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A Cell Cycle-Regulated Inhibitor of Cyclin-Dependent Kinases LUDGER HENGST*, VJEKOSLAV DULIC *T, JOYCE M

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A Cell Cycle-Regulated Inhibitor of Cyclin-Dependent Kinases LUDGER HENGST*, VJEKOSLAV DULIC *T, JOYCE M Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5291-5295, June 1994 Cell Biology A cell cycle-regulated inhibitor of cyclin-dependent kinases LUDGER HENGST*, VJEKOSLAV DULIC *t, JOYCE M. SLINGERLAND*t, EMMA LEES§¶, AND STEVEN I. REED* *Department of Molecular Biology, MB-7, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037; and §Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center, Charlestown, MA 02129 Communicated by Peter K. Vogt, March 10, 1994 ABSTRACT Cyclin-dependent kinases (Cdks) previously Cells were synchronized by a thymidine/nocodazole block have been shown to drive the major cell cycle transitions in release protocol using 2 mM thymidine for 19 hr, release for eukaryotic organisms ranging from yeast to humans. We 3 hr, and treatment with nocodazole (75 ng/ml) for 12 hr. report here the identification of a 28-kDa protein, p2$1'* Aliquots ofthe synchronized cells were harvested every hour (inhibitor of cyclin-dependent kinase), that binds to and after release from the nocodazole block and the percentage of inhibits the kinase activity of preformed Cdk/cyclin com- cells in different phases of the cell cycle was determined by plexes from human cells. p28 Inhibitory activity fluctuates FACS analysis as described (16). Fibroblasts derived from during the cell cycle with maximal levels in G, and accumu- neonatal foreskin (NHF1) were cultured as described (17) and lates in GI- and G-e d cells. These results suggest that were made quiescent by contact inhibition (17). control of the G1/S transition may be influenced by a family Immunoprecipitation, Immunoblots, and Inhibition Analy- of Cdk inhibitors that include p28I' and the recently de- sis. Immunoblots and immunoprecipitations of cyclin and scribed inhibitors p2lciPl/w'Al/caP2 and pl6Ink. Cdk complexes from whole cell extracts were performed with polyclonal (anti-cyclins A, B1, E, D1, anti-cdc2, and anti- Cell cycle progression is controlled at several irreversible Cdk2) or monoclonal (anti-cyclin E and anti-PSTAIRE) an- transition points. Passage through these transitions is based tibodies as described (18). Antibodies against human Cdk4 on regulation of the activity of a family of related protein were a gift from S. Hanks (Vanderbilt University). kinases (cyclin-dependent kinases or Cdks). Cyclins have Inhibition experiments were performed either by adding been demonstrated to promote cell cycle transitions by inhibitor-containing fractions to immunoprecipitated kinase binding and activating specific Cdks (1-3). Progression complexes or by mixing crude extracts or inhibitor- from G1 to S phase in mammalian cells involves activation containing fractions before immunoprecipitation. In mixing ofcyclin E/Cdk2, cyclin D/Cdk4, and cyclin A/Cdk2 (4, 5). experiments, extracts containing active kinase complexes Cyclins D1 and E accumulate and activate distinct Cdk were incubated in lysis buffer [50 mM Tris-HCl, pH 7.5/150 proteins in G1, while the kinetics of cyclin A accumulation mM NaCl/0.1% Nonidet P-40/1 mM phenylmethylsulfonyl and Cdk activation suggest an S-phase role (5). However, fluoride (PMSF)/2 ug of leupeptin per ml/2 jug of pepstatin cyclin accumulation and Cdk binding do not constitute the per ml/10 pug of aprotinin per ml] with inhibitor-containing only levels of Cdk regulation. Additional modes of regula- extracts for 30 min at 300C followed by immunoprecipitation tion of Cdks include positive and negative phosphorylation of Cdk/cyclin complexes and analysis of their associated events and accumulation or activation of inhibitory pro- kinase activity. Alternatively, cycin/Cdk immune com- teins. Initially demonstrated for the yeast Cdk Cdc28 (6, 7), plexes were immobilized on protein A-Sepharose beads and Cdk inhibitory proteins have recently been implicated in G1 incubated with inhibitor-containing extracts or fractions for control of mammalian cells. Whereas p21ciPl/wan/caP2o is a 30 min at 300C before they were washed several times in lysis p53-induced inhibitor of a broad range of Cdk/cyclin com- buffer and the associated kinase activity was determined. The plexes (8-11), pl6"n4 specifically inhibits cyclin D/Cdk4 assay for cyclin Di-associated kinase activity was performed (12). Another inhibitory protein, p27KiP1, has been identi- as described (19). fied in transforming growth factor 1-arrested and in contact- To release inhibitory activity from complexes, protein inhibited cells (13, 14). extracts were incubated at 100TC for 5 min followed by In the course of investigating the mechanism of lovastatin centrifugation, and the inhibitory activity was recovered in arrest in HeLa cells, we found high levels of inactive cyclin the supernatant containing heat stable proteins. Proteinase K E/Cdk2 and cyclin A/Cdk2 complexes, suggesting the pres- (0.1 mg/ml) digests of gel-purified p28Ick were performed in ence of a Cdk inhibitor. This paper describes the identifica- lysis buffer for 80 min at room temperature. The protease was tion of a protein present in lovastatin-arrested and cycling G1 inactivated by PMSF treatment (2.5 mM PMSF for 15 min at cells or Go cells that inhibits a broad range of cyclin/Cdk room temperature). complexes. Fractionation of Protein Extrats. Superose 12 chromatog- raphy. Protein extracts in lysis buffer were spun at 100,000 x g for 20 min. Supernatant containing 2 mg of protein was MATERIALS AND METHODS either boiled or directly applied to a Superose 12 column Cell Culture and Sychronizations. HeLa cells were grown in connected to an FPLC system (Pharmacia); 0.5-ml fractions suspension culture in Dulbecco's modified Eagle's medium were collected and 50-pl aliquots were assayed for cyclin supplemented with 10% newborn calf serum at a density of A/Cdk2 inhibitory activity. 2-6 x 105 cells per ml. Asynchronous growing cells were arrested in G1 by treatment with 66 puM lovastatin for 33 hr Abbreviation: PMSF, phenylmethylsulfonyl fluoride. as described (15). Lovastatin was kindly provided by A. W. tPresent address: Center of Biochemistry, Centre National de la Alberts (Merck Sharp & Dohme). Recherche Scientifique, University of Nice, Parc Valrose, F-06108 Nice, cedex 2, France. tPresent address: Department of Molecular and Medical Genetics, The publication costs ofthis article were defrayed in part by page charge University of Toronto, Toronto, ON Canada M5S 1A8. payment. This article must therefore be hereby marked "advertisement" VPresent address: DNAX Research Institute, 901 California Avenue, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Palo Alto, CA 94304. 5291 Downloaded by guest on October 1, 2021 5292 Cell Biology: Hengst et al. Proc. Natl. Acad. Sci. USA 91 (1994) -d a > >, a o Cd 0 (d - Cd {jcu 50c ..,A Cd Cd, ns c ..- Cyc A Cyc E 80-60c. v1 ' Cd C- Cd b >d >, a)a o u 2 0 U) ..Y>0 -Cdk 2 = zzw Cdk 2 vR 20%- IPa Cyc A Pa Cyc E C CdU .0 0~~~~~~ 1 2 3 4 5 6 8 9 10 12 11a hOlr5 after rnocodazole release 0 4- + + b c Cd c c pad a Cd Cd >, C, Cd Cd ahC > 10 )2ICd501LL1L~C~ ~ ~ ~ ~ >otd I '~~~~~~~~~~~~~6" ;?3dA7 8, .-4 1 Or 1 f-1 20> CD d mz > M > I O o a V0 -4 + + _j 5 6 -,8 as1vr ftacLior- _40 Cdk 2 4rn. Cdk 2 FIG. 2. Cyclin/Cdk inhibitory activity varies during the cell cycle IP Cyc E Pcu Cyc A and is maximal in G1. (a) HeLa cells were synchronized by using a thymidine/nocodazole arrest release protocol. (b) Human erythro- FIG. 1. Lovastatin-arrested HeLa cells contain Cdk2-bound cy- leukemia-derived HL-60 cells were fractionated by elutriation as clin A and cyclin E proteins with low associated kinase activities and described (22). Percentages of cells in the different phases ofthe cell a heat stable cyclin/Cdk inhibitory activity. (a) Western blot analysis cycle [Gi (e), S (u), G2/M (A)] are shown for each synchronized of protein extracts from lovastatin-arrested (Lova.) and asynchro- population or the asynchronous cells used for the elutriation exper- nous (asyn.) HeLa cells. Cyclin A and cyclin E proteins were iment. Protein extract (50 pg) from the different synchronized detected by using polyclonal antibodies (17). (b) Cdk2 protein bound populations was boiled and mixed with 50 jug of protein from to cyclin A and cyclin E was detected after immunoprecipitation asynchronous HeLa cells. Cyclin A-associated kinase activity (bars) from extracts of lovastatin-arrested or asynchronous HeLa cells was then determined and is shown as percentage of activity obtained followed by immunoblotting. Cyclin E-associated Cdk2 was sepa- from 50 pg of protein from asynchronous cells (asyn.). rated into the unphosphorylated inactive form (upper band) and the active faster-migrating phosphorylated form (18, 21). (c) Cyclin E- the supernatants were used for depletion experiments. All and cyclin A-associated histone H1 kinase activities from lovastatin- at arrested or asynchronous HeLa cells were measured after immuno- samples were incubated six times for 15 min 300C with precipitation (IPa) (18). To detect cyclin/Cdk inhibitory activity, protein A-Sepharose beads: x = 0-6 times with protein extracts from asynchronous cells were mixed with boiled or non- A-Sepharose beads containing cyclin A immune complexes boiled extracts from lovastatin-arrested cells followed by immuno- from 50 pg ofprotein from asynchronous HeLa cells and 6 - precipitation of cyclin A- or E-associated kinases. Kinase activities x times with protein A-Sepharose beads only. The samples are shown as percentage of activity obtained from immunoprecipi- (Ox-6x) were analyzed for inhibitory activity by mixing with tation of asynchronous cells. (d) After mixing protein extracts from additional cyclin A immune complexes from 50 pg of extract asynchronous cells with boiled extracts from lovastatin-arrested from cells. After an incubation at for 30 cells (Lovab.), cyclin A or cyclin E bound to Cdk2 was detected as asynchronous 300C described above.
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