Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4651-4655, May 1995 Cell Biology Cdc37 is required for association of the kinase Cdc28 with G1 and mitotic cyclins (/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 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 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 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 , 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. This start codon is preceded by a stop histone H., 2 mM MgC12, 1 mM EGTA, 0.3 mM ATP, 2.5 ,ACi codon 138 bp further upstream. The new start codon is found of [y-32P]ATP, and 5 mM reduced glutathione. Clb2- in the context 5'-AAG-TCA-AAA-ATG-GCC-ATT-GAT- associated kinase activity was measured by the same method TAC-TCT-AAG-TGG-GAT-AAA-ATT-3', where the begin- in anti-Clb2 immunoprecipitates. ning of the published sequence is underlined. The revised open Binding of Cdc28 and Cyclins. To assess Cdc28-Cln2 bind- reading frame encodes a protein of 506 aa, with a predicted ing, lysates were subjected to immunoaffinity chromatography molecular mass of 58.4 kDa. Galactose-induced overexpres- on a column of monoclonal antibody 12CA5 (15). To assess sion of the revised open reading frame rescued the growth Cdc28-Clb2 binding, lysates (500 ,tg) were loaded on columns defect of the cdc37-1 mutant at 37°C and resulted in the containing 50 Atg of anti-Clb2 antibodies covalently coupled to overproduction of a Cdc37 protein that migrated in polyacryl- 100 ,tl of protein A-Sepharose. Columns were washed with 2.5 amide gels at the same anomalous position (-68 kDa) as the ml of 20 mM Hepes, pH 7.6/250 mM NaCl/5 mM EDTA/ wild-type protein (data not shown). Thus, the amino-terminal 0.1% Triton X-100 and 1.5 ml of 10 mM sodium phosphate, pH 57 residues are required for Cdc37 function. 7.4/75 mM NaCl. Bound were eluted with 100 mM We also analyzed the Cdc37 coding sequence in the cdc37-1 triethylamine at pH 11. mutant. The mutant cdc37gene from cdc37-1 cells was isolated by standard gap-repair methods (16), and sequencing of the RESULTS coding region revealed that codon 360 (CAA in wild-type CDC37) was changed to a stop codon (TAA; data not shown). CDC37 Encodes an Essential 58-kDa Protein. To begin our Premature termination at this codon would be consistent with analysis of Cdc37 function, we characterized the Cdc37 protein the smaller size of the mutant protein (Fig. 1). Interestingly, by immunoblot analysis of yeast lysates with an anti-Cdc37 overexpression of the mutant gene on a low-copy plasmid antibody. Lysates of wild-type cells contained an immunore- (resulting in expression of the mutant protein at levels 2- to active protein with an apparent mobility of -68 kDa (Fig. 1A). 3-fold higher than normal mutant levels) allowed slow but Lysates from the cdc37-1 mutant contained a less abundant significant growth of cdc37-1 cells at 37°C (data not shown). protein of -44 kDa. The level of Cdc37 protein was similar in Thus, the carboxyl-terminal region is not strictly required for exponentially growing and stationary-phase cells (data not Cdc37 function, but truncation of this region appears to cause shown) and did not change significantly during the cell cycle a decrease in the steady-state concentration of Cdc37 protein (see below, Fig. 3A). We also examined the Cdc37 protein by (Fig. 1A). Low Cdc37 levels may be the primary cause of the immunoprecipitation from cells metabolically labeled with growth defect in cdc37-1 cells. [35S]methionine (Fig. 1B). No labeled proteins consistently To confirm that CDC37 is an essential gene, we constructed associated with wild-type or mutant Cdc37 under these con- a diploid strain in which one chromosomal copy of the CDC37 ditions. gene was replaced with the LEU2 gene. Analysis of spores During the course of this work, we have found that the produced by this diploid indicated that CDC37 is essential at previously reported CDC37 sequence (4) is incomplete. We 30°C (data not shown). Spores bearing the CDC37 deletion constructed a low-copy expression vector in which the pub- formed microcolonies containing between 4 and =60 cells. lished CDC37 open reading frame was placed under the The terminal phenotype of these cells was heterogeneous: control of the galactose-inducible GALl promoter. This plas- many cells were enlarged and elongated with multiple projec- mid failed to complement the cdc37-1 mutant, and immuno- tions, suggesting that Cdc37 may be required at multiple cell blotting of cell lysates revealed a galactose-induced Cdc37 cycle stages. Downloaded by guest on September 30, 2021 Cell Biology: Gerber et at. Proc. NatL Acad Sci. USA 92 (1995) 4653

A WT cdc28-4 cdc37-1 vations). In cdc37-1 extracts, however, most of the Cln2 protein I is present in a hypophosphorylated form (Fig. 2B). 24"C2437370C24124"C 3737"C 24c24"c 3737"c The amount of Cdc28 protein in lysates from cdc37-1 mutants was severalfold lower than in wild-type lysates (Fig. Histone H1 VI AMi * 2B). In addition, the electrophoretic mobility of Cdc28 was altered in mutant cells. Cdc28 migrated at two positions in 1 2 3 4 5 6 polyacrylamide gels; in wild-type extracts the faster migrating form was predominant, whereas the slower form was more B WT abundant in extracts of cdc37-1 cells grown at 37°C (Fig. 2B). I I Icdc37-11 Further studies will be required to determine whether these 240C 370C 240C 370C mobility shifts result from differences in the phosphorylation of Cdc28. Several lines of evidence suggest that the reduction in Cdc28 CLN2 .. levels in mutant cells was not solely responsible for decreased Cln2-associated kinase activity. First, the loss of kinase activity CDC28 was temperature-dependent, whereas the reduction in Cdc28 levels was not. Second, additional experiments indicated that Cdc28 was present in excess over Cln2 in both wild-type and 1 2 3 4 cdc37-1 lysates: when virtually all Cln2 was immunodepleted from lysates of wild-type or mutant cells (as in Fig. 3B), there FIG. 2. Cln2-associated kinase activity in cdc37-1 cells. (A) Histone was no decrease in Cdc28 in these H1 kinase activity in immunoprecipitates of epitope-tagged Cln2 from significant protein lysates. wild-type (WT, lanes 1 and 2), cdc28-4 (lanes 3 and 4), and cdc37-1 Finally, the cdc37-1 growth defect was not completely rescued (lanes 5 and 6) cells grown at 24°C or 37°C. (B) Immunoblots of by overexpression of Cdc28. Mutant cells overexpressing epitope-tagged Cln2 (Upper) and Cdc28 (Lower) in extracts from Cdc28 (from a multicopy plasmid), which contained Cdc28 wild-type (lanes 1 and 2) and cdc37-1 (lanes 3 and 4) cells grown at levels equal to those in wild-type cells, grew slowly at 35°C but 24°C or 37°C. Note that the cdc28-4 and cdc37-1 mutants are partially not at 37°C. Mutant cells transformed with a vector control did defective at 24°C. not grow at either temperature (data not shown). Thus, reduced levels of Cdc28 protein cannot completely account for Cln2-Associated Cdc28 Kinase Activity Is Reduced in the the growth defect ofthe cdc37-1 mutant, although the presence cdc37-1 Mutant. To determine whether Cdc37 is involved in of additional Cdc28 can partially rescue the defect. the regulation of Cdc28, we analyzed Cdc28 activity in wild- To examine further the effect of the cdc37-1 mutation on type and cdc37-1 mutant cells. To assess Cdc28 activity spe- Cln2-associated kinase activity, we analyzed Cdc28-Cln2 ac- cifically during G1, we measured the histone HI kinase activity tivity in cells synchronized in G1. Cells growing at the permis- in immunoprecipitates of epitope-tagged Cln2. We found that sive temperature were arrested in G1 by incubation with the Cln2-associated kinase activity in lysates of cdc37-1 cells at mating pheromone a-factor. Cultures were then shifted to 37°C was 2- to 3-fold lower than in lysates of wild-type cells 37°C for 1 hr, after which the a-factor was washed out and cells (Fig. 2A). Diminished Cln2-associated kinase activity in were returned to 37°C. When wild-type cells were released cdc37-1 extracts was not a consequence of lower levels of Cln2; from a-factor arrest, Cln2 was rapidly synthesized and then indeed, cdc37-1 cells grown at the restrictive temperature degraded, consistent with exit from G1 (Fig. 3A). Increases in contained about 7-fold more Cln2 protein than wild-type cells Cln2 levels were accompanied by parallel increases in Cln2- (Fig. 2B). In wild-type cells, the association of Cln2 and Cdc28 associated kinase activity. Similarly, cdc37-1 mutant cells is accompanied by extensive phosphorylation ofCln2, resulting rapidly synthesized Cln2 protein after removal of pheromone in decreased Cln2 mobility (12) (R.J.D., unpublished obser- (Fig. 3A). Cln2 continued to accumulate, in an underphos- A WT cdc37-1

time (min) c B 0 10 20 30 60 120 C B 0 10 20 30 60 120

Histone H1 i.. ..:: I *___4_.__0 'IO_* B WT cdc37-1 24°C 37°C 24°C 37°C CLN2 * CDC28 .

CDC37 _. CDC28 _,! _i,

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 FIG. 3. (A) Cln2-associated kinase activity following release from a-factor arrest. Cells were arrested with a-factor for 2 hr at 24°C, shifted to 37°C for 1 hr, washed free of pheromone, and returned to 37°C. Samples were taken before addition of a-factor (lanes 1, marked C), immediately before release (lanes 2, marked B), and at the indicated times after release (lanes 3-8). Top panels show histone H1 kinase activity of Cln2 immunoprecipitates from lysates of wild-type (WT) (Left) and cdc37-1 cells (Right). Lower panels show immunoblots of Cln2, Cdc28, and Cdc37. (B) Binding of Cln2 and Cdc28. Epitope-tagged Cln2 was isolated by immunoaffinity chromatography from lysates ofwild-type (lanes 1 and 2) and cdc37-1 (lanes 3 and 4) cells grown at 24°C (lanes 1 and 3) or 37°C (lanes 2 and 4) for 3 hr. Column eluates were immunoblotted to detect Cln2 and Cdc28. Downloaded by guest on September 30, 2021 4654 Cell Biology: Gerber et at Proc. Natl. Acad Sci. USA 92 (1995) phorylated form, throughout the 2-hr experiment. Despite A increasing Cln2 levels, there was only a minor increase in WT cdc37-1 kinase /1 Cln2-associated activity. 000 000 o o o~ t, oo o inLO co 00 - - We addressed the possibility that decreased Cln2-associated Time (min) O coO a) -- O o o o-N kinase activity in mutant cells is caused by the failure of Cln2 to bind Cdc28. Cln2 was purified from cell lysates by immuno- affinity chromatography and then immunoblotted to determine Histone H1 the amount of associated Cdc28 (Fig. 3B). After incubation at 37°C, cdc37-1 mutants exhibited a 6-fold decrease in the amount ofCdc28 bound to Cln2 relative to the amount bound in wild-type GST-CLB2 -l-m.m cells (Fig. 3B, lanes 2 and 4). These results suggest that the reduced Cln2-associated kinase activity in the cdc37-1 mutant results from a defect in the association of Cdc28 and Cln2. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Cdc37 Is Required for Cdc28 Activation by the Mitotic Cyclin Clb2. To determine whether the cdc37-1 mutation B WT cdc37-1 causes a defect in Cdc28 we next general activation, analyzed 000 000 °- the activation of Cdc28 by the mitotic cyclin Clb2. We trans- Time (min) m c -o-oC- cH formed yeast with a plasmid encoding a GST-Clb2 fusion protein under the control of the GALl promoter. Cells were synchronized in mitosis by treatment with the microtubule Histone H1 _ ..._ . _--- polymerization inhibitors benomyl and nocodazole. Cultures were shifted to the restrictive temperature, and after 1 hr the expression of GST-Clb2 was induced by addition of galactose. CLB2 ,___ _-_...___, ' Immunoblotting revealed that GST-Clb2 levels in wild-type and mutant cells rose to equivalent levels after 3 hr of induction (Fig. 4A). In wild-type cells, the increase in GST- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Clb2 protein was accompanied by a parallel increase in 24° 37° associated histone HI kinase activity (Fig. 4A). In cdc37-1 C cells, however, the increase in kinase activity was dramatically inhibited. Cl) CY) To explore further the activation of Cdc28 by Clb2, wild-type 3: 'a 'LO) and mutant cells were arrested in G1 with a-factor at 24°C and then released from the arrest in the presence of benomyl and nocodazole. To avoid G1 arrest in mutant cells, the tempera- CLB2 ture was not shifted to 37°C until budding occurred (indicating entry into S phase). After the temperature shift, Clb2 con- centrations increased to similar levels in wild-type and mutant less histone HI cells (Fig. 4B). However, there was -5-fold ~ .. kinase activity associated with Clb2 in cdc37-1 cells. CDC28 To assess the binding of Cdc28 and Clb2, Clb2 was purified by immunoaffinity chromatography and then immunoblotted to determine the amount of associated Cdc28. At 37°C, the 1 2 3 4 amount of Cdc28 associated with Clb2 in mutant cells was FIG. 4. Activation of Cdc28 by Clb2 in cdc37-1 cells. (A) Wild type severalfold lower than the amount associated with Clb2 in (WT) and cdc37-1 cells carrying a plasmid encoding a GST-Clb2 fusion wild-type cells (Fig. 4 C, lanes 3 and 4). The decreased amount protein under the control of the GALl promoter were arrested in of Cdc28 associated with Clb2 in mutant cells was not simply mitosis by treatment with benomyl and nocodazole, shifted to 37°C for due to the reduced level of total Cdc28 in these cells: 1 hr, and treated with galactose. Samples were taken before mitotic depletion arrest (lanes 1 and 8, marked C) and at the indicated times after of Clb2 from cell lysates in these experiments did not result in addition of galactose. The upper panel shows histone H1 kinase decreased Cdc28 concentrations (data not shown). activity associated with the GST-Clb2 protein; the lower panel shows an immunoblot of GST-Clb2. (B) Wild-type and cdc37-1 cells were DISCUSSION released from a-factor arrest into medium containing benomyl and nocodazole. After 1 hr, when budding had occurred in most cells, Our studies indicate that Cdc37 is required for the association temperature was shifted to 37°C. Samples were taken immediately of Cdc28 with at least two cyclin subunits, the G1 cyclin Cln2 following a-factor release (lanes 1 and 8, marked B) and at the and the mitotic Clb2. As formation of active Cdc28-Cln indicated times following the temperature shift. The upper panel cyclin shows histone H1 kinase activity in Clb2 immunoprecipitates; the complexes is necessary for cells to progress through G1 (1, 2, lower panel shows an immunoblot of Clb2. (C) Wild-type and cdc37-1 6), these observations can account for defects in progression cells were synchronized in mitosis at 24°C or 37°C for 3 hr, as described from G1 to S phase in cdc37-1 mutant cells. In addition, our in B. Clb2 was purified from cell lysates by immunoaffinity chroma- analysis of Clb2 suggests that Cdc37 is required for passage tography and immunoblotted with antibodies against Clb2 and Cdc28. through mitosis, although further studies will be required to determine whether the cdcqj7-1 mutant exhibits mitotic defects. personal communication) and casein kinase II (R. McCann The molecular mechanisms underlying the cdc37-1 defect and C. Glover, personal communication). are not clear. Because the cdc37-1 mutation results in de- The activity of CDK-cyclin complexes is controlled by a creased Cdc28 levels as well as impaired cyclin binding, it is wide range of mechanisms (7-9). For example, several proteins possible that Cdc37 is required for the folding, localization, or have been identified that inhibit CDK activity (8, 17), and in stability of Cdc28. Cdc37 may play a general role in kinase some cases (e.g., p16'NK4 in mammalian cells) (18) these regulation: overproduction of Cdc37 has recently been ob- inhibitors may act by interfering with CDK-cyclin binding. served to suppress temperature-sensitive mutations in other Cdc37 could negatively regulate such an inhibitor. Another protein kinases, including Mpsl (A. Schutz and M. Winey, possibility is that Cdc37 positively regulates Cdc28 activity, Downloaded by guest on September 30, 2021 Cell Biology: Gerber et aL Proc. Nat. Acad Sci. USA 92 (1995) 4655 perhaps by directly catalyzing Cdc28-cyclin binding or by 2. Nasmyth, K. (1993) Curr. Opin. Cell Biol. 5, 166-179. stimulating the activity of the CDK-activating kinase (CAK). 3. Reed, S. I. (1980) Genetics 95, 561-577. In some cases (e.g., human CDC2 and cyclin A), the binding 4. Ferguson, J., Ho, J., Peterson, T. A. & Reed, S. I. (1986) Nucleic of CDK and cyclin requires phosphorylation by CAK at a Acids Res. 14, 6681-6697. conserved threonine residue (Thr161 in CDC2) (19, 20). Thus, 5. Neiman, A. M., Chang, F., Komachi, K. & Herskowitz, I. (1990) if at the Cell Regul. 1, 391-401. Cdc28-cyclin binding requires phosphorylation equiv- 6. Reed, S. I. (1992) Annu. Rev. Cell Biol. 8, 529-561. alent site (Thr169), then mutation of CDC37 could inhibit 7. Draetta, G. (1993) Trends Cell Biol. 3, 287-289. Cdc28-cyclin binding by reducing CAK activity. Biochemical 8. Morgan, D. 0. (1995) Nature (London) 374, 131-134. explorations of these issues, both in yeast and in vertebrate 9. Solomon, M. (1993) Curr. Opin. Cell Biol. 5, 180-186. cells, could lead to the identification of novel mechanisms in 10. Cutforth, T. & Rubin, G. M. (1994) Cell 77, 1027-1036. CDK regulation. 11. Tyers, M., Tokiwa, G., Nash, R. & Futcher, B. (1992) EMBO J. 11, 1773-1784. We thank Yong Gu, Doug Kellogg, Marc Kirschner, Steve Murphy, 12. Tyers, M., Tokiwa, G. & Futcher, B. (1993) EMBO J. 12, Andrew Murray, Matthias Peter, Steve Reed, Aaron Straight, and 1955-1968. Mike Tyers for reagents and helpful advice and Rob Fisher for 13. Kellogg, D. R. & Alberts, B. M. (1992) Mol. Biol. Cell 3, 1-11. comments on the manuscript. This work was supported by grants to 14. Gu, Y., Rosenblatt, J. & Morgan, D. 0. (1992) EMBO J. 11, I.H. from the National Institutes of Health and to D.O.M. from the 3995-4005. National Institutes of Health, the Markey Charitable Trust, the March 15. Gu, Y., Turck, C. W. & Morgan, D. 0. (1993) Nature (London) of Dimes Birth Defects Foundation, and the Rita Allen Foundation. 366, 707-710. R.J.D. is a Lucille P. Markey Scholar. M.R.G. was supported by 16. Rothstein, R. (1991) Methods Enzymol. 194, 281-301. National Institutes of Health training grant to the Medical Scientist 17. Peter, M. & Herskowitz, I. (1994) Cell 79, 181-184. Training Program of the University of California, Los Angeles. A.F. 18. Serrano, M., Hannon, G. J. & Beach, D. (1993) Nature (London) is supported by a postgraduate scholarship from the Natural Sciences 366, 704-707. and Engineering Research Council of Canada. 19. Ducommun, B., Brambilla, P., Felix, M.-A., Franza, B. R., Karsenti, E. & Draetta, G. (1991) EMBO J. 10, 3311-3319. 1. Forsburg, S. L. & Nurse, P. (1991) Annu. Rev. Cell Biol. 7, 20. Desai, D., Wessling, H. C., Fisher, R. P. & Morgan, D. 0. (1995) 227-256. Mol. Cell. Biol. 15, 345-350. Downloaded by guest on September 30, 2021