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Cdc37 Is Required for Association of the Protein Kinase Cdc28 with G1 and Mitotic Cyclins (Cell Cycle/Cyclin-Dependent Kinase) MONICA R

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Cdc37 Is Required for Association of the Protein Kinase Cdc28 with G1 and Mitotic Cyclins (Cell Cycle/Cyclin-Dependent Kinase) MONICA R Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4651-4655, May 1995 Cell Biology Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins (cell cycle/cyclin-dependent kinase) MONICA R. GERBER*t, ALISON FARRELL*t, RAYMOND J. DESHAIEStI, IRA HERSKOWITZt, AND DAVID 0. MORGAN*t Departments of *Physiology and tBiochemistry and Biophysics, University of California, San Francisco, CA 94143-0444 Contributed by Ira Herskowitz, January 30, 1995 ABSTRACT Studies of the temperature-sensitive cdc37-1 demonstrate that mutation of CDC37 results in reduced acti- mutant of Saccharomyces cerevisiae suggest that Cdc37 is vation of Cdc28, apparently due to a defect in Cdc28-cyclin required for passage through the G1 phase ofthe cell cycle, but binding. These studies demonstrate that in wild-type cells its precise function is not known. We have investigated the role Cdc37 positively regulates Cdc28-cyclin binding. of Cdc37 in the regulation of the cyclin-dependent protein kinase Cdc28. We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated MATERIALS AND METHODS with the G1 cyclin Cln2. This defect appears to be caused by Yeast Strains and Manipulations. All experiments were a decrease in the binding of Cdc28 and Cln2. cdc37-1 mutants performed with strains of the A364a background. Mutant also exhibit a defect in the binding and activation of Cdc28 by strains were extensively backcrossed into this background. the mitotic cyclin Clb2. Thus Cdc37 may be a regulator that Characterization of Cdc37 was performed in wild-type is required for the association of Cdc28 with multiple cyclins. (RD204-4C) and cdc37-1 (RD249-2D) strains. Experiments in Figs. 2-4 were performed with wild-type (RD219-2C), cdc37-1 In the budding yeast Saccharomyces cerevisiae, commitment to (RD249-4B), and cdc28-4 (RD705) strains in which the en- a new cell division cycle in G1 is dependent on nutritional dogenous CLN2 gene was replaced with a version in which conditions and other extracellular signals. Cells arrest in G1 Cln2 is fused to a C-terminal triple epitope tag from influenza when starved of nutrients or when treated with mating pher- hemagglutinin (11, 12). The experiment in Fig. 4A was per- omone. During nutrient arrest, cells stop growing and are formed with strains carrying an expression vector (pDK11, a unable to mate, whereas in pheromone arrest the cell division gift of D. Kellogg, University of California, San Francisco) cycle is blocked but cell growth continues and mating can occur encoding the entire Clb2 sequence fused to glutathione S- (1, 2). transferase (GST) and driven by the GALl promoter. Our understanding of cell cycle control has been greatly For a-factor arrest, 120-ml cultures were grown at 24°C to enhanced by the analysis of temperature-sensitive mutants that an OD600 of 0.5. a-Factor (1.0 tLg/ml) was added for 2 hr. For exhibit a G1 arrest like that seen during pheromone treatment mitotic arrest, 25-ml cultures were grown at 24°C to an OD600 (3). Screens for such mutants have led to the identification of of 0.3. Cells were transferred to medium containing benomyl several genes, including CDC28, CDC36, CDC37, and CDC39 (60 ,ug/ml) and nocodazole (20 ,g/ml) for 2 hr at 24°C. (3, 4). Mutations in CDC36 and CDC39 result in constitutive A 5.8-kb genomic DNA fragment containing the CDC37 activation of the mating pheromone pathway (5), whereas gene was a gift of S. Reed (Scripps Institute, La Jolla, CA). To CDC28 is more directly involved in cell cycle control (1, 2, 6). construct the cdc37A allele, a fragment of CDC37 (coding for The function of CDC37 is unknown. aa 48-506) was replaced with LEU2 in plasmid pMG1. Wild- The product of the CDC28 gene is a member of the highly type diploid cells were transformed with linearized pMG1, and conserved family of cyclin-dependent kinases (CDKs), whose gene replacement was checked by Southern blot analysis. activation at specific cell cycle stages requires association with Antibodies. Rabbit antiserum was raised against a GST- cyclin regulatory subunits (2, 7-9). The commitment to a new Cdc37 fusion protein (containing aa 48-506 of Cdc37) and cell division cycle in G1 is controlled by complexes of Cdc28 affinity purified on antigen columns (13). Polyclonal antibod- and the G1 cyclins Clnl, -2, and -3. G1 arrest by mating ies were raised against a C-terminal Cdc28 peptide (R.J.D., pheromone involves inhibition of specific Cdc28-Cln com- unpublished work). Monoclonal antibody (12CA5) against the plexes and decreased synthesis of Clnl and Cln2. Cdc28 influenza hemagglutinin epitope was obtained from BabCO. function is also required later in the cell cycle: progress Affinity-purified anti-Clb2 antiserum was a gift from D. through S phase and mitosis requires activation of Cdc28 by Kellogg. S-phase cyclins (Clb5 and -6) and mitotic cyclins (Clbl, -2, -3, Lysate Preparation. In Figs. 1-3, logarithmic-phase cells and -4), respectively (see ref. 2 for review). were resuspended in 700 ptl of ice-cold 20 mM Tris-HCl, pH Unlike CDC28, CDC37 is poorly understood. Cell cycle 7.4/0.1% Triton X-100/100 mM NaCl/5 mM EDTA/50 mM defects in cdc37 mutants have not been extensively character- P-glycerophosphate/50 mM NaF/1 mM phenylmethylsulfonyl ized, and little is known about the Cdc37 protein. Its predicted fluoride/1 mM dithiothreitol with aprotinin (2 ,tg/ml) and amino acid sequence does not display any significant homol- leupeptin (1 tLg/ml). One milliliter of glass beads was added, ogies that might suggest a function (4, 10). To explore the role and cells were lysed at 4°C by two pulses (60 s) in a mini- of Cdc37, we have begun a biochemical analysis of the Cdc37 BeadBeater (Biospec Products, Bartlesville, OK). Lysates were protein in wild-type cells and in the temperature-sensitive clarified by centrifugation at 14,000 x g for 10 min at 4°C. In cdc37-1 mutant. Because Cdc28 plays a key role at or near the Fig. 4, lysates were prepared by lysis with glass beads in 50 mM G1 arrest point of cdc37-1 cells, we have also analyzed the Hepes, pH 7.6/1 M NaCl/1 mM EGTA/0.2% Tween 20/1 effect of the cdc37-1 mutation on Cdc28 activity. Our findings Abbreviations: CDK, cyclin-dependent kinase; GST, glutathione S- The publication costs of this article were defrayed in part by page charge transferase. payment. This article must therefore be hereby marked "advertisement" in tPresent address: Division of Biology 156-29, California Institute of accordance with 18 U.S.C. §1734 solely to indicate this fact. Technology, Pasadena, CA 91125. 4651 Downloaded by guest on September 30, 2021 4652 Cell Biology: Gerber et al, Proc. Natl. Acad Sci. USA 92 (1995) mM phenylmethylsulfonyl fluoride with aprotinin (2 ,tg/ml) A 24°C 37°C B WT cdc37-1 and +I leupeptin (1 Lag/ml). _ +I-_ + Immunoblotting. Immunoblots ofcell lysates were probed as Ki" K described (14) with anti-Cdc37 antibodies (1:1000), anti-Cdc28 antibodies (1:1000), monoclonal antibody 12CA5 (14 uag/ml), kDa kDa or anti-Clb2 antibodies (1:2000). In some experiments, blots were incubated with 125I-labeled secondary antibodies and 143- 97- quantified with a PhosphorImager (Molecular Dynamics). 97- Metabolic Labeling. Cells growing at 30°C (wild type) or 66- 24°C (cdc37-1) were transferred to minimal medium lacking methionine (SD-Met) at an OD600 of 0.15, grown to an OD600 50- I "..' of 0.6, harvested by centrifugation, and resuspended in 3 ml of 45- 4'5 -' SD-Met containing 50taCi of [35S]methionine per OD unit of .0 cells (1 ,tCi = 37 kBq). Cells were cultured for 1 hr at 30°C 35- (wild type) or 24°C (cdc37-1). Lysates (50 t,g of protein) were 31- incubated for 2 hr at 4°C with 1 ,jg of anti-Cdc37 antibodies 21- and protein A-Sepharose. Immune complexes were washed three times with buffer and and lysis analyzed by SDS/PAGE 14- autoradiography. 1 2 3 4 Histone H1 Kinase Assays. For analysis of Cln2-associated 1 2 3 4 kinase activity, cell lysates (200 jig) were incubated with 1 ,tg of monoclonal antibody 12CA5 and protein A-Sepharose for FIG. 1. Characterization of Cdc37 protein in wild-type and cdc37-1 3 hr at mutant cells. (A) Anti-Cdc37 immunoblots of total protein isolated 4°C. Immune complexes were washed three times and from wild-type (WT, lanes 1 and 3) and cdc37-1 (lanes 2 and 4) cells incubated for 10 min at 24°C in a 30-,ul kinase reaction mixture grown at 24°C (lanes 1 and 2) or 37°C (lanes 3 and 4). (B) Immuno- containing 10 mM Hepes (pH 7.6), 1 mM dithiothreitol, 10 precipitation of extracts from wild-type (lanes 1 and 2) and cdc37-1 mM MgCl2, 5 ,tg of histone HI, 50 ,tM ATP, and 1.0 tCi of (lanes 3 and 4) cells metabolically labeled with [35S]methionine, with [y-32P]ATP. Reaction products were analyzed by SDS/PAGE anti-Cdc37 (+, lanes 2 and 4) or no antibody (-, lanes 1 and 3). and autoradiography. To measure GST-Clb2-associated ki- nase activity, 50 Ag of lysate was incubated for 2 hr at 4°C with protein -5 kDa smaller than the wild-type protein. We 30 ,tl of glutathione-agarose beads. The beads were washed sequenced the CDC37 gene 5' of the published sequence and four times and incubated for 30 min at 24°C in a 20-tul kinase found an upstream start codon that added 57 aa to the reaction mixture containing 50 mM Hepes (pH 7.6), 5 ,Ag of predicted sequence.
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