pathway, associates with Ste20 protein (11Leeuw T. Fourest A. Wu C. Chenevert J. Clark K. Whiteway M. Thomas D.Y. Leberer E. Science. 1995; 270: 1210-1213Crossref PubMed Scopus (176) Google Scholar) and so does the Bem1 protein (11Leeuw T. Fourest A. Wu C. Chenevert J. Clark K. Whiteway M. Thomas D.Y. Leberer E. Science. 1995; 270: 1210-1213Crossref PubMed Scopus (176) Google Scholar). The Ste4 protein, which encodes the β-subunit of the heterotrimeric G-protein, interacts with Ste20p in a mating pheromonedependent manner (12Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-194Crossref PubMed Scopus (183) Google Scholar). Ultimately, processes that localize or activate Ste20p in a cell cycle-dependent manner must be controlled, directly or indirectly, by the cell cycle regulatory machinery. The central regulator of the yeast cell cycle engine consists of the cyclin-dependent kinase (CDK) encoded by theCDC28 gene, and a series of cyclin molecules: the three G1 cyclins encoded by CLN1, CLN2, and CLN3 and six mitotic cyclins encoded by CLB1, CLB2, CLB3, CLB4, CLB5, and CLB6. Through physical association, these cyclins are required for the activation of Cdc28p at different stages of the cell cycle (see Refs. 13Nasmyth K. Trends Genet. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (295) Google Scholar and 14Cross F.R. Curr. Opin. Cell Biol. 1995; 7: 790-797Crossref PubMed Scopus (105) Google Scholar for reviews). These cyclins themselves are somewhat specialized; current evidence suggests that CLN3 is important in transcriptional activation of SBF (Swi4p-Swi6p)- and MBF (Mbp1p-Swi6p)-dependent gene expression at Start (13Nasmyth K. Trends Genet. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 15Stuart D. Wittenberg C. Genes Dev. 1995; 9: 2780-2794Crossref PubMed Scopus (174) Google Scholar, 16Tyers M. Tokiwa G. Futcher B. EMBO J. 1993; 12: 1955-1968Crossref PubMed Scopus (393) Google Scholar, 17Dirick L. Bohm T. Nasmyth K. EMBO J. 1995; 14: 4803-4813Crossref PubMed Scopus (279) Google Scholar), while CLN1 and CLN2 are required for bud emergence (18Lew D.J. Marini N.J. Reed S.I. Cell. 1992; 69: 317-327Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 19Lew D.J. Reed S.I. Curr. Opin. Genet. Dev. 1995; 5: 17-23Crossref PubMed Scopus (197) Google Scholar, 20Schwob E. Nasmyth K. Genes Dev. 1993; 7: 1160-1175Crossref PubMed Scopus (406) Google Scholar). Within theCLB family of cyclins, CLB5 and CLB6play a role in DNA replication (20Schwob E. Nasmyth K. Genes Dev. 1993; 7: 1160-1175Crossref PubMed Scopus (406) Google Scholar, 21Epstein C.B. Cross F.R. Genes Dev. 1992; 6: 1695-1706Crossref PubMed Scopus (299) Google Scholar), CLB3 and CLB4 in the formation of mitotic spindles (22Fitch I. Dahmann C. Surana U. Amon A. Nasmyth K. Goetsch L. Byers B. Futcher B. Mol. Biol. Cell. 1992; 3: 805-818Crossref PubMed Scopus (235) Google Scholar, 23Richardson H. Lew D.J. Henze M. Sugimoto K. Reed S.I. Genes Dev. 1992; 6: 2021-2034Crossref PubMed Scopus (208) Google Scholar), and CLB1 and CLB2 in isometric bud growth and nuclear division (24Lew D.J. Reed S.I. J. Cell Biol. 1993; 120: 1305-1320Crossref PubMed Scopus (402) Google Scholar, 25Surana U. Robitsch H. Price C. Schuster T. Fitch I. Futcher A.B. Nasmyth K. Cell. 1991; 65: 145-161Abstract Full Text PDF PubMed Scopus (350) Google Scholar). The yeast pheromone response is tightly integrated with cell cycle regulation. Treatment of responsive cells with mating pheromones ultimately causes cell cycle arrest at Start (1Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 59-66Crossref PubMed Scopus (189) Google Scholar, 26Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (865) Google Scholar, 27Schultz J. Ferguson B. Sprague Jr., G.F. Curr. Opin. Genet. Dev. 1995; 5: 31-37Crossref PubMed Scopus (48) Google Scholar). This arrest requires Far1p, a G1 CDK inhibitor (28Chang F. Herskowitz I. Cell. 1990; 63: 999-1011Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 29Peter M. Gartner A. Horecka J. Ammerer G. Herskowitz I. Cell. 1993; 73: 747-760Abstract Full Text PDF PubMed Scopus (275) Google Scholar), whose expression and stability are normally under control of the cell cycle machinery. Far1p is abundant throughout mitosis and G1, and undetectable from S until the next M phase (30McKinney J.D. Chang F. Heintz N. Cross F.R. Genes Dev. 1993; 7: 833-843Crossref PubMed Scopus (123) Google Scholar). The activation of the pheromone pathway leads to increased expression and stability of Far1p (30McKinney J.D. Chang F. Heintz N. Cross F.R. Genes Dev. 1993; 7: 833-843Crossref PubMed Scopus (123) Google Scholar, 31Henchoz S. Chi Y. Catarin B. Herskowitz I. Deshaies R.J. Peter M. Genes Dev. 1997; 11: 3046-3060Crossref PubMed Scopus (180) Google Scholar). In addition, the basal activities (in the absence of mating pheromone) of other components such as Fus3 and Ste7 fluctuate in a cell cycle-regulated manner with a regulation profile similar to that of Far1p (32Wassmann K. Ammerer G. J. Biol. Chem. 1997; 272: 13180-13188Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The cell cycle machinery also plays a role in pheromone responsiveness. Only cells that are in the M/G1 phase of the cell cycle are responsive to pheromone, whereas overexpression of the G1 cyclin CLN2, or of CLN1 with the combination of a far1 deletion, creates a nonresponsive phenotype (14Cross F.R. Curr. Opin. Cell Biol. 1995; 7: 790-797Crossref PubMed Scopus (105) Google Scholar, 33Oehlen L.J. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Although the mechanism for this remains unclear, the repressing effect by G1 cyclins on the regulation of mating pheromone signal transduction has been placed at a level that is upstream of the mitogen-activated protein kinase, and downstream of the mating pheromone receptor and the α-subunit of the heterotrimeric G-protein (14Cross F.R. Curr. Opin. Cell Biol. 1995; 7: 790-797Crossref PubMed Scopus (105) Google Scholar, 32Wassmann K. Ammerer G. J. Biol. Chem. 1997; 272: 13180-13188Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 33Oehlen L.J. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Here we show that Ste20p is post-translationally modified in a cell cycle-regulated manner, and this modification requires Cdc28p and Cln2p. Furthermore, Ste20p is an in vitro substrate of Cdc28p-Cln2p kinase complex. Restriction endonucleases and DNA-modifying enzymes were obtained from Boehringer Mannheim, Life Technologies, Inc., Amersham Pharmacia Biotech, and New England Biolabs.Taq thermostable DNA polymerase was purchased from Cetus. [γ-32P]ATP was obtained from ICN. Acid-washed glass beads (450–600 mm), synthetic α-factor, myelin basic protein, protease inhibitors, and bovine serum albumin were purchased from Sigma. α-Factor was dissolved in 90% methanol at a concentration of 1.0 mg/ml and stored at −20 °C. Histone H1 was obtained from Boehringer Mannheim. Plasmid pGEX-4T-3, glutathione-Sepharose beads, glutathione, and protein A/G Sepharose beads were obtained from Amersham Pharmacia Biotech. Alkaline phosphatase-conjugated and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad. Protein phosphatase 2A, okadaic acid, and anti-p34cdc2 antibodies were from Upstate Biotechnology Inc. Nitrocellulose membranes were from Xymotech. Specific Ste20p antibodies were produced and used as described previously (34Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Monoclonal anti-hemagglutinin (HA) and polyclonal anti-HA antibodies were from Babco (Richmond, VA). Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad. The enhanced chemiluminescence (ECL) assay system was purchased from Amersham Pharmacia Biotech. Standard protocols were used for all recombinant DNA techniques (35Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982Google Scholar). Yeast media, culture conditions, and manipulations of yeast strains were as described (36Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Yeast transformations with circular or linearized plasmid DNA were carried out after treatment of yeast cells with lithium acetate (36Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Plasmids pVTU-STE20, pVTU-STE20K649R, and pVTU-STE20K649A, which contain a wild-type Ste20p or catalytically inactive versions of Ste20p created by changing lysine at position 649 to either arginine or alanine, respectively, were constructed as described (34Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Plasmid pCWGEX-1 expressing a GST fusion of the catalytically inactive version of Ste20p, Ste20pK649R, was constructed as follows: The mutant version of ste20 was first amplified by the polymerase chain reaction (37Saiki R.J. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13495) Google Scholar) using 5′-TCAGATCTATGAGCAATGATCCATCT-3′ (the addedBglII site is underlined) and 5′-CCGCTCGAGTTTACTTTTGTTTATCATC-3′ (the addedXhoI is underlined) as primers and pVTU-STE20K649R as template. The polymerase chain reaction product was digested withBglII-XhoI, and then cloned intoBamHI-XhoI sites of pGEX-4T-3. Plasmids with HA epitope-tagged version of CLN2, CLB5, and CLB2 under the control of GAL1 promoter were kindly provided by Dr. M. Tyers of University Toronto. The integrating plasmid withGAL1::CLN2::LEU2 was kindly provided by Drs. L. J. Oehlen and F. R. Cross of Rockefeller University. Plasmid pRL116, carrying the GFP::STE20 under the control of the STE20 promoter, was described previously (9Leberer E. Wu C. Leeuw T. Fourest-Lieuvin A. Segall J.E. Thomas D.Y. EMBO J. 1997; 16: 83-97Crossref PubMed Scopus (166) Google Scholar). To synchronize cells with temperature-sensitive (ts) alleles, cells grown to early log phase at room temperature were filtered out using a sterile filtration apparatus (Nalgene) and resuspended into pre-warmed (37 °C) media and then incubated at restrictive temperature (37 °C) for 2.5 h. The synchronized cells were examined by microscopy and released from restrictive to permissive temperature by being filtered out and resuspended into medium at the permissive temperature. The cells were then incubated at permissive temperature; samples were taken at indicated time intervals and immediately mixed with approximately equal volume of crushed ice to quickly cool the sample to approximately 0–4 °C. Cells were then collected by centrifugation and washed once with ice-cold sterile water, and the cell pellets were then quickly frozen and stored at –80 °C until preparation of total cell extracts. The GST fusion proteins were expressed in Escherichia coli strain UT5600 (New England Biolabs), bound to glutathione-Sepharose beads, and eluted with glutathione as described previously (34Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The eluted proteins were then concentrated by centrifigation using the Centricon (Amicon Inc.) and stored at −80 °C. Total cell extract was prepared essentially as described previously (34Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Briefly, cells were harvested by centrifugation at an OD600 of 0.5–1.0 unless otherwise indicated, and resuspended at a concentration approximately 5 × 108cells/ml in lysis buffer containing 50 mmTris-HCl (pH 7.5), 100 mm NaCl, 50 mm NaF, 5 mm EDTA, 1 mm dithiothreitol, 1% Triton X-100 (unless otherwise indicated), 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin, and 5 μg/ml antipain. Cells were then disrupted using a bead beater (Bio-spec products) for 3 min at 4 °C. The extracts were clarified by centrifugation in a microcentrifuge at 10,000 × g for 10 min at 4 °C, and total protein concentration was determined by the Bradford reaction using the Bio-Rad protein determination kit following the manufacturer's instructions (Bio-Rad). The supernatant fractions were supplemented with 10% glycerol and stored at –80 °C. For Ste20p immunoprecipitation, aliquots of cell extracts containing approximately 1 mg of total cellular protein were incubated with 3 μl of anti-Ste20p antibodies for 1 h at 4 °C in a final volume of 0.5–1 ml of lysis buffer supplemented with 0.1% bovine serum albumin. The antibody-antigen complexes were then incubated with 30 μl of protein A-Sepharose beads in lysis buffer (50% v/v) for 1 h at 4 °C. After washing four times in lysis buffer and then twice in kinase buffer (50 mm Tris-HCl buffer, pH 7.5, containing 20 mmmagnesium chloride, 1 mm dithiothreitol, 0.5 mmsodium orthovanadate, 5 μg/ml aprotinin, and 5 μg/ml leupeptin), or washing twice with protein phosphatase 2A buffer (50 mmTris-HCl, pH 7.0, 1 mm EDTA, 50 mmβ-mercaptoethanol, 1 mm manganese chloride, 5 μg/ml aprotinin, and 5 μg/ml leupeptin), the kinase reactions were then started by addition of 30 μl of prewarmed (30 °C) kinase buffer supplemented with 5 μm ATP and 1 μl of [γ-32P]ATP (4,500 Ci/mmol, 10 mCi/ml). The reaction mixture was incubated for 20 min at 30 °C with indicated protein as substrate, and then boiled for 5 min after the addition of 30 μl of 2× Laemmli buffer (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). Aliquots (10–30 μl) of the solubilized immune complexes were then subjected to SDS-PAGE (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). Gels were either dried and autoradiographed directly, or the samples were transferred to nitrocellulose, then autoradiographed and subjected to immunoblot analysis when needed. For the phosphatase experiment, the Ste20p immune complexes were incubated for 30 min at 37 °C in 100 μl of phosphatase buffer containing 0.5 units of protein phosphatase 2A. Control incubations were performed in the presence of phosphatase inhibitor okadaic acid at 30 nm to inhibit protein phosphatase 2A, or in the absence of phosphatases. For the immunoprecipitation of HA-tagged protein complexes, cell extracts, prepared as described above except for the addition of 0.1% Triton X-100, were incubated for 1 h at 4 °C with 2 μl of anti-HA antibody pre-absorbed onto 20 μl of protein G-Sepharose beads in lysis buffer with 1% bovine serum albumin. The immune complexes were washed three times with immunoprecipitation buffer and twice with kinase buffer. The kinase reactions were started by adding 50 μl of kinase buffer containing 5 μm ATP and 1 μl of [γ-32P]ATP (4, 500 Ci/mmol, 10 mCi/ml), 2 μg of GST-Ste20pK649R, or 2 μg of histone H1 as substrates, incubated for 20 min at 30 °C, and subjected to SDS-PAGE, autoradiography, and immunoblot analyses. Relative phosphorylation levels were determined either by densitometric analyses of the autoradiogram or by scintillation counting the radioactivity, and normalized to the amount of the relevant proteins which were determined by densitometric analyses of immunoblot blots using the ECL system. Cells were grown as indicated, sonicated, fixed with formaldehyde at a final concentration of 3.7% with 150 mm NaCl, and viewed with a microscope equipped with Nomarski optics, and photographs were taken with a 100× objective. GFP fluorescence was visualized with a Leitz photomicroscope equipped with UV light source, and microscopic photographs were acquired with a 100× objective using a Micro Max camera (Princeton Instruments Inc.) and Northern Eclipse imaging software (Empix Imaging Inc.), and processed using Adobe Photoshop for MacIntosh. In response to pheromone treatment, yeast cells arrest as unbudded, G1 phase cells (39Bucking-Throm E. Duntze W. Hartwell L.H. Manney T.R. Exp. Cell Res. 1973; 76: 99-110Crossref PubMed Scopus (199) Google Scholar, 40Hartwell L.H. Exp. Cell Res. 1973; 76: 111-117Crossref PubMed Scopus (90) Google Scholar). We observed that immunoblots of Ste20p isolated from log phase and pheromone-treated cultures revealed differences in the migration pattern of the protein. Ste20p from cells of pheromone-arrested cultures migrated as a relatively compact, fast-migrating band, while Ste20p from log phase cells appeared as a slower-migrating smear (Fig. 1 A). Removal of pheromone resulted in the appearance of slow-migrating Ste20p within 30 min; this change in the modification of Ste20p correlated with the timing of bud emergence. The diminution of the slow-migrating form of Ste20p caused by pheromone treatment could result either from the cells containing an activated pheromone response pathway, or from the cells being blocked in cell cycle progression. Blocking the signal transduction pathway by mutations in either STE4 or STE5 (see Table I for list of strains) prevented the disappearance of the slow-migrating form of Ste20p (Fig. 1,B and D). Mutation in STE11 also prevented the disappearance of the slow-migrating species of Ste20p (data not shown). Most significantly, mutations in FAR1 also eliminated the reduction in the slow-migrating form of Ste20p (Fig. 1 C). Therefore, cell cycle arrest, and not simply activation of the pheromone pathway, is critical for the reduction of the slow-migrating form of Ste20p. Nutritional limitation also causes the arrest of yeast cells in G1 (41Wittenberg C. Reed S.I. Curr. Opin. Cell Biol. 1996; 8: 223-230Crossref PubMed Scopus (41) Google Scholar, 42Pringle J.R. Hartwell L.H. Broach J. Strathern J. Jones E. Molecular Biology of the Saccharomyces cerevisiae: Life Cycle and Inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1981: 97-142Google Scholar). Analysis of Ste20p from saturated cultures also showed the diminution of the slow-migrating version of the protein (Fig. 2). Thus cycling cells, even in the presence of an activated pheromone response pathway, contain the modified form of Ste20p, while arrested cells contain the fast-migrating form of the protein.Table IStrainsW303–1AMAT a ade2 ura3 his3 leu2 trp1 can1R. RothsteinW303–1BMATα ade2 ura3 his3 leu2 trp1 can1R. RothsteinA364AMAT a ade1 ade2 ura3 his7 lys2 tyr1 gal1F. CrossY157A364Acdc7–1F. CrossY158A364Acdc13–1F. CrossY159A364Acdc20–1F. CrossYEL206W303–1Aste20Δ::TRP1(34Wu C. Whiteway M. Thomas D.Y. Leberer E. J. Biol. Chem. 1995; 270: 15984-15992Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar)YEL196W303–1Afar1Δ::TRP1This studyYEL187W303–1Aste5Δ::TRP1This studyYEL121W303–1Aste4::LEU2(59Leberer E. Dignard D. Harcus D. Hougan L. Whiteway M. Thomas D.Y. Mol. Gen. Genet. 1993; 241: 241-254Crossref PubMed Scopus (49) Google Scholar)YCW232W303–1AGAL1::CLN2::LEU2This studyYCW232BW303–1BGAL1::CLN2::LEU2This studyK1993MAT a ura3 leu2 trp1 cdc15ts-2K. NasmythBY1365MAT a ade2 ade3 ura3 leu2 trp1 cdc28ts-13L. BreedenK4727MAT a ade2 ura3 leu2 his3 trp1 can1 cdc34tsK. NasmythYCW287K4727GAL1::CLN2::LEU2This studyYCW303W303–1A ste20Δ::TRP1 sst1::hisG GAL1::CLN2::LEU2This studyYCW304MAT a ura3 leu2 trp1cdc15ts-2 GAL1::CLN2::LEU2(segregant from cross K1993 × YCW232B)This studyYCW306MATa ura3 leu2 trp1 ste20Δ::TRP1 cdc15ts-2This studyYCW307MAT a ura3 leu2 trp1 cdc28ts-13 GAL1::CLN2::LEU2(segregant from cross BY1365 × YCW232B)This study1227–2CSMAT a ade1 his2 leu2 trp1 ura3 cln1Δ cln3Δ sst1::LEU2This study Open table in a new tab We asked whether blocking cells at points in the cell cycle other than Start would cause the switch to the fast-migrating form of Ste20p. The mobility status of Ste20p from yeast strains with temperature-sensitiveCDC alleles was checked at both permissive and restrictive temperatures. As shown in Fig. 3, cells blocked at the cdc13–1 and to some extent thecdc20–1 arrest points contained Ste20p predominantly in the fast-moving state. Cdc13p is a telomere DNA-binding protein; lack of Cdc13p blocks at G2/M, after DNA replication, but before mitosis (43Nugent C.I. Hughes T.R. Lue N.F. Lundblad V. Science. 1996; 274: 249-252Crossref PubMed Scopus (513) Google Scholar, 44Hartwell L.H. Smith D. Genetics. 1985; 110: 381-395Crossref PubMed Google Scholar). Cdc20p is a WD-40 repeat protein and a component of the anaphase-promoting complex that targets Pds1 for degradation, so lack of Cdc20p prevents sister chromatid separation (45Lim H.H. Surana U. Mol. Gen. Genet. 1996; 253: 138-148Crossref PubMed Scopus (26) Google Scholar, 46Lim H.H. Goh P.Y. Surana U. Curr Biol. 1998; 8: 231-234Abstract Full Text Full Text PDF PubMed Google Scholar, 47Sethi N. Monteagudo M.C. Koshland D. Hogan E. Burke D.J. Mol. Cell. Biol. 1991; 11: 5592-5602Crossref PubMed Google Scholar). However, mutants blocked at the cdc7–1 arrest point contained the slow migrating form of Ste20p. Cdc7p is a protein kinase, and lack of Cdc7p blocks at G1/S boundary, after spindle pole body duplication, but before initiation of chromosomal DNA replication (48Patterson M. Sclafani R.A. Fangman W.L. Rosamond J. Mol. Cell. Biol. 1986; 6: 1590-1598Crossref PubMed Scopus (70) Google Scholar, 49Yoon H.J. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3574-3578Crossref PubMed Scopus (42) Google Scholar, 50Hereford L.M. Hartwell L.H. J. Mol. Biol. 1974; 84: 445-461Crossref PubMed Scopus (168) Google Scholar). Therefore, the fast-migrating, presumably less modified species of Ste20p was not due simply to nonspecific blockage of cell cycle progression. To monitor Ste20p modification throughout the cell cycle, a yeast strain with cdc28–13, which arrests cells at Start at the restrictive temperature, was used in a block and release experiment. As shown in Fig. 4, Ste20p from cells that had been blocked for 2.5 h at the restrictive temperature (37 °C) migrated as a compact, fast-migrating band. Ste20p remained as a fast-migrating species up to 30 min after release, during which time the cells were unbudded. However, Ste20p from cells released for 60 min appeared as a smeary band with the majority of the protein migrating slowly. This shift in the migration state of Ste20p correlated with the onset of budding, consistent with the trapping of Ste20p in the modified state in the cdc7–1 arrested cells, which are arrested prior to the onset of S-phase at restrictive temperature. This slow-migrating state was maintained until 90 min and then started to disappear at 120 min, at the point of transition from G2/M. The slow-migrating species of Ste20p, however, reappeared at 180 min after the cells were released from the arrest point. Cdc15p is a protein kinase in the Cdc20p-dependent pathway and required for the exit from mitosis; lack of Cdc15p blocks cells at late mitotic phase (51Irniger S. Piatti S. Michaelis C. Nasmyth K. Cell. 1995; 81: 269-278Abstract Full Text PDF PubMed Scopus" @default.
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• W2074088249 title "Cell Cycle- and Cln2p-Cdc28p-dependent Phosphorylation of the Yeast Ste20p Protein Kinase" @default.
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• W2074088249 doi "https://doi.org/10.1074/jbc.273.43.28107" @default.
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