Differential Interaction of the Cyclin-dependent Kinase (Cdk) Inhibitor p27Kip1 with Cyclin A-Cdk2 and Cyclin D2-Cdk4

Artigo Acesso aberto Revisado por pares

Differential Interaction of the Cyclin-dependent Kinase (Cdk) Inhibitor p27Kip1 with Cyclin A-Cdk2 and Cyclin D2-Cdk4

1997; Elsevier BV; Volume: 272; Issue: 41 Linguagem: Inglês

10.1074/jbc.272.41.25863

ISSN

1083-351X

Autores

Stacy W. Blain, Ermelinda Montalvo, Joan Massagué,

Tópico(s)

Cancer Research and Treatments

Resumo

Although p27Kip1 has been considered a general inhibitor of G1 and S phase cyclin-dependent kinases, we report that the interaction of p27 with two such kinases, cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu cells containing a p27 inducible system, a 6-fold increase over the basal p27 level completely inhibited Cdk2 and cell cycle progression. In contrast, the same or a larger increase in p27 levels did not inhibit Cdk4 or its homologue Cdk6, despite extensive binding to these kinases. A p27-cyclin A-Cdk2 complex formed in vitro was essentially inactive, whereas a p27-cyclin D2-Cdk4 complex was active as a retinoblastoma kinase and served as a substrate for the Cdk-activating kinase Cak. High concentrations of p27 inhibited cyclin D2-Cdk4, apparently by conversion of active complexes into inactive ones by the binding of additional p27 molecules. In contrast to their differential interaction, cyclin A-Cdk2 and cyclin D2-Cdk4 were similarly inhibited by bound p21Cip1/Waf1. Roles of cyclin A-Cdk2 as a p27 target and cyclin D2-Cdk4 as a p27 reservoir may result from the differential ability of bound p27 to inhibit the kinase subunit in these complexes. Although p27Kip1 has been considered a general inhibitor of G1 and S phase cyclin-dependent kinases, we report that the interaction of p27 with two such kinases, cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu cells containing a p27 inducible system, a 6-fold increase over the basal p27 level completely inhibited Cdk2 and cell cycle progression. In contrast, the same or a larger increase in p27 levels did not inhibit Cdk4 or its homologue Cdk6, despite extensive binding to these kinases. A p27-cyclin A-Cdk2 complex formed in vitro was essentially inactive, whereas a p27-cyclin D2-Cdk4 complex was active as a retinoblastoma kinase and served as a substrate for the Cdk-activating kinase Cak. High concentrations of p27 inhibited cyclin D2-Cdk4, apparently by conversion of active complexes into inactive ones by the binding of additional p27 molecules. In contrast to their differential interaction, cyclin A-Cdk2 and cyclin D2-Cdk4 were similarly inhibited by bound p21Cip1/Waf1. Roles of cyclin A-Cdk2 as a p27 target and cyclin D2-Cdk4 as a p27 reservoir may result from the differential ability of bound p27 to inhibit the kinase subunit in these complexes. Cell cycle transitions are controlled by the action of the cyclin-dependent kinases (Cdk) 1The abbreviations used are: Cdk, cyclin-dependent kinase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; Cak, Cdk-activating kinase; Rb, retinoblastoma. and their activating subunits, the cyclins (1Hunter T. Pines J. Cell. 1994; 79: 573-582Abstract Full Text PDF PubMed Scopus (2160) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2594) Google Scholar). In mammalian cells, cyclin D-Cdk4 or -Cdk6, cyclin E-Cdk2, and cyclin A-Cdk2 act sequentially during the G1/S transition and are required for cell cycle progression through this period. Cdk activity is tightly regulated by a combination of mechanisms, including changes in the cyclin or Cdk levels, phosphorylation of positive and negative regulatory sites, and interaction with stoichiometric inhibitors (3Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar). The latter in particular act as mediators of a wide range of antimitogenic signals. However, their specific functions are still poorly understood. Two families of stoichiometric Cdk inhibitors have been described (4Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar). The Ink4 family, which includes p16Ink4a (5Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3392) Google Scholar), p15Ink4b (6Hannon G.J. Beach D. Nature. 1994; 371: 257-261Crossref PubMed Scopus (1893) Google Scholar), p18Ink4c (7Guan K.-L. Jenkins C.W. Li Y. Nichols M.A. Wu X. O'Keefe C.L. Matera A.G. Xiong Y. Genes Dev. 1994; 8: 2939-2952Crossref PubMed Scopus (728) Google Scholar), and p19Ink4d (8Chan F.K.M. Zhang J. Cheng L. Shapiro D.N. Winoto A. Mol. Cell. Biol. 1995; 15: 2682-2688Crossref PubMed Scopus (337) Google Scholar, 9Hirai H. Roussel M.F. Kato J.-Y. Ashmun R.A. Sherr C.J. Mol. Cell. Biol. 1995; 15: 2672-2681Crossref PubMed Scopus (587) Google Scholar), specifically inhibits the cyclin D-dependent kinases, Cdk4 and Cdk6, by binding to the Cdk subunit either free or in complex with cyclin D. Ink4 proteins are structurally unrelated at the amino acid level to the other family of inhibitors, which include p21Cip1/Waf1 (10El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar, 11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (709) Google Scholar, 12Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar, 13Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1317) Google Scholar), p27Kip1 (14Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar, 15Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar), and p57Kip2 (16Lee M.-H. Reynisdóttir I. Massagué J. Genes Dev. 1995; 9: 639-649Crossref PubMed Scopus (856) Google Scholar, 17Matsuoka S. Edwards M. Bai C. Parker S. Zhang P. Baldini A. Harper J.W. Elledge S.J. Genes Dev. 1995; 9: 650-662Crossref PubMed Scopus (911) Google Scholar). In contrast to the Ink4 proteins, the Cip/Kip proteins can interact with many different cyclin-Cdk complexes. This interaction is mediated by a homologous domain (18Luo Y. Hurwitz J. Massagué J. Nature. 1995; 375: 159-161Crossref PubMed Scopus (516) Google Scholar, 19WaRbrick E. Lane D.P. Glover D.M. Cox L.S. Curr. Biol. 1995; 5: 275-282Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 20Chen J. Jackson P.K. Kirschner M.W. Dutta A. Nature. 1995; 374: 386-388Crossref PubMed Scopus (512) Google Scholar, 21Nakanishi M. Rovetorye R.S. Adamai G.R. Pereira-Smith O.M. Smith J.R. EMBO J. 1995; 14: 555-563Crossref PubMed Scopus (172) Google Scholar, 22Chen J. Saha P. Kornbluth S. Dynlacht B.D. Dutta A. Mol. Cell. Biol. 1996; 16: 4673-4682Crossref PubMed Scopus (278) Google Scholar) that contacts both subunits in the cyclin A-Cdk2 complex (23Russo A.A. Jeffrey P.D. Patten A. Massagué J. Pavletich N. Nature. 1996; 382: 325-331Crossref PubMed Scopus (801) Google Scholar). Cip/Kip proteins have higher affinity for G1 and S phase Cdks than for mitotic Cdks, and their overexpression causes G1 arrest, suggesting that they primarily regulate G1 and S phase Cdks in vivo. Cdk regulation by inhibitors is an important step in linking mitogenic or antimitogenic signals to cell cycle progression. Cdk inhibitors are thought to set thresholds for cyclin-Cdk activity by setting levels that cyclin-Cdk complexes must surpass to become active (4Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar). According to this model, cell cycle progression or arrest would depend on the relative concentration of inhibitors and Cdks; a decrease in cyclin-Cdk components or an increase in inhibitor levels would prevent the accumulation of inhibitor-free cyclin-Cdk complexes, thus inhibiting cell cycle progression. Evidence for this type of mechanism has emerged. For example, p53 inhibits G1 progression following radiation-induced DNA damage in part by elevating p21 expression (10El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar,24El-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. Pietenpol J.A. Burrell M. Hill D.E. Wang Y. Wiman K.G. Mercer W.E. Kastan M.B. Kohn K.W. Elledge S.J. Kinzler K.W. Vogelstein B. Cancer Res. 1994; 54: 1169-1174PubMed Google Scholar), and mitogens facilitate emergence from G0 in part by inducing p27 down-regulation (25Kato J. Matsuoka M. Polyak K. Massagué J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar, 26Nourse J. Firpo E. Flanagan M.W. Meyerson M. Polyak K. Lee M.-H. Massagué J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar, 27Hengst L. 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Swindell E. Fox M.P. Wei N. Mol. Biol. Cell. 1995; 6: 387-400Crossref PubMed Scopus (862) Google Scholar). In vivo, p27 mediates inhibition of cyclin E-Cdk2 in cells that are exposed to transforming growth factor-β, lovastatin, rapamycin, vitamin D3, cell-to-cell contact, or lack of anchorage (26Nourse J. Firpo E. Flanagan M.W. Meyerson M. Polyak K. Lee M.-H. Massagué J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar, 27Hengst L. Dulic V. Slingerland J.M. Lees E. Reed S.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5291-5295Crossref PubMed Scopus (283) Google Scholar, 28Poon R.Y.C. Toyoshima H. Hunter T. Mol. Biol. Cell. 1995; 6: 1197-1213Crossref PubMed Scopus (232) Google Scholar, 34Polyak K. Kato J.-Y. Solomon M.J. Sherr C.J. Massagué J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1837) Google Scholar, 35Slingerland J.M. Hengst L. Pan C. Alexander D. Stampfer M. Reed S.I. Mol. Cell. 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However, the notion that Cdks are equivalent targets of p27 proteins and the concept that inhibitor-Cdk interactions are simply governed by their relative abundance in the cell are challenged by various observations. In vitro, p27 is a more effective inhibitor of cyclin E-Cdk2 than of cyclin D-Cdk4 (14Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar, 15Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1938) Google Scholar, 33Harper J.W. Elledge S.J. Keyomarsi K. Dynlacht B. Tsai L.-H. Zhang P. Dobrowolski S. Bai C. Connell-Crowley L. Swindell E. Fox M.P. Wei N. Mol. Biol. Cell. 1995; 6: 387-400Crossref PubMed Scopus (862) Google Scholar). During periods of proliferation as well as during exit from the cell cycle, it has been observed that p27 shuttles between Cdk4/6 and Cdk2 complexes even though the levels of p27, Cdk2, and Cdk4 may remain constant (28Poon R.Y.C. Toyoshima H. Hunter T. Mol. Biol. Cell. 1995; 6: 1197-1213Crossref PubMed Scopus (232) Google Scholar, 37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar,41Soos T.J. Kiyokawa H. Yan J.S. Rubin M.S. Giordano A. DeBlasio A. Bottega S. Wong B. Mendelsohn J. Koff A. Cell Growth Differ. 1996; 7: 135-146PubMed Google Scholar). Furthermore, although biochemical and structural evidence (23Russo A.A. Jeffrey P.D. Patten A. Massagué J. Pavletich N. Nature. 1996; 382: 325-331Crossref PubMed Scopus (801) Google Scholar) argues that the p27-cyclin A-Cdk2 complex is inactive, p27 immunoprecipitated from proliferating human B cell lymphoma was shown to be associated with retinoblastoma protein (Rb) kinase activity that could be significantly depleted with antibodies against Cdk6 (41Soos T.J. Kiyokawa H. Yan J.S. Rubin M.S. Giordano A. DeBlasio A. Bottega S. Wong B. Mendelsohn J. Koff A. Cell Growth Differ. 1996; 7: 135-146PubMed Google Scholar). These observations have raised the possibility that in some conditions at least, p27 may interact differently with Cdk2 and Cdk4/6 complexes, with p27 binding not necessarily causing Cdk4/6 inhibition. In the present work, we have investigated the idea whether p27-associated Cdk4 or Cdk6 complexes might exist as active kinases in vitro as well as in vivo. Here we report on the existence of p27-cyclin D-Cdk4 complexes that are largely active, whereas similar complexes of p27 with cyclin A-Cdk2 are essentially inactive. Mv1Lu cells expressing a tetracycline-regulated p27 expression system (37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar) were grown to 80% confluency in the presence of 1 μg/ml tetracycline. The culture medium was then switched to medium containing different tetracycline concentrations. After 18 h, the cells were lysed in hypotonic buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 10% glycerol, 1 mm dithiothreitol, 0.1% Tween 20, 10 mmβ-glycerophosphate, 1 mm NaF, 0.1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin) as described previously (42Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1027) Google Scholar). Cell lysates were normalized based on protein content (Bio-Rad). Precleared lysates were immunoprecipitated with the appropriate antibody for 3–16 h at 4 °C. Complexes bound to protein A-Sepharose were washed 4 times with hypotonic buffer (42Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1027) Google Scholar) and separated by SDS-PAGE. 1.5 mg of total cell extract protein was used for immunoprecipitation followed by Western analysis, and 0.5 mg was used for immunoprecipitation followed by kinase analysis. p27-associated complexes were recovered by immunoprecipitation with p27 antibodies (37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar) and Cdk2-associated complexes by immunoprecipitation with Cdk2 antibodies (43Reynisdóttir I. Massagué J. Genes Dev. 1997; 11: 492-503Crossref PubMed Scopus (308) Google Scholar). Immunocomplexes were analyzed by Western immunoblot analysis with p27, Cdk4 (Pharmigen), or Cdk6 (Santa Cruz) antibodies by standard techniques. Total cell extract was subjected to SDS-PAGE analysis, followed by Western immunoblot analysis with p27 or Rb antibodies (Pharmigen). Immunocomplexes were assayed for Rb kinase activity as described previously (42Matsushime H. Quelle D.E. Shurtleff S.A. Shibuya M. Sherr C.J. Kato J.-Y. Mol. Cell. Biol. 1994; 14: 2066-2076Crossref PubMed Scopus (1027) Google Scholar). Where indicated, histone H1 was used as a substrate instead of GST-Rb C-terminal domain (pRb amino acids 773–928). For immunodepletion, extracts were subjected to four cycles of immunoprecipitation with Cdk4 and Cdk6 antibodies or to four cycles with normal rabbit serum. The depleted extract was then immunoprecipitated with p27 antibodies, split, and assayed by Western immunoblotting with Cdk4 and Cdk6 antibodies, and assayed for Rb kinase activity. Parallel cell cultures were assayed in triplicate for125I-deoxyuridine incorporation (44Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massagué J. Cell. 1990; 62: 175-185Abstract Full Text PDF PubMed Scopus (679) Google Scholar) after 18 h of incubation in the indicated tetracycline concentrations. Data are averages of triplicate determinations and are plotted as percentage relative to the cpm incorporated in the presence of 1 μg/ml tetracycline. Fixed amounts of insect cell lysates containing baculovirally expressed cyclin A and Cdk2 or cyclin D2 and Cdk4 (45Kato J.-Y. Matsushime H. Hiebert S. Ewen M. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1095) Google Scholar) were incubated with the indicated concentrations of bacterially expressed p27 or p21 and assayed (18Luo Y. Hurwitz J. Massagué J. Nature. 1995; 375: 159-161Crossref PubMed Scopus (516) Google Scholar) in kinase buffer (20 mm Tris-HCl, pH 7.5, 7.5 mm MgCl2), containing 30 μm ATP, 5 μCi of [γ-32P]ATP and a GST-Rb fusion protein bound to glutathione-agarose beads. Radioactivity incorporated into GST-Rb was quantified and plotted as a percentage relative to controls without p27. The baculoviral cyclin-Cdk complexes were prepared as described (46Desai D. Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: 571-582Crossref PubMed Scopus (193) Google Scholar). The p27 and p21 proteins were tagged at their C termini with a hexahistidine sequence and purified from Escherichia coli as described (14Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J.M. Tempst P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2057) Google Scholar). Cdk2-associated complexes were recovered by immunoprecipitation either with Cdk2 antibodies or via a HA epitope tag at the C terminus of Cdk2 (46Desai D. Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: 571-582Crossref PubMed Scopus (193) Google Scholar), and Cdk4 or cyclin A-associated complexes with Cdk4 (Santa Cruz) or cyclin A (Santa Cruz) antibodies, respectively. p27-associated complexes were recovered by immunoprecipitation with p27 antibodies (37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar), and p21-associated complexes were recovered by immunoprecipitation with p21 antibodies (Santa Cruz). Immunoprecipitations were performed in modified LSLD buffer (50 mm HEPES, pH 7.5, 50 mm NaCl, 10% glycerol, 0. 1% Tween 20, 80 μm β-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 10 μg/ml antipain, 10 μg/ml leupeptin, 100 μg/ml soybean trypsin inhibitor, 100 μg/ml benzamidine) (37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar), with the indicated antibody for 1–2 h at 4 °C with gentle agitation. Immunocomplexes were assayed for kinase activity as described above. The same antibodies and cyclin D2 antibodies (Santa Cruz) were used for Western immunoblotting. The amount of complex used in the specific activity assays was normalized by immunoblotting for the component that was not used for immunoprecipitation. Cyclin D2-Cdk4 complexes expressed in insect cell lysates were immunoprecipitated with p27 or Cdk4 antibodies. The immunocomplexes were incubated in a 50-μl reaction volume of Cak activation buffer (50 mm HEPES, pH 7.4, 15 mmMgCl2, 20 mm EGTA, 5 mmdithiothreitol, 80 μm β-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 10 μg/ml antipain, 10 μg/ml leupeptin, 100 μg/ml soybean trypsin inhibitor, 100 μg/ml benzamidine) with or without 50 ng of purified baculovirally expressed cyclin H-Cdk7, for 1 h at 22 °C, essentially as described (25Kato J. Matsuoka M. Polyak K. Massagué J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar). Cak was prepared as described (47Russo A.A. Jeffrey P.D. Pavletich N. Nat. Struct. Biol. 1996; 3: 696-700Crossref PubMed Scopus (505) Google Scholar). The immunocomplexes were washed three times in LSLD buffer and then subjected to Rb kinase assays as described above. p27 was tagged at the N terminus with the Flag epitope sequence, expressed in E. coli, and purified on Flag-agarose beads (Eastman Kodak Co.) as described (16Lee M.-H. Reynisdóttir I. Massagué J. Genes Dev. 1995; 9: 639-649Crossref PubMed Scopus (856) Google Scholar, 29Lee M.-H. Nikolic M. Baptista C.A. Lai E. Tsai L.-H. Massagué J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3259-3263Crossref PubMed Scopus (116) Google Scholar). p27His-cyclin D2-Cdk4 complexes were isolated by binding to metal-agarose beads (Talon beads,CLONTECH). 260 nm p27Flagwas added to these immobilized complexes and incubated on ice, with constant mixing for 30 min. The complexes were extensively washed three times with LSLD buffer, subjected to SDS-PAGE analysis, and Western analysis with Cdk4 (Santa Cruz) and Flag antibodies (Kodak). Cyclin D2-Cdk4, cyclin D2, or Cdk4-containing baculoviral extracts were centrifuged at 50,000 rpm for 30 min, and the supernatant was injected onto a Superose 12 column, HR 10/30 (Pharmacia Biotech Inc.), pre-equilibrated with column buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA), and using fast protein liquid chromatography 0.5-ml fractions (0.35 ml/min) were collected. The void volume of the column was 8.3 ml. Molecular weight markers (Sigma) were subjected to gel filtration separately and monitored by UV absorption and SDS-PAGE analysis. Fractions 1–50 were subjected to SDS-PAGE analysis and Western immunoblotting with Cdk4 (Santa Cruz) or cyclin D2 (Santa Cruz) antibodies. Densitometric quantitation of the Cdk4 or cyclin D2 band was plotted as a percent of maximal immunoreactivity. Radioactivity incorporated into GST-Rb was quantified and plotted as a percentage of maximal phosphorylation. We compared the ability of p27 to inhibit Cdk2 and Cdk4 in vivo by examining its interaction with these Cdks in the Tet-p27 cell line. Tet-p27 is a derivative of the mink lung epithelial cell line (Mv1Lu) that expresses human p27 under the control of a tetracycline transactivator (37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar, 48Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4268) Google Scholar). In media containing a high concentration of tetracycline (1 μg/ml), Tet-p27 cells do not express exogenous p27, and their basal level of endogenous p27 is similar to the level in parental Mv1Lu cells (37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar). In proliferating Mv1Lu and Tet-p27 cells, p27 is bound to cyclin D-Cdk4 (Fig. 1 c) and, to a lesser extent, cyclin D-Cdk6 (Fig. 1 d) and cyclin A-Cdk2 complexes (Fig. 1 e). By lowering the tetracycline concentration, the p27 level in Tet-p27 cells can be gradually increased up to 25-fold over basal (Fig. 1 b), with a concomitant increase in the level of p27-bound Cdk2 or Cdk2-bound p27 (Fig. 1 e; Ref.37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar), a complete loss of Cdk2-associated histone H1 kinase activity and Rb kinase activity (Fig. 1, f and g), and inhibition of DNA synthesis (Fig. 1 a; Ref. 37Reynisdóttir I. Polyak K. Iavarone A. Massagué J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (893) Google Scholar). A 6-fold increase in p27 levels is sufficient to completely inhibit Cdk2-associated kinase activity on both substrates (Fig. 1,f and g). As cyclin E-Cdk2 levels remain constant during this treatment, 2I. Reynisdóttir and J. Massagué, unpublished results. the loss of Cdk2 kinase activity is due to its increased association with p27. p27 immunoprecipitates did not contain histone H1 kinase activity even when derived from cells not overexpressing p27 (Fig.1 i), in which the level of Cdk2-associated kinase activity was high. Among G1 Cdks, Cdk2, Cdk4, and its close isoform Cdk6, all have Rb kinase activity in vitro, whereas only Cdk2 has histone H1 kinase activity (49Matsushime H. Ewen M.E. Strom D.K. Kato J.-Y. Hanks S.K. Roussel M.F. Sherr C.J. Cell. 1992; 71: 323-334Abstract Full Text PDF PubMed Scopus (780) Google Scholar). This suggested that p27-associated Cdk2 is largely inactive, an observation consistent with previous results (23Russo A.A. Jeffrey P.D. Patten A. Massagué J. Pavletich N. Nature. 1996; 382: 325-331Crossref PubMed Scopus (801) Google Scholar, 41Soos T.J. Kiyokawa H. Yan J.S. Rubin M.S. Giordano A. DeBlasio A. Bottega S. Wong B. Mendelsohn J. Koff A. Cell Growth Differ. 1996; 7: 135-146PubMed Google Scholar). A marked increase in p27 levels led only to a small (less than 2-fold) increase in the amount of p27-bound Cdk4 in the Tet-p27 cells (Fig.1 c), suggesting that most of the cyclin D-Cdk4 present in proliferating Mv1Lu cells is already bound to p27. A larger increase was observed in the level of p27-bound Cdk6 (Fig. 1 d), indicating an interesting difference in the ability of Cdk4 and Cdk6 to interact with p27. p27 immunoprecipitated from Tet-p27 cells was associated with Rb kinase activity (Fig. 1 j), which declined only partially at the highest p27 concentrations. Due to a lack of suitable antibodies against the mink proteins (the available immunprecipitating antibodies do not yield catalytically active complexes), it was not possible to directly assay cyclin D- or Cdk4/6-associated Rb kinase activity in these cells. However, immunodepletion of both kinases from cell lysates prevented the subsequent recovery of p27-associated Rb kinase activity (Fig.1 k), whereas immunodepletion with normal rabbit serum did not have any effect on p27-associated Rb kinase activity. This suggests that the majority of the Rb kinase activity in p27 immunocomplexes is due to bound Cdk4 and/or Cdk6. p27 was associated with Rb kinase activity even when precipitated from cells that contained enough p27 to cause a complete inhibition of Cdk2 (Fig. 1, f and i, lanes 2–7). The presence of Rb kinase activity in p27 complexes from these cells correlated with the presence of hyperphosphorylated Rb protein in the cells, as indicated by the levels of the slow migrating Rb band in Western immunoblotting of cell lysates with Rb antibodies (Fig. 1 h). Rb hyperphosphorylation did decrease at the highest p27 concentration, and this correlated with a decrease in p27-associated Rb kinase activity. These results therefore suggest that a large portion of Cdk4 in the exponentially growing cells is bound to p27, that p27-bound Cdk4 or Cdk6 can be active as kinases, and that p27 may not effectively inhibit these kinases in vivo. In an attempt to explain the above described phenomena, we analyzed the ability of p27 to inhibit Cdk2 and Cdk4. We measured the ability of bacterially expressed p27 to inhibit the Rb kinase activity of baculovirally expressed cyclin A-Cdk2 or cyclin D2-Cdk4 in insect cell extracts (46Desai D. Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: 571-582Crossref PubMed Scopus (193) Google Scholar). Both cyclin A-Cdk2 and cyclin D2-Cdk4 extracts contained a similar amount of catalytically inactive complexes, as determined by their ability to be further activated by the addition of exogenous Cak (see below). Rb kinase assays were conducted under conditions of Rb substrate excess, in the linear range of the kinase reaction, and using two different concentrations of the kinases. The use of the higher concentration allowed the visualization of p27- and Cdk-associated complexes by immunoblotting analysis. Although a 100-fold higher p27 concentration range was needed to achieve the same level of inhibition when the 100-fold higher cyclin-Cdk concentration was assayed, the inhibition profile was the same in both cases (Fig.2 a). The p27 inhibition profile was also the same when Cdk immunoprecipitated complexes were assayed (Fi

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Differential Interaction of the Cyclin-dependent Kinase (Cdk) Inhibitor p27Kip1 with Cyclin A-Cdk2 and Cyclin D2-Cdk4