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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 41, Issue of October 10, pp. 25863–25872, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Differential Interaction of the Cyclin-dependent Kinase (Cdk) Kip1 with Cyclin A-Cdk2 and Cyclin D2-Cdk4* Inhibitor p27 (Received for publication, June 13, 1997, and in revised form, July 30, 1997) Stacy W. Blain‡, Ermelinda Montalvo, and Joan Massague ´§ From the Cell Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Cip1/Waf1 Kip1 Although p27 has been considered a general inhib- the other family of inhibitors, which include p21 (10 – Kip1 Kip2 itor of G and S phase cyclin-dependent kinases, we 13), p27 (14, 15), and p57 (16, 17). In contrast to the report that the interaction of p27 with two such kinases, Ink4 proteins, the Cip/Kip proteins can interact with many cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu different cyclin-Cdk complexes. This interaction is mediated by cells containing a p27 inducible system, a 6-fold increase a homologous domain (18 –22) that contacts both subunits in over the basal p27 level completely inhibited Cdk2 and the cyclin A-Cdk2 complex (23). Cip/Kip proteins have higher cell cycle progression. In contrast, the same or a larger affinity for G and S phase Cdks than for mitotic Cdks, and increase in p27 levels did not inhibit Cdk4 or its homo- their overexpression causes G arrest, suggesting that they logue Cdk6, despite extensive binding to these kinases. primarily regulate G and S phase Cdks in vivo. A p27-cyclin A-Cdk2 complex formed in vitro was essen- Cdk regulation by inhibitors is an important step in linking tially inactive, whereas a p27-cyclin D2-Cdk4 complex mitogenic or antimitogenic signals to cell cycle progression. was active as a retinoblastoma kinase and served as a Cdk inhibitors are thought to set thresholds for cyclin-Cdk substrate for the Cdk-activating kinase Cak. High con- activity by setting levels that cyclin-Cdk complexes must sur- centrations of p27 inhibited cyclin D2-Cdk4, apparently pass to become active (4). According to this model, cell cycle by conversion of active complexes into inactive ones by the binding of additional p27 molecules. In contrast to progression or arrest would depend on the relative concentra- their differential interaction, cyclin A-Cdk2 and cyclin tion of inhibitors and Cdks; a decrease in cyclin-Cdk compo- Cip1/Waf1 D2-Cdk4 were similarly inhibited by bound p21 . nents or an increase in inhibitor levels would prevent the Roles of cyclin A-Cdk2 as a p27 target and cyclin D2- accumulation of inhibitor-free cyclin-Cdk complexes, thus in- Cdk4 as a p27 reservoir may result from the differential hibiting cell cycle progression. Evidence for this type of mech- ability of bound p27 to inhibit the kinase subunit in anism has emerged. For example, p53 inhibits G progression these complexes. following radiation-induced DNA damage in part by elevating p21 expression (10, 24), and mitogens facilitate emergence from G in part by inducing p27 down-regulation (25–28). Cell cycle transitions are controlled by the action of the p27 is one of the most widely distributed Cdk inhibitors, cyclin-dependent kinases (Cdk) and their activating subunits, being expressed both in proliferating as well as differentiated the cyclins (1, 2). In mammalian cells, cyclin D-Cdk4 or -Cdk6, cells (14, 15, 29 –32) and is an inhibitor of a broad range of G cyclin E-Cdk2, and cyclin A-Cdk2 act sequentially during the Cdk complexes. Thus, p27 can inhibit cyclin D-Cdk4, cyclin G /S transition and are required for cell cycle progression E-Cdk2, and cyclin A-Cdk2 in vitro (14, 15, 33). In vivo, p27 through this period. Cdk activity is tightly regulated by a mediates inhibition of cyclin E-Cdk2 in cells that are exposed to combination of mechanisms, including changes in the cyclin or transforming growth factor-b, lovastatin, rapamycin, vitamin Cdk levels, phosphorylation of positive and negative regulatory D3, cell-to-cell contact, or lack of anchorage (26 –28, 34 – 40). sites, and interaction with stoichiometric inhibitors (3). The p27 may also inhibit cyclin D-dependent kinases in vivo,as latter in particular act as mediators of a wide range of antimi- shown with macrophages containing elevated levels of cAMP togenic signals. However, their specific functions are still (25). poorly understood. However, the notion that Cdks are equivalent targets of p27 Two families of stoichiometric Cdk inhibitors have been de- Ink4a proteins and the concept that inhibitor-Cdk interactions are scribed (4). The Ink4 family, which includes p16 (5), Ink4b Ink4c Ink4d simply governed by their relative abundance in the cell are p15 (6), p18 (7), and p19 (8, 9), specifically inhib- challenged by various observations. In vitro, p27 is a more its the cyclin D-dependent kinases, Cdk4 and Cdk6, by binding effective inhibitor of cyclin E-Cdk2 than of cyclin D-Cdk4 (14, to the Cdk subunit either free or in complex with cyclin D. Ink4 15, 33). During periods of proliferation as well as during exit proteins are structurally unrelated at the amino acid level to from the cell cycle, it has been observed that p27 shuttles between Cdk4/6 and Cdk2 complexes even though the levels of * This work was supported by a National Institutes of Health grant. p27, Cdk2, and Cdk4 may remain constant (28, 37, 41). Fur- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked thermore, although biochemical and structural evidence (23) “advertisement” in accordance with 18 U.S.C. Section 1734 solely to argues that the p27-cyclin A-Cdk2 complex is inactive, p27 indicate this fact. immunoprecipitated from proliferating human B cell lym- ‡ Leukemia Society of America fellow. phoma was shown to be associated with retinoblastoma protein § Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Memorial Sloan-Kettering Cancer (Rb) kinase activity that could be significantly depleted with Center, Box 116, 1275 York Ave., New York, NY 10021. Tel.: 212-639- antibodies against Cdk6 (41). These observations have raised 8975; Fax: 212-717-3298; E-mail: [email protected]. the possibility that in some conditions at least, p27 may inter- The abbreviations used are: Cdk, cyclin-dependent kinase; PAGE, act differently with Cdk2 and Cdk4/6 complexes, with p27 polyacrylamide gel electrophoresis; GST, glutathione S-transferase; Cak, Cdk-activating kinase; Rb, retinoblastoma. binding not necessarily causing Cdk4/6 inhibition. In the pres- This paper is available on line at http://www.jbc.org 25863 This is an Open Access article under the CC BY license. Kip1 25864 p27 Inhibition of Cdk2 and Cdk4 100 mg/ml soybean trypsin inhibitor, 100 mg/ml benzamidine) with or ent work, we have investigated the idea whether p27-associ- without 50 ng of purified baculovirally expressed cyclin H-Cdk7, for 1 h ated Cdk4 or Cdk6 complexes might exist as active kinases in at 22 °C, essentially as described (25). Cak was prepared as described vitro as well as in vivo. Here we report on the existence of (47). The immunocomplexes were washed three times in LSLD buffer p27-cyclin D-Cdk4 complexes that are largely active, whereas and then subjected to Rb kinase assays as described above. Flag similar complexes of p27 with cyclin A-Cdk2 are essentially p27 Association Assays—p27 was tagged at the N terminus with inactive. the Flag epitope sequence, expressed in E. coli, and purified on Flag- His agarose beads (Eastman Kodak Co.) as described (16, 29). p27 -cyclin EXPERIMENTAL PROCEDURES D2-Cdk4 complexes were isolated by binding to metal-agarose beads Flag (Talon beads, CLONTECH). 260 nM p27 was added to these immo- Analysis of Tet-p27 Cells—Mv1Lu cells expressing a tetracycline- bilized complexes and incubated on ice, with constant mixing for 30 regulated p27 expression system (37) were grown to 80% confluency in min. The complexes were extensively washed three times with LSLD the presence of 1 mg/ml tetracycline. The culture medium was then buffer, subjected to SDS-PAGE analysis, and Western analysis with switched to medium containing different tetracycline concentrations. Cdk4 (Santa Cruz) and Flag antibodies (Kodak). After 18 h, the cells were lysed in hypotonic buffer (50 mM HEPES, pH Purification of Cyclin-Cdk Complexes—Cyclin D2-Cdk4, cyclin D2, or 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 1 mM Cdk4-containing baculoviral extracts were centrifuged at 50,000 rpm dithiothreitol, 0.1% Tween 20, 10 mM b-glycerophosphate, 1 mM NaF, for 30 min, and the supernatant was injected onto a Superose 12 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 column, HR 10/30 (Pharmacia Biotech Inc.), pre-equilibrated with col- mg/ml aprotinin, 1 mg/ml leupeptin) as described previously (42). Cell umn buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA), and lysates were normalized based on protein content (Bio-Rad). Precleared using fast protein liquid chromatography 0.5-ml fractions (0.35 ml/min) lysates were immunoprecipitated with the appropriate antibody for were collected. The void volume of the column was 8.3 ml. Molecular 3–16 h at 4 °C. Complexes bound to protein A-Sepharose were washed weight markers (Sigma) were subjected to gel filtration separately and 4 times with hypotonic buffer (42) and separated by SDS-PAGE. 1.5 mg monitored by UV absorption and SDS-PAGE analysis. Fractions 1–50 of total cell extract protein was used for immunoprecipitation followed were subjected to SDS-PAGE analysis and Western immunoblotting by Western analysis, and 0.5 mg was used for immunoprecipitation with Cdk4 (Santa Cruz) or cyclin D2 (Santa Cruz) antibodies. Densito- followed by kinase analysis. p27-associated complexes were recovered metric quantitation of the Cdk4 or cyclin D2 band was plotted as a by immunoprecipitation with p27 antibodies (37) and Cdk2-associated percent of maximal immunoreactivity. Radioactivity incorporated into complexes by immunoprecipitation with Cdk2 antibodies (43). Immu- GST-Rb was quantified and plotted as a percentage of maximal nocomplexes were analyzed by Western immunoblot analysis with p27, phosphorylation. Cdk4 (Pharmigen), or Cdk6 (Santa Cruz) antibodies by standard tech- niques. Total cell extract was subjected to SDS-PAGE analysis, followed RESULTS by Western immunoblot analysis with p27 or Rb antibodies (Pharmi- gen). Immunocomplexes were assayed for Rb kinase activity as de- Cdk4 Resistance to Inhibition by p27 in Vivo—We compared scribed previously (42). Where indicated, histone H1 was used as a the ability of p27 to inhibit Cdk2 and Cdk4 in vivo by examin- substrate instead of GST-Rb C-terminal domain (pRb amino acids 773– ing its interaction with these Cdks in the Tet-p27 cell line. 928). For immunodepletion, extracts were subjected to four cycles of Tet-p27 is a derivative of the mink lung epithelial cell line immunoprecipitation with Cdk4 and Cdk6 antibodies or to four cycles (Mv1Lu) that expresses human p27 under the control of a with normal rabbit serum. The depleted extract was then immunopre- cipitated with p27 antibodies, split, and assayed by Western immuno- tetracycline transactivator (37, 48). In media containing a high blotting with Cdk4 and Cdk6 antibodies, and assayed for Rb kinase concentration of tetracycline (1 mg/ml), Tet-p27 cells do not activity. Parallel cell cultures were assayed in triplicate for I-de- express exogenous p27, and their basal level of endogenous p27 oxyuridine incorporation (44) after 18 h of incubation in the indicated is similar to the level in parental Mv1Lu cells (37). In prolifer- tetracycline concentrations. Data are averages of triplicate determina- ating Mv1Lu and Tet-p27 cells, p27 is bound to cyclin D-Cdk4 tions and are plotted as percentage relative to the cpm incorporated in (Fig. 1c) and, to a lesser extent, cyclin D-Cdk6 (Fig. 1d) and the presence of 1 mg/ml tetracycline. In Vitro Kinase Assays—Fixed amounts of insect cell lysates contain- cyclin A-Cdk2 complexes (Fig. 1e). By lowering the tetracycline ing baculovirally expressed cyclin A and Cdk2 or cyclin D2 and Cdk4 concentration, the p27 level in Tet-p27 cells can be gradually (45) were incubated with the indicated concentrations of bacterially increased up to 25-fold over basal (Fig. 1b), with a concomitant expressed p27 or p21 and assayed (18) in kinase buffer (20 mM Tris- increase in the level of p27-bound Cdk2 or Cdk2-bound p27 HCl, pH 7.5, 7.5 mM MgCl ), containing 30 mM ATP, 5 mCi of [g- P]ATP (Fig. 1e; Ref. 37), a complete loss of Cdk2-associated histone H1 and a GST-Rb fusion protein bound to glutathione-agarose beads. Ra- kinase activity and Rb kinase activity (Fig. 1, f and g), and dioactivity incorporated into GST-Rb was quantified and plotted as a percentage relative to controls without p27. The baculoviral cyclin-Cdk inhibition of DNA synthesis (Fig. 1a; Ref. 37). A 6-fold increase complexes were prepared as described (46). The p27 and p21 proteins in p27 levels is sufficient to completely inhibit Cdk2-associated were tagged at their C termini with a hexahistidine sequence and kinase activity on both substrates (Fig. 1, f and g). As cyclin purified from Escherichia coli as described (14). Cdk2-associated com- E-Cdk2 levels remain constant during this treatment, the loss plexes were recovered by immunoprecipitation either with Cdk2 anti- of Cdk2 kinase activity is due to its increased association with bodies or via a HA epitope tag at the C terminus of Cdk2 (46), and Cdk4 p27. p27 immunoprecipitates did not contain histone H1 kinase or cyclin A-associated complexes with Cdk4 (Santa Cruz) or cyclin A (Santa Cruz) antibodies, respectively. p27-associated complexes were activity even when derived from cells not overexpressing p27 recovered by immunoprecipitation with p27 antibodies (37), and p21- (Fig. 1i), in which the level of Cdk2-associated kinase activity associated complexes were recovered by immunoprecipitation with p21 was high. Among G Cdks, Cdk2, Cdk4, and its close isoform antibodies (Santa Cruz). Immunoprecipitations were performed in mod- Cdk6, all have Rb kinase activity in vitro, whereas only Cdk2 ified LSLD buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 10% glycerol, 0. has histone H1 kinase activity (49). This suggested that p27- 1% Tween 20, 80 mM b-glycerophosphate, 1 mM phenylmethylsulfonyl associated Cdk2 is largely inactive, an observation consistent fluoride, 5 mg/ml aprotinin, 10 mg/ml antipain, 10 mg/ml leupeptin, 100 mg/ml soybean trypsin inhibitor, 100 mg/ml benzamidine) (37), with the with previous results (23, 41). indicated antibody for 1–2 h at 4 °C with gentle agitation. Immunocom- A marked increase in p27 levels led only to a small (less than plexes were assayed for kinase activity as described above. The same 2-fold) increase in the amount of p27-bound Cdk4 in the Tet- antibodies and cyclin D2 antibodies (Santa Cruz) were used for Western p27 cells (Fig. 1c), suggesting that most of the cyclin D-Cdk4 immunoblotting. The amount of complex used in the specific activity present in proliferating Mv1Lu cells is already bound to p27. A assays was normalized by immunoblotting for the component that was larger increase was observed in the level of p27-bound Cdk6 not used for immunoprecipitation. Cak Activation—Cyclin D2-Cdk4 complexes expressed in insect cell (Fig. 1d), indicating an interesting difference in the ability of lysates were immunoprecipitated with p27 or Cdk4 antibodies. The Cdk4 and Cdk6 to interact with p27. p27 immunoprecipitated immunocomplexes were incubated in a 50-ml reaction volume of Cak from Tet-p27 cells was associated with Rb kinase activity (Fig. activation buffer (50 mM HEPES, pH 7.4, 15 mM MgCl ,20mM EGTA, 5mM dithiothreitol, 80 mM b-glycerophosphate, 1 mM phenylmethylsul- fonyl fluoride, 5 mg/ml aprotinin, 10 mg/ml antipain, 10 mg/ml leupeptin, I. Reynisdo ´ ttir and J. Massague ´ , unpublished results. Kip1 p27 Inhibition of Cdk2 and Cdk4 25865 in these cells. However, immunodepletion of both kinases from cell lysates prevented the subsequent recovery of p27-associ- ated Rb kinase activity (Fig. 1k), 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 pre- cipitated from cells that contained enough p27 to cause a com- plete inhibition of Cdk2 (Fig. 1, f and i, lanes 2–7). The presence of Rb kinase activity in p27 complexes from these cells corre- lated 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 anti- bodies (Fig. 1h). 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 grow- ing 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. Different Susceptibility of Cdk2 and Cdk4 to Inhibition by p27 in Vitro—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 (46). 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 exoge- nous 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 visu- alization of p27- and Cdk-associated complexes by immunoblot- ting 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. 2a). The p27 FIG.1. Analysis of Cdk2- and p27-associated kinase activity in inhibition profile was also the same when Cdk immunoprecipi- p27 inducible Mv1Lu cells. Tet-p27 cells were grown in media con- tated complexes were assayed (Fig. 3b). taining the indicated concentration of tetracycline for 18 h before they An important qualitative difference between Cdk2 and Cdk4 were assayed as follows. a,[ I]iododeoxyuridine incorporation into complexes was observed when the extent of Cdk inhibition by DNA at the end of the incubation (data are averages of triplicate determinations and are plotted as percent relative to the incorporation p27 was compared with the extent of Cdk association with p27 in the presence of 1 mg/ml tetracycline). b, immunoblotting of total cell (Fig. 2, b and d). With cyclin A-Cdk2, the inhibition of kinase extract (100 mg of protein) with p27 antibodies. c–e, p27 immunopre- activity was proportional to p27 binding; Cdk2 inhibition and cipitation followed by immunoblotting with anti-Cdk4 (c), anti-Cdk6 binding to p27 were both half-maximal at similar p27 concen- (d), or anti-Cdk2 (e). For reasons unknown, the recovery of p27-bound Cdk was low in extracts from cells incubated without tetracycline (data trations. In contrast to this proportionality, p27 did not signif- not shown). f and g, Cdk2 immunoprecipitation followed by histone H1 icantly inhibit Cdk4 until it bound a near-maximal amount of (f)orRb(g) kinase assays. h, immunoblotting of total cell extract (200 Cdk4 (Fig. 2, a, b, and d). The concentration of p27 required for phos mg of protein) with Rb antiserum. Rb indicates the hyperphospho- maximal Cdk4 inhibition was approximately 4-fold higher than rylated form of Rb. i and j, p27 immunoprecipitation followed by histone H1 (i)orRb(j) kinase assays. NRS, normal rabbit serum. Cdk2, Cdk2 the concentration of p27 required for a maximal level of Cdk4 immunoprecipitation, followed by kinase assay. k, left, in a separate binding. When separately expressed, cyclin D2 and Cdk4 experiment, Mv1Lu cell extracts were subjected to four cycles of immu- bound to p27 very weakly compared with the cyclin D2-Cdk4 nodepletion with Cdk4 and Cdk6 antisera. These extracts were then complex (data not shown). Under our experimental conditions, immunoprecipitated with p27 antiserum (lane 5), followed by Cdk6 and Cdk4 immunoblotting. Lanes 1– 4 are the immunocomplexes from each p27 did not promote the assembly of cyclin D2-Cdk4 complexes depletion round, analyzed by Cdk4 and Cdk6 immunoblotting. k, right, from free components as reported by others (50), as a constant p27 was immunoprecipitated from lysates that had been subjected to amount of cyclin D2 was bound to Cdk4 in the presence of four cycles of immunodepletion with Cdk4/Cdk6 or normal rabbit se- increasing amounts of p27 (Fig. 2c). Thus, whereas the cyclin rum. The Rb kinase activity associated with the p27 immunocomplexes was then determined. A-Cdk2 complex is inhibited if p27 is bound, p27 can bind to cyclin D2-Cdk4 and not cause significant inhibition. 1j), which declined only partially at the highest p27 concentra- Different Specific Activity of p27-bound Cdk2 and Cdk4 — tions. Due to a lack of suitable antibodies against the mink These results were consistent with the observation made with proteins (the available immunprecipitating antibodies do not Tet-p27 cells that once bound, p27 may inhibit cyclin D-Cdk4 yield catalytically active complexes), it was not possible to less effectively than it inhibits cyclin A-Cdk2. To confirm this directly assay cyclin D- or Cdk4/6-associated Rb kinase activity phenomenon, we immunoprecipitated p27-cyclin-Cdk com- Kip1 25866 p27 Inhibition of Cdk2 and Cdk4 FIG.2. In vitro inhibition of cyclin A-Cdk2 and cyclin D2-Cdk4 complexes. a, inhibition of cyclin A-Cdk2 (left) and cyclin D2-Cdk4 (right) Rb kinase activity by p27. The indicated low range (top scale) and high range (bottom scale) of p27 concentrations were added to low or high concentrations of cyclin A-Cdk2 and cyclin D2-Cdk4, respectively, and assayed for phosphorylation of GST-Rb. Radioactivity incorporated into GST-Rb was quantified and plotted as a percentage relative to controls without p27 and is the average of four separate experiments. The lower range of kinase was inhibited at p27 concentrations similar to those previously published (12, 14, 15). The profile of inhibition between the different concentrations of kinase was the same, enabling the higher concentration range to be used in immunoblot analysis of the complexes in b. b, the amount of p27-bound Cdk2 or Cdk4 at the high concentration range was determined by immunoblot analysis of p27 immunoprecipitates with Cdk2 (left) and Cdk4 (right) antiserum. c, the amount of Cdk2-bound cyclin A (left) or Cdk4-bound cyclin D2 (right) at the high concentration range in the presence of increasing concentrations of p27 was determined by immunoblot analysis of Cdk2 or Cdk4 immunoprecipitates with cyclin A (left) and cyclin D2 (right) antiserum. d, inhibition of cyclin A-Cdk2 and cyclin D2-Cdk4 kinase activity by p27 (Rb phosphorylation) plotted against the amount of p27-bound Cdk2 or Cdk4 detected at different concentrations of p27. Rb phosphorylation data taken from a (closed squares and circles) and densitometric quantitation of the Cdk2 or Cdk4 band in b (bars) are shown in the same panel for comparison. The p27-bound Cdk2 and Cdk4 are expressed as a percent of the maximal p27 association with each respective complex. plexes using anti-p27 antibodies and assayed their Rb kinase (data not shown) and required the presence of both cyclin A and activity (Fig. 3). The p27-cyclin D2-Cdk4 complexes had Rb Cdk2. Interestingly, cyclin D2-Cdk4 did not phosphorylate p27. kinase activity (Fig. 3c) when obtained from mixtures that were p27 phosphorylation by Cdk2 was partially inhibited byaTto not fully inhibited by the added p27 (Fig. 3, a and b, middle A mutation in the Cdk consensus site TPKK present near the C lanes). As the amount of added p27 was increased, so did the terminus of p27 (14) (data not shown). We did not determine amount of p27-bound cyclin D2-Cdk4 and p27-associated Rb the stoichiometry of the p27 phosphorylation, and thus it may kinase activity. However, a p27 concentration was eventually constitute a small percentage of the total bound p27. reached beyond which p27 inhibited the Cdk4-associated Rb The presence of kinase activity in a p27-cyclin A-Cdk2 com- kinase activity (Fig. 3, a and b and Fig. 2a). This pattern is plex was in discordance with our observation that Cdk2 inhi- consistent with the phenomenon illustrated in Fig. 2: p27- bition is proportional to p27 binding. This discrepancy was associated kinase activity increases in direct proportion to the resolved by comparing the kinase activity of free and p27- amount of p27-bound Cdk4 up to a certain level after which the bound cyclin A-Cdk2. When normalized by Western immuno- amount of p27-associated Cdk4 plateaus and p27-associated blotting with anti-Cdk2 antibodies, p27-bound cyclin A-Cdk2 kinase activity decreases. was found to be 50-fold less active than free cyclin A-Cdk2, as Weak kinase activity was also present in the p27-cyclin visualized by different film exposures of the same gel (Fig. 4a A-Cdk2 complex as manifested by its ability to phosphorylate (left panel) and data not shown). p27-cyclin A-Cdk2 complexes not only Rb but also the associated p27 (Fig. 3c). p27 phospho- generated in the presence of 10, 20, or 40 nM p27 all had the rylation was observed in kinase assays using complexes pre- same low specific activity (data not shown). The three-dimen- cipitated with p27 antibodies (Fig. 3c) or with Cdk2 antibodies sional structure of the p27-cyclin A-Cdk2 complex reveals that Kip1 p27 Inhibition of Cdk2 and Cdk4 25867 FIG.3. Rb kinase activity of cyclin A-Cdk2 and cyclin D2-Cdk4 in the presence of p27. Activity was deter- mined by analyzing Rb kinase activity of the incubation mixtures in solution (a), immunocomplexes after precipitation with Cdk antibodies (b), or after precipi- tation with p27 antibodies (c). The posi- tion of phosphorylated p27 is noted in c. The higher concentrations of kinase and inhibitor were used in these assays. separate regions of p27 interact with the cyclin and the Cdk with insect cell lysates, we wanted to eliminate possible inter- (23). On the Cdk, p27 interacts with the active site in a manner ference of cyclin D2-Cdk4 aggregates or free components in the that is incompatible with catalytic activity. Although p27 could reactions. To this end, we subjected the lysates to gel filtration bind to cyclin A without contacting the Cdk2 subunit, the chromatography on Superose 12 and analyzed fractions by structure argues that Cdk2 inhibition is strongly favored upon anti-cyclin D2 immunoblotting, anti-Cdk4 immunoblotting, p27 binding to the cyclin subunit (23). This is consistent with and Rb kinase assays (Fig. 5, a and b). To determine the elution our result that the p27-cyclin A-Cdk2 complex is largely profile of free cyclin D2 and Cdk4, we separately subjected inactive. cyclin D2 and Cdk4 proteins produced by single baculovirus The opposite result was obtained with the p27-cyclin D2- infections to gel filtration. When cyclin D2 was expressed Cdk4 complex. When normalized by Western immunoblotting alone, the majority of the protein migrated as an aggregate with anti-cyclin D2 antibodies, this complex retained a level of (.440 kDa) in the void volume (Fig. 5, a and b; void volume of kinase activity that was comparable to that of free cyclin D2- 8.3 ml ended at fraction 16). Less than 10% of the total cyclin Cdk4 (Fig. 4a, right panel). This was confirmed by performing D2 eluted at the size expected of free cyclin. On the contrary, a time course of Rb phosphorylation with equal amounts of free Cdk4 eluted at the expected size in fraction 27. Cyclin D2 or or p27-associated cyclin D2-Cdk4 complex (Fig. 4b). To rule out Cdk4 expressed alone had no significant Rb kinase activity the possibility that the Cdk4 antibody itself was inhibitory, we (data not shown). When derived from coinfected lysate, both compared the kinase activity of free and antibody-bound ki- cyclin D2 and Cdk4 eluted with the size expected of the binary nases (Fig. 4c). When normalized by Western immunoblotting complex (66 kDa), in fraction 25, and coeluted with peak Rb with anti-cyclin D2 antibodies, antibody-bound cyclin D2-Cdk4 kinase activity (Fig. 5, a and b). Other proteins were present in was as active as free cyclin D2-Cdk4. p27 was not dissociating this fraction, as judged by SDS-PAGE and Coomassie Blue from the cyclin D2-Cdk4 complex during the kinase assay, as staining (data not shown). Some monomeric Cdk4 was detected analysis of the supernatant following the assay did not reveal in the coinfected lysates, but this limited amount was separa- the presence of free cyclin D2 or Cdk4 (data not shown). Col- ble from the binary complex. Note that all of the cyclin D2 lectively, these results suggest that p27 binding to cyclin A- appeared in the fractions containing the binary complex, sug- Cdk2 results in a 50-fold inhibition of this complex, whereas gesting that cyclin D2 expression was limiting for cyclin D2- under similar conditions, p27 can bind to cyclin D2-Cdk4 with- Cdk4 complex formation in our insect cell infection conditions, out causing a discernible decrease in the Rb kinase activity of and suggesting also that cyclin D2 did not form aggregates this complex. when bound to Cdk4. Importantly, addition of p27 to the cyclin To determine if the ability of p27 to bind to cyclin D2-Cdk4 D2-Cdk4 sample followed by analysis of the resulting p27- complexes without causing inhibition could be extended to cyclin D2-Cdk4 complexes demonstrated that these complexes other Cip/Kip inhibitors, we analyzed p21 in a similar assay had Rb kinase activity (Fig. 5c). When normalized by immuno- (Fig. 4d), using non-inhibitory concentrations of p21 for cyclin blotting with anti-cyclin D2 antibodies, the p27-cyclin D2-Cdk4 A-Cdk2 or cyclin D2-Cdk4, respectively (data not shown). complex formed at low p27 concentrations had a level of kinase When normalized by Western immunoblotting with anti-Cdk2 activity comparable to that of the cyclin D2-Cdk4 complex (Fig. antibodies, the p21-cyclin A-Cdk2 complex only retained 25% of 5c). This confirmed that the presence of Rb kinase activity in the activity observed with the free complex (Fig. 4d, left panel), p27-cyclin D2-Cdk4 was intrinsic to this ternary complex and consistent with previous results (33, 51). Thus, while this com- not the result of p27 binding to cyclin D2-Cdk4 aggregates. plex has kinase activity, it is significantly impaired as com- Activation of p27-cyclin D2-Cdk4 by Cak—In addition to the pared with the free complex. However, when normalized by effect of p27 on activated cyclin-Cdk complexes, it has been Western immunoblotting with anti-cyclin D2 antibodies, p21- reported that p27 binding may prevent activation of these bound cyclin D2-Cdk4 was found to be about 30-fold less active complexes by the kinase Cak (14, 25). To investigate whether than free cyclin D2-Cdk4 (Fig. 4d, right panel). p21 association p27 binding has this effect on cyclin D2-Cdk4 in vitro,we with cyclin D2-Cdk4 in vitro appears to cause inhibition of this examined the ability of Cak (cyclin H-Cdk7) (52–54) to activate complex at all concentrations and thus is more similar to the cyclin D2-Cdk4 in the presence or absence of p27. Baculovirally p27-cyclin A-Cdk2 interaction. Therefore, the ability of p27 to expressed cyclin D2-Cdk4 and cyclin A-Cdk2 are not fully ac- bind to cyclin D2-Cdk4 in a non-inhibitory fashion does not tivated, presumably because of insufficient Cak activity in the appear to be a hallmark of all Cip/Kip inhibitors but rather is insect cells. Cyclin D2-Cdk4 preparations were incubated with specific for the p27-cyclin D2-Cdk4 interaction. no p27, a low, non-inhibitory concentration of p27, or a high, Analysis of Cyclin D2-Cdk4 Complexes Purified by Gel Fil- inhibitory concentration of p27 (Fig. 6; refer to Fig. 2). Aliquots tration—As the above described experiments were performed of these mixtures were precipitated with anti-Cdk4 or anti-p27 Kip1 25868 p27 Inhibition of Cdk2 and Cdk4 antibodies and the immunocomplexes incubated with or with- out purified Cak. Incubation with Cak increased 10-fold the Rb kinase activity of Cdk4 complexes (Fig. 6; compare top lanes 1 and 4), suggesting that a significant fraction of the original complexes had not undergone activation by Cak. A similar 10-fold increase in Rb kinase activity was seen when cyclin A-Cdk2 complexes were treated with Cak (data not shown). p27 complexes formed with a high concentration of p27 lacked Rb kinase activity and did not gain activity upon incubation with Cak (Fig. 6), either because this high concentration of p27 prevented activation by Cak or inhibited Cak-activated com- plexes. However, incubation with a low concentration of p27 did not decrease Cdk4 activation by Cak (Fig. 6, compare top lanes 2 and 5). Furthermore, the Rb kinase activity of the p27 complexes formed under these conditions was also increased 10-fold when these complexes were incubated with Cak (Fig. 6; compare bottom lanes 2 and 5). Thus, p27 can bind to cyclin D2-Cdk4 in a manner that interferes neither with the activa- tion of this complex by Cak nor with the Rb kinase activity of the activated complex. Cdk4 Inhibition by Supra-stoichiometric Binding of p27— Although it appeared that p27-cyclin D2-Cdk4 complexes could be active both in vivo and in vitro, the above results also suggested that two different types of p27-cyclin D2-Cdk4 com- plexes can be formed in vitro, one having Rb kinase activity and the other, which is obtained at higher p27 concentrations, lacking Rb kinase activity. Therefore, we analyzed the compo- sition of the inactive p27-cyclin D2-Cdk4 complexes formed at high concentrations of p27 in vitro. We determined the levels of p27-bound cyclin D2 and Cdk4-bound cyclin D2 and p27 achieved over a range of p27 concentrations added to a fixed amount of cyclin D2 and Cdk4 (Fig. 7a). p27 did not promote assembly of cyclin D2-Cdk4 complexes, as Cdk4 bound cyclin D2 was constant in the presence of increasing p27 concentra- tions (Fig. 7a, top panel). The level of Cdk4-associated p27 (Fig. 7a, middel panel) continued to increase well after the level of p27-associated cyclin D2 (Fig. 7a, bottom panel) had reached a plateau. This suggested that the same amount of cyclin D2- Cdk4 was binding more p27. The progressive increase in p27 binding to Cdk4 (Fig. 7a, middle) eventually reached a plateau when complete inhibition of Cdk4 was achieved (refer to Fig. 2a). The simplest interpretation of this phenomenon is that a p27-cyclin D2-Cdk4 complex can become inhibited in vitro by binding additional p27 molecules. A similar conclusion was FIG.4. Kinase activity of free and p27-bound Cdk2 and Cdk4 previously reached in studies on the related Cdk inhibitor Cip1/Waf1 complexes. a, left panel, an amount of p27-bound cyclin A-Cdk2 p21 which can form p21-cyclin A-Cdk2 complexes that (formed with 20 nM p27) was compared with the same amount of the retain kinase activity (33, 51). To obtain direct evidence for the corresponding p27-free cyclin-Cdk complex. The free cyclin A-Cdk2 binding of multiple p27 molecules to a cyclin D2-Cdk4 complex, complexes was isolated by immunoprecipitation of cyclin A. Western immunoblot analysis of the component that was not used for immuno- we used two recombinant forms of p27, one tagged with the Flag precipitation (Cdk2)(bottom) served as a measure of the amount of Flag epitope (p27 ) and the other tagged with hexahistidine complex present in the kinase reactions (top). a, right panel, an amount His His (p27 ). p27 -cyclin D2-Cdk4 complexes were formed at in- of p27-bound cyclin D2-Cdk4 (formed with 160 nM p27) was compared His creasing concentrations of p27 and isolated by binding to with the same amount of the corresponding p27-free cyclin-Cdk com- metal-agarose beads. The ability of these immobilized com- plex. The free cyclin D2-Cdk4 complex was isolated by immunoprecipi- tation of Cdk4. Western immunoblot analysis of the component that plexes to bind additional p27 was then assayed by incubation was not used for immunoprecipitation (cyc D2)(bottom) served as a Flag with a fixed concentration of p27 followed by detection by measure of the amount of complex present in the kinase reactions (top). His anti-Flag Western immunoblotting (Fig. 7b). p27 -cyclin D2- b, equivalent amounts of free or p27-bound cyclin D2-Cdk4 complexes Cdk4 complexes formed in the presence of low concentrations of (normalized by cyclin D2 immunoblotting as in a) were subjected to Rb His Flag kinase assays for the indicated times. c, an amount of Cdk4 antibody- p27 were able to bind to p27 , whereas complexes formed bound cyclin D2-Cdk4 was compared with the same amount of anti- His at higher concentrations of p27 were not (Fig. 7b). The abil- body-free cyclin D2-Cdk4 complex. Western immunoblot analysis of ity of an additional molecule of p27 to bind to the immunopre- cyclin D2 (bottom) served as a measure of the amount of complex present in the kinase reactions (top). The cyclin D2-Cdk4 used in this panel was isolated from free components by gel filtration as described in with the same amount of the corresponding p21-free cyclin-Cdk com- Fig. 7. d, left panel, an amount of p21-bound cyclin A-Cdk2 (formed with 20 nM p27) was compared with the same amount of the corresponding plex. The free cyclin D2-Cdk4 complex was isolated by immunoprecipi- tation of Cdk4. As above, Western immunoblot analysis of the compo- p21-free cyclin-Cdk complex. The free cyclin A-Cdk2 complexes were isolated by immunoprecipitation of cyclin A. d, right panel, an amount nent that was not used for immunoprecipitation (bottom) served as a measure of the amount of complex present in the kinase reactions (top). of p21-bound cyclin D2-Cdk4 (formed with 250 nM p21) was compared Kip1 p27 Inhibition of Cdk2 and Cdk4 25869 FIG.5. Activity of cyclin D2-Cdk4 complexes purified by gel filtration. a, fractions from gel filtration of Cdk4 alone, cyclin D2 alone, or cyclin D2-Cdk4 from coinfected baculoviral extracts were analyzed by Western immunoblot analysis with Cdk4 or cyclin D2 antibodies. Fractions were assayed for phosphorylation of GST-Rb. b, densitometric quantitation of the Cdk4 or cyclin D2 band in a was plotted as a percent of maximal immunoreactivity. Open circles correspond to immunoreactivity from Cdk4 or cyclin D2 from single baculoviral infections, and closed circles correspond to immunoreactivity detected in fractions from coinfected cyclin D2-Cdk4. Radioactivity incorporated into GST-Rb by cyclin D2-Cdk4 coinfected complexes was quantified and plotted as a percentage of maximal phosphorylation (closed triangles). Molecular mass standards are indicated at the top of the panel. c, left, Rb kinase activity of cyclin D2-Cdk4 complexes partially purified by gel filtration in the presence of p27, as determined by analyzing Rb kinase activity in solution (top) or after precipitation with p27 antibodies (middle). The amount of Cdk4-bound cyclin D2 in the presence of p27 was determined by immunoblot analysis of Cdk4 immunoprecipitates with cyclin D2 antiserum (bottom). c, right, an amount of p27-bound cyclin D2-Cdk4 was compared with the same amount of the corresponding p27-free cyclin-Cdk complex, isolated by immunoprecipitation of Cdk4. Western immunoblot analysis of the component that was not used for immunoprecipitation (cyclin D2) (bottom) served as a measure of the amount of complex present in the kinase reactions (top). crystallographic studies (23) appears to be inhibited by binding of a single p27 molecule, an active p27-cyclin D2-Cdk4 can bind and be inhibited by an additional p27 molecule under our in vitro assay conditions. DISCUSSION The ability of p27 and related proteins to act as Cdk inhibi- tors and as repressors of cell proliferation is well established. Overexpression of p27 leads to cell cycle arrest (14, 15), and antisense inhibition of p27 expression can prevent quiescence upon growth factor withdrawal (55, 56). p27 levels increase during cell cycle arrest in response to cell-cell contact (34), growth inhibitory agents (25–27) inducers of terminal differen- tiation (38, 57), loss of anchorage (39, 40), and during neuronal FIG.6. Cak activation of cyclin D2-Cdk4 complexes. Cyclin D2- differentiation in vivo (29). Further evidence for a physiological Cdk4 insect cell lysates were incubated with the indicated concentra- role of p27 as a negative regulator of growth is provided by the tions of p27 and immunoprecipitated with Cdk4 antiserum (top)orp27 phenotype of generalized organomegaly and increased body antiserum (bottom). The precipitates were incubated with or without size observed in p27 null mice (58 – 60). Cak (cyclin H-Cdk7) before they were assayed for Rb kinase activity. Targets of this inhibitory action of p27 include the cyclin- cipitated complexes only occurred within those complexes that Cdk2 complexes. p27 inhibits cyclin E-Cdk2, whose function is essential for G progression, and also cyclin A-Cdk2, whose contained submaximal Cdk4 binding (Fig. 7b, lanes 2– 4). Im- His Flag mobilized p27 alone did not bind p27 (data not shown), function is essential in S phase (14, 15, 18, 20, 33, 51). The Flag indicating that under these conditions, p27 binding was three-dimensional structure of the p27-cyclin A-Cdk2 complex His mediated by p27 -associated cyclin D2-Cdk4. Thus, although formed in vitro shows that one molecule of p27 bound to this the cyclin A-Cdk2 complex in Tet-p27 cells, in vitro, and in complex interacts with the Cdk subunit in a manner that Kip1 25870 p27 Inhibition of Cdk2 and Cdk4 FIG.7. Inhibition of cyclin D2-Cdk4 by binding of multiple p27 molecules. a, levels of Cdk4-associated cyclin D2 (top), Cdk4-associated p27 (middle), and p27-associated cyclin D2 (bottom) after incubation of a fixed concentration of cyclin D2-Cdk4 with increasing concentrations of p27. At the end of the incubations, the mixtures were immunoprecipitated with p27 antiserum, followed by cyclin D2 Western immunoblotting, or immunoprecipitated with Cdk4 antiserum followed by cyclin D2 or p27 Western immunoblotting. Immunoprecipitations were always performed in antibody excess. Densitometric quantitation of the Cdk4-associated p27 (middle) and p27-associated cyclin D2 (bottom) bands are plotted in the His same panel for comparison. b, as depicted in the scheme, cyclin D2-Cdk4 was incubated with increasing concentrations of p27 followed by binding His Flag to metal-agarose beads to isolate p27-associated complexes. To these immobilized p27 -cyclin D2-Cdk4 complexes, 260 nM p27 was added, Flag followed by extensive washing. Levels of bound p27 and Cdk4 were determined by Flag and Cdk4 Western immunoblotting. precludes catalytic activity (23). In the cell, even a modest be possible to raise the level of exogenous p27 high enough to increase in p27 levels can completely inhibit all measurable inhibit cyclin D-Cdk4, this concentration may not normally be Cdk2 kinase activity (Refs. 37, 41, 61, and present work). available in the parental cells. We therefore conclude that in Furthermore, a p27-cyclin A-Cdk2 complex produced in vitro is these cells p27 normally acts as a cyclin A-Cdk2 inhibitor but 50-fold less active as an Rb kinase than a cyclin A-Cdk2 com- not as a cyclin D-Cdk4/6 inhibitor. The latter role may fall on plex. These present and previous results therefore support the the Ink4 family of selective Cdk4 inhibitors (4, 5, 37, 43). conclusion that Cdk2 complexes, and the cyclin A-Cdk2 com- The results of our experiments using recombinant proteins plex in particular, are prime targets for inhibition by p27. provide further evidence that p27 interacts with and inhibits The Cdk inhibitory activity of p27 extends to cyclin D-de- cyclin D-Cdk4 and cyclin A-Cdk2 differently. p27-cyclin A- pendent kinases. Cell cycle entry in macrophages is prevented Cdk2 complexes are essentially inactive, whereas p27-cyclin by cyclic AMP which maintains the high level of p27 present in D2-Cdk4 complexes formed at low concentrations of p27 retain the quiescent state and inhibits cyclin D-Cdk4 activation by Rb kinase activity. Furthermore, the specific activity of these Cak (25). Transient transfection of p27 in U2OS human tumor p27-cyclin D2-Cdk4 complexes is similar to that of cyclin D2- cells causes inhibition of cotransfected cyclin D1-Cdk4 (50). p27 Cdk4. As obtained from baculovirally infected insect lysates, can inhibit cyclin D-Cdk prepared from recombinant sources cyclin D2-Cdk4 is not fully activated by Cak since a 10-fold (14, 15, 33, and present work). This notwithstanding, recent further activation is achieved by incubation with Cak in vitro. reports and the present results provide evidence that in other A similar increase in activity is obtained by incubation of cell types or under other in vitro conditions, p27 is not an p27-cyclin D2-Cdk4 complexes with Cak, implying that p27- effective inhibitor of cyclin D-Cdk4 even though it binds to this cyclin D2-Cdk4 not only remains active but it also remains complex. In proliferating mink lung epithelial cells, human susceptible to Cdk4 activation by Cak. keratinocytes (37), mouse fibroblasts (15, 28), rat fibroblasts The p27-cyclin A-Cdk2 complex contains residual kinase ac- (61), and Manca B cell lymphoma (41), cyclin D-Cdk4 is asso- tivity and can phosphorylate the associated p27 at a Cdk con- ciated with a large proportion of the p27 present in the cell. sensus site. This activity may result from an incomplete block Further evidence comes from the present analysis of the effect of the Cdk2 binding site by bound p27. However, while meas- of p27 on cyclin D-dependent kinases in the Tet-p27 inducible urable, the level of kinase activity in p27-cyclin A-Cdk2 com- cell line. In these cells, p27 levels that cause full inhibition of plexes appears negligible when compared with the activity of Cdk2 fail to inhibit Cdk4/6. Tet-p27 cells that have been free cyclin A-Cdk2. Yet, the underlying phenomenon, incom- growth-arrested by p27 with full inhibition of Cdk2 still yield plete inhibition of a cyclin-Cdk by bound p27, is a prevalent high levels of p27-associated Rb kinase activity and contain feature in the interaction of p27 with cyclin D2-Cdk4. Higher phosphorylated Rb protein. The p27-associated Rb kinase ac- concentrations of p27 can inhibit cyclin D2-Cdk4 complexes tivity under these conditions is attributable to bound Cdk4 and (12, 14, 15, and present work). Under our conditions, this Cdk6, as determined by Cdk4/6 immunodepletion experiments. appears to involve the conversion of an active p27-cyclin D2- The concentration of p27 needed to inhibit cyclin D-Cdk4/6 in Cdk4 complex into an inactive one by the binding of additional Tet-p27 cells is significantly higher than that needed to inhibit p27 molecules. A similar conclusion was previously reached in Cip1/Waf1 Cdk2. Although under certain experimental conditions it may studies on the related Cdk inhibitor p21 which can Kip1 p27 Inhibition of Cdk2 and Cdk4 25871 may represent two extremes of the interaction between a Cip/ Kip inhibitor and a cyclin-Cdk complex. Each individual inter- action of p21, p27, and p57 with various cyclin-Cdks may be characterized by a different balance between the two states represented in Fig. 8. Indeed, p21-cyclin A-Cdk2 complexes are not completely inactive but retain approximately 25% of the activity seen with the free cyclin A-Cdk2 complex, consistent with previous results (33, 51). However, p21 association with cyclin D2-Cdk4 in vitro appears to cause extensive inhibition of this complex at all concentrations, consistent with the in vivo observations reported by others (64, 65) concerning the ability of p21 to inhibit cyclin D-Cdk4 complexes. Thus, the p21-cyclin D2-Cdk4 complex is more similar to the p27-cyclin A-Cdk2 interaction. Our present results stand in contrast with the previously observed ability of p27 to inhibit cyclin D-Cdk4 in macrophages stimulated with agents that raised cyclic AMP levels (25), in FIG.8. Schematic representation of the model for CKI associ- which p27 was able to block Cak activation of cyclin D-Cdk4. ation and inhibition of cyclin A-Cdk2 and cyclin D2-Cdk4 com- One potential explanation for this discrepancy is that in cyclic plexes. In the three-dimensional structure of the p27-cyclin A-Cdk2 complex, p27 anchors on the cyclin via the conserved LFG motif and AMP-stimulated macrophages p27 levels might be sufficiently inserts into the ATP binding site of Cdk2 via the FY region (23). For high to achieve binding of multiple molecules to cyclin D-Cdk4, simplicity, only the cyclin-Cdk interaction domain of p27 is depicted. as observed in our in vitro conditions. Alternatively, an as yet These contacts are schematically represented in State B. Binding of p27 unknown factor might modify the ability of p27 to bind cyclin to the cyclin subunit in State C is not sufficient for Cdk inhibition. This D-Cdk4 in an inhibitory mode. Such a factor might enhance the state is not favored in the p27-cyclin A-Cdk2 complex but may be prevalent in the p27-cyclin D2-Cdk4 complex. As a result, p27-cyclin ability of bound p27 to block the Cdk4 subunit in a p27-cyclin A-Cdk2 complexes are essentially inactive, whereas p27-cyclin D2- D-Cdk4 complex. There is precedent for the idea that diverse Cdk4 complexes are largely active. In contrast, p21-cyclin D2-Cdk4 proteins associate with cyclin D-Cdk4 (42, 66 – 68). complexes are essentially inactive and would be represented by State B. An implication of the present findings is that, in the cell, the Inactivation of a p27-cyclin D2-Cdk4 complex at high p27 concentra- tions involves the binding of additional p27 molecules to State C. Thus, outcome of an interaction between p27 and G /S cyclin-Cdk the relative activity of a CKI-cyclin-Cdk complex would be determined complexes will be determined by the distribution of p27 be- by the type of interaction (State B or C) with the respective CKI. tween inhibitable targets such as cyclin A-Cdk2 and a reservoir such as cyclin D-Cdk4, which sequesters p27 while remaining form p21-cyclin A-Cdk2 complexes that retain kinase activity active. The differential interaction of p27 with cyclin D-Cdk4 (33, 51). Evidence for the conversion of p27-cyclin D2-Cdk4 and cyclin A-Cdk2 may fulfill several purposes. It may help complexes from an active to an inactive form is provided by maintain the two types of Cdk complexes in an active state saturation analysis of these complexes and the use of differ- during periods of growth. It may also allow a concerted inhibi- ently tagged p27 molecules. A similar phenomenon has been tion of cyclin A-Cdk2 by mobilization of p27 from cyclin D-Cdk4 observed in the interaction of p27 with cyclin D2-Cdk6 in complexes. This could occur when the cellular levels of cyclin D vitro. and/or Cdk4 decline upon mitogen deprivation (69 –72), contact A possible basis for these different interactions is provided inhibition (28, 37), or loss of anchorage in non-transformed by the three-dimensional structure of p27 in complex with cells (39, 73). Likewise, a displacement of p27 from cyclin cyclin A-Cdk2. This structure suggests that binding of a single D-Cdk4 complexes by elevated Ink4 inhibitors during a re- molecule of p27 is sufficient for inhibition of Cdk2 kinase ac- sponse to transforming growth factor-b (37, 43) or by other tivity (23), which is consistent with our results. 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Cell 7, 101–111 )
Journal
Journal of Biological Chemistry – Unpaywall
Published: Oct 1, 1997