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The p21Cip1 and p27Kip1 CDK ‘inhibitors’ are essential activators of cyclin D‐dependent kinases in murine fibroblasts

The p21Cip1 and p27Kip1 CDK ‘inhibitors’ are essential activators of cyclin D‐dependent kinases... The EMBO Journal Vol.18 No.6 pp.1571–1583, 1999 Cip1 Kip1 The p21 and p27 CDK ‘inhibitors’ are essential activators of cyclin D-dependent kinases in murine fibroblasts 1 2,3 has not been defined. Finally, proteasomal degradation of Mangeng Cheng , Paul Olivier , 1,3 2,3 cyclin D1 is triggered by its phosphorylation on a single J.Alan Diehl , Matthew Fero , 1 2,3 threonine residue (Thr286) by glycogen synthase kinase- Martine F.Roussel , James M.Roberts and 1,3,4 3β (Diehl et al., 1997, 1998), a process antagonized Charles J.Sherr by signaling through a separate Ras-dependent pathway Department of Tumor Cell Biology, St Jude Children’s Research involving phosphatidylinositol 3-kinase (PI3K) and protein Hospital, 332 N. Lauderdale, Memphis, TN 38105, Division of Basic kinase B (also called Akt) (Boudewijn et al., 1995; Cross Sciences, Fred Hutchinson Cancer Center, Seattle, WA 98104 and et al., 1995; Franke et al., 1995, 1997; Klinghoffer et al., Howard Hughes Medical Institute, USA 1996; Dudek et al., 1997; Vanhaesebroeck et al., 1997). Corresponding author In the continued presence of mitogenic signals, cyclin D1– e-mail: [email protected] CDK4 complexes assemble and accumulate throughout G phase, enter the nucleus and undergo phosphorylation by The widely prevailing view that the cyclin-dependent CDK-activating kinase (CAK) to yield active holoen- kinase inhibitors (CKIs) are solely negative regulators zymes. One key function of the cyclin D-dependent kinases of cyclin-dependent kinases (CDKs) is challenged here is to initiate phosphorylation of the retinoblastoma protein by observations that normal up-regulation of cyclin D– (Rb), thereby helping to cancel its activity as a transcrip- CDK4 in mitogen-stimulated fibroblasts depends tional repressor of a bank of genes, including cyclins E Cip1 Kip1 redundantly upon p21 and p27 . Primary mouse and A, whose activities are required for S phase entry embryonic fibroblasts that lack genes encoding both (Weinberg, 1995; Sherr, 1996). p21 and p27 fail to assemble detectable amounts of A separate, non-catalytic action of cyclin D-dependent cyclin D–CDK complexes, express cyclin D proteins at Kip1 kinases is the sequestration of CKIs, including p27 much reduced levels, and are unable to efficiently Cip1 and p21 (Sherr and Roberts, 1995). The Cip/Kip direct cyclin D proteins to the cell nucleus. Restoration proteins interact with a variety of cyclin–CDK complexes of CKI function reverses all three defects and thereby through a conserved N-terminal domain that contains both restores cyclin D activity to normal physiological levels. cyclin and CDK binding sites (Toyoshima and Hunter, In the absence of both CKIs, the severe reduction in 1994; Chen et al., 1995, 1996; Luo et al., 1995; Nakanishi cyclin D-dependent kinase activity was well tolerated et al., 1995; Lin et al., 1996; Russo et al., 1996). Cyclin and had no overt effects on the cell cycle. Cip1 D-dependent CDKs isolated from mammalian cells appear Keywords: CDK4/cell cycle/D-type cyclins/p21 / Kip1 to be less susceptible to Cip/Kip-mediated inhibition than p27 are other classes of cyclin–CDKs (Soos et al., 1996; Blain et al., 1997; LaBaer et al., 1997), and sequestration of Cip1 Kip1 p21 and p27 into higher order complexes with Introduction cyclin D-dependent kinases during G phase helps to relieve cyclin E–CDK2 from their constraint, thereby Regulation of mammalian cell proliferation by extracellu- facilitating its activation later in G phase. This ability lar mitogens is governed through receptor-mediated sig- 1 to ‘titrate’ CKIs therefore sets a dependency of cyclin naling circuits which ultimately converge on the cell cycle E-dependent kinase on the mitogen-stimulated assembly of machinery driven by cyclin-dependent kinases (CDKs) cyclin D-dependent kinases. Cyclin E–CDK2 collaborates and opposed by CDK inhibitors (CKIs) (Sherr and Roberts, with cyclin D-dependent kinases to phosphorylate Rb 1995). One important example is receptor-activated Ras (Hatakeyama et al., 1994; Mittnacht et al., 1994; Lee signaling, which governs the accumulation of cyclin D1– et al., 1996; Kelly et al., 1998; Lundberg and Weinberg, CDK4 complexes by at least three independent but comple- Kip1 1998), phosphorylates p27 to trigger its degradation mentary pathways: gene transcription, protein association (Sheaff et al., 1997; Vlach et al., 1997), and may target and protein stabilization. First, Ras signaling promotes other proteins whose modifications trigger origin firing transcription of the cyclin D1 gene via a kinase cascade and DNA replication per se (Stillman, 1996; Krude that depends upon the sequential activities of Ras, Raf-1, et al., 1997). mitogen-activated protein kinase kinase (MEK1) and Although it is generally assumed that CKIs act solely mitogen-activated protein kinases (MAPKs), also referred to retard G progression, the fact that they can be found to as extracellular signal-regulated protein kinases (ERKs) in complexes with active cyclin–CDKs (Zhang et al., (Albanese et al., 1995; Lavoie et al., 1996; Winston et al., 1994; Soos et al., 1996; Blain et al., 1997; LaBaer et al., 1996; Aktas et al., 1997; Kerkhoff and Rapp, 1997; Weber 1997) raises the possibility that they may also act as et al., 1997). Signaling through this same pathway is also sufficient to promote assembly of cyclin D1–CDK4 positive regulators. Intriguingly, LaBaer et al. (1997) Cip1 complexes (Cheng et al., 1998), although the physiological demonstrated that p21 could promote the assembly of target of ERK phosphorylation that mediates this process active cyclin D1–CDK4 complexes and, in addition, could © European Molecular Biology Organization 1571 M.Cheng et al. provide a localization signal for their nuclear import. However, the fact that p21 nullizygous mice undergo normal development and do not seem to have a significant deficiency in cyclin D-dependent kinase function (Brugarolas et al., 1995; Deng et al., 1995) leaves open the question of whether the CKIs are normal physiological regulators of cyclin D–CDK assembly. In this study, we have used primary mouse embryo fibroblast (MEF) strains deficient in p21, p27 or both to study their roles in governing the activities of cyclin D–CDK holoenzymes. Results Impaired assembly of cyclin D–CDK4 complexes in MEFs lacking p21 and p27 Cell lysates from asynchronously proliferating MEFs derived from wild-type mice, p21- and p27-null mice and from animals lacking both genes were precipitated with antibodies to cyclin D1 or CDK4. Precipitated proteins were resolved on denaturing polyacrylamide gels, trans- ferred to nitrocellulose membranes and blotted with the cognate antibodies to quantitate cyclin D1 and CDK4 levels, respectively, or with the reciprocal antibodies to score for the presence of cyclin D1–CDK4 complexes (Figure 1A). In early passage (p5) wild-type MEFs, ~40% of the total CDK4 (lane 1, K4 blot) co-precipitated with antibodies to D1 (lane 2, K4 blot). A smaller percentage of the total D1 pool (lane 2, D1 blot) co-precipitated with CDK4 (lane 1, D1 blot). However, antibodies to full-length recombinant CDK4 used in this experiment preferentially detect free versus cyclin D1-bound catalytic subunits, so the amount of D1 detected in CDK4 immunoprecipitates underestimates the extent of complex formation. Generally equivalent levels of CDK4 were expressed in MEF strains lacking one or both CKIs (Figure 1A, lanes 3, 5 and 7). In contrast, the overall levels of cyclin D1 were significantly lowered in cells lacking either p21 or p27 (D1 blot, lanes 6 and 8 versus lane 2) and were decreased at least 10- fold in lysates of cells lacking both CKIs (lane 4). Cyclin Fig. 1. Impaired assembly of cyclin D–CDK4 complexes in MEFs D1–CDK4 complexes were recovered at similarly reduced lacking p21 and p27. (A) Cell lysates (500 μg total protein per lane) levels from lysates of p21-null and p27-null MEFs (lanes from MEFs of the indicated genotypes were immunoprecipitated (IP) 5–8), but at this level of resolution, no cyclin D1–CDK4 with antibodies to CDK4 (K4) or cyclin D1 and the separated proteins were blotted with the cognate or reciprocal antibodies. (B) Cell lysates complexes were detected in immune precipitates from were normalized for cyclin D1 abundance, and D1 immune cells lacking both CKIs (lanes 3 and 4). precipitates were blotted with antibodies to cyclin D1 or CDK4. Two Reduced cyclin D1–CDK4 complex formation in electrophoretic forms of cyclin D1 detected in this experiment can be double-null MEFs may have simply reflected the lower routinely observed when separation conditions are sufficiently stringent (Matsushime et al., 1991); both are phosphoproteins and the nature of levels of cyclin D1 expressed in these cells. However, the differences between them remains unclear (Diehl et al., 1998). several lines of evidence indicate that this is not the (C) Wild-type or p21/p27 double-null MEFs were infected with explanation. First, when cell lysates were normalized so retrovirus encoding Flag-tagged cyclin D1. Cell lysates prepared 48 h that each contained comparable amounts of cyclin D1 post-infection were precipitated with a control monoclonal antibody protein, the levels of CDK4 that co-precipitated with (C) or with antibodies to the Flag epitope (M2), and the separated proteins were blotted with antibodies to cyclin D1 or CDK4. (D) Cell cyclin D1 were again found to be decreased in p21-null lysates from MEFs with the indicated genotypes were precipitated or p27-null cells (Figure 1B, lanes 2 and 3) and were very with antibodies to cyclin D2 and the separated proteins were blotted much reduced in p21/p27 double-null MEFs, whether the with antibodies to cyclin D2 or CDK4. All immunoblots were cells were in early (p7) or late (p15) passage (Figure 1B, visualized using enhanced chemiluminescence. lanes 4 and 6). We estimated that p7 double-null cells contained 10% of the D1–CDK4 complexes detected in age-matched wild-type MEFs. Secondly and more the p21/p27 double-null cells do not account for the defect importantly, infection of p21/p27 double-null MEFs for observed in cyclin D1–CDK4 assembly. 48 h with a retrovirus encoding Flag epitope-tagged cyclin MEFs also express cyclin D2 (Figure 1D, lane 1) but D1 restored high levels of D1 expression but not D1– little detectable cyclin D3 (data not shown). Compared CDK4 complex formation (Figure 1C). This demonstrated with wild-type MEFs, cyclin D2 levels were reduced by directly that the lower levels of cyclin D1 expressed in ~30% in cells lacking either p21 (lane 2) or p27 (lane 3) 1572 CDK inhibitors promote cyclin D–CDK assembly p27 double-null MEFs (lanes 5 and 9), consistent with observations that few cyclin D–CDK4 complexes were formed (Figure 1). Cell lysates from wild-type MEFs were subjected to two rounds of immunodepletion using antisera to p21, p27 or to both, and CDK4 kinase activity was measured using glutathione S-transferase (GST)-Rb as the substrate. Removal of p27 (Figure 2B, lane 3) or p21 (lane 4) from lysates of wild-type MEFs partially reduced CDK4 kinase activity, whereas elimination of both p21 and p27 (lane 5) completely depleted the activity. Similarly, removal of p27 from lysates of p21-null MEFs (lane 7) or vice versa (lane 9) depleted all CDK4 kinase activity from these lysates. Therefore, both p21- and p27-associated cyclin D–CDK complexes retain activity (Zhang et al., 1994; Soos et al., 1996; Blain et al., 1997; LaBaer et al., 1997), consistent with results that either p21 or p27 is required for efficient assembly of active cyclin D–CDK4 complexes. p21 or p27 promotes assembly of stable cyclin D1–CDK4 complexes in double-null MEFs One prediction is that reintroduction of p21 or p27 into double-null MEFs should increase the assembly of cyclin Fig. 2. Cyclin D1 and CDK4-dependent Rb kinase activity in MEFs D1–CDK4 complexes. We infected these cells either with lacking p21 and p27. (A) Lysates from MEFs of the indicated a control retrovirus encoding the T-cell co-receptor CD8 genotypes were precipitated with non-immune rabbit serum (NRS) or or with retroviruses encoding either p21 or p27 (Figure with antibodies to cyclin D1 or CDK4. Resulting complexes were 3A). Lysates prepared from MEFs infected for 48 h were assayed for kinase activity using GST-Rb as the substrate. (B) Lysates from cells of the indicated genotype were depleted of p21, p27 or precipitated with antibodies to cyclin D1 or CDK4, and both, and then precipitated with antibodies to CDK4 or control NRS. assayed for complex formation. Ectopic expression of Washed immune complexes were assayed for Rb kinase activity. either p21 (Figure 3A, lane 3) or p27 (lane 4) but not CD8 (lane 1) increased cyclin D1–CDK4 complex and, like D1, were significantly decreased in cells lacking formation in p21/p27 double-null MEFs. The N-terminal both CKIs (lane 4). Despite the fact that the D2 signal in portion of p27, which contains the cyclin and CDK binding Figure 1D exceeds the D1 signal in Figure 1A and sites, is sufficient to promote the stable association of B, quantitation of the two cyclins by comparison with cyclin D1 and CDK4, whereas the C-terminal half of p27 recombinant protein standards indicated that the level of is inactive in this assembly assay (data not shown). Ectopic INK4a D1 exceeds that of D2 by 2- to 3-fold (data not shown). expression of another CDK inhibitor, p16 , which Cyclin D2–CDK4 complexes were readily detected in binds to CDK4 or CDK6 but not to D cyclins (Serrano wild-type MEFs and in those lacking either CKI but et al., 1993), did not promote assembly of cyclin D1– were significantly reduced in cells lacking both inhibitors CDK4 in these cells (data not shown). Ectopic expression (Figure 1D, K4 blot). In agreement with previous data of p21 or p27 not only increased the assembly of cyclin showing that CDK4 is the predominant partner of D-type D–CDK4 complexes but also increased the overall levels cyclins in rodent fibroblasts (Matsushime et al., 1994), of cyclin D1 in p21/p27 double-null MEFs to levels that virtually no cyclin D1–CDK6 or D2–CDK6 complexes approached those in wild-type MEFs (Figure 3A, lanes 3 were detected in MEFs. and 4 versus lane 2). D-type cyclin binding is essential for activating CDK4 Cyclin D1 is a labile protein (Matsushime et al., kinase activity (Matsushime et al., 1992). Since association 1992), and its rapid proteolytic degradation is triggered of D-type cyclins with CDK4 was significantly compro- by phosphorylation on Thr286 (Diehl et al., 1997). By mised in the p21/p27 double null MEFs, both cyclin D1- interacting with both cyclin D1 and CDK4, p21 and p27 dependent and total CDK4 kinase activity were measured might slow cyclin D1 turnover, possibly by promoting in these cells (Figure 2A). Cell lysates from proliferating nuclear localization of the complexes (see below) and/or MEFs were precipitated with either non-immune rabbit by interfering with cyclin D1 phosphorylation. Double- serum (NRS), antibody to cyclin D1 or antibody to CDK4, null MEFs were infected for 48 h with a retrovirus and the resulting immune complexes were assayed for encoding p27, and cells were metabolically labeled with kinase activity using recombinant GST-Rb as the substrate. [ S]methionine for 30 min. Medium containing labeled Note that CDK4-dependent kinase activity should include methionine was removed, and cells were incubated in contributions from both D1- and D2-containing holo- complete medium containing a 100-fold excess of un- enzymes (Figure 1). Active cyclin D1- and CDK4-depend- labeled methionine. Cell lysates prepared after different ent Rb kinase activities were detected in precipitates from periods of ‘chase’ were precipitated with the monoclonal wild-type MEFs (Figure 2A, lanes 2 and 6), p21-null antibody to cyclin D1, and the labeled proteins were MEFs (lanes 3 and 7) and p27-null MEFs (lanes 4 and resolved on a denaturing gel (Figure 3B). The half-life of 8). In contrast, only background levels of kinase activity cyclin D1 in p21/p27 double-null MEFs in several such were detected in immune complexes recovered from p21/ experiments was calculated to be 15 min (Figure 3B, lanes 1573 M.Cheng et al. are very low (Dai et al., 1996; Stepanova et al., 1996). Similarly, the vast majority of CDK6 subunits are bound to other molecules (Mahony et al., 1998). CDK4 requires association with Hsp90/Cdc37 for stabilization, suggesting that the latter acts as a chaperone for the proper folding of kinase subunits (Dai et al., 1996; Stepanova et al., 1996). High molecular weight complexes containing Hsp90, Cdc37 and CDK4 or CDK6 are cytoplasmic and do not contain D-type cyclins, so assembly of cyclin D with CDK4, the nuclear translocation of these complexes, and their activation by CAK presumably occur as later steps. We considered the possibility that in p21/p27 double- null cells, the lack of complexes between CDK4 and cyclins D1 and D2 might result in a greater association of CDK4 subunits with Cdc37. Instead, the level of Cdc37- bound CDK4 was lower in double-null cells than in wild- type cells (Figure 4D), suggesting that most CDK4 was complexed with other molecules or remained monomeric. Apart from interacting with D-type cyclins, CDK4 and CDK6 can independently associate with INK4 proteins (Serrano et al., 1993; Guan et al., 1994; Hannon and Beach, 1994; Chan et al., 1995; Hirai et al., 1995). CDK– INK4 complexes are stable, lack Cdc37, cannot assemble with cyclins, and therefore appear inaccessible for enzym- atic activation (Parry et al., 1995; Stepanova et al., 1996). It is therefore presumed that CDK4 can achieve alternative fates after release from the chaperone complex, either assembling with D-type cyclins or being inactivated through INK4 binding. Possibly, p21 and p27 might facilitate assembly of CDK4 and D-type cyclins by blocking the ability of INK4 proteins to sequester CDK4 Fig. 3. Reconstitution of cyclin D1–CDK4 complexes in vivo and in an inactive pool. We therefore studied the expression restabilization of cyclin. (A) Wild-type MEFs or those lacking both INK4a p21 and p27 were infected for 48 h with control virus (CD8) or with of the four different INK4 family members (p16 , INK4b INK4c INK4d viruses encoding p21 or p27. Cells were lysed and immunoprecipitated p15 , p18 and p19 ) and compared their with antibodies to cyclin D1 or CDK4. Separated immune complexes associations with CDK4 in wild-type and p21/p27 double- were then blotted with the cognate or reciprocal antibodies, and sites null MEFs. of antibody binding were detected by enhanced chemiluminescence. Individual INK4 proteins were depleted from cell lysates (B) MEFs lacking both p21 and p27 were infected with a control retrovirus encoding CD8 or with a virus encoding p27. Two days post- by two sequential immunoprecipitations, and the levels of infection, cells were pulse-labeled for 30 min with [ S]methionine CDK4 associated with each INK4 family member were and then ‘chased’ in the presence of 100-fold excess of unlabeled determined by blotting the precipitated proteins with methionine for the indicated times. Lysates normalized for protein antibodies to CDK4 (Figure 4A, lanes 1 and 2). Lysates concentration were precipitated with a monoclonal antibody to cyclin depleted of individual INK4 proteins were then precipit- D1, and the labeled proteins were resolved on a denaturing gel, which was dried and subjected to autoradiography. ated and blotted with antibodies to CDK4, in order to estimate the levels of residual CDK4 that remained 2–5), which is shorter than that in wild-type cells (t  unassociated with INK4 proteins (Figure 4A, lanes 3). 1/2 25 min) (Matsushime et al., 1992; Diehl et al., 1997, Wild-type MEFs expressed significant amounts of CDK4 INK4a INK4b INK4c 1998). In contrast, in cells infected with p27-virus, the in complexes with p16 , p15 and p18 (Figure half-life of D1 exceeded 40 min (Figure 3B, lanes 6– 4A). In cells lacking both p21 and p27, CDK4 associated 9). Metabolic labeling experiments indicated that the with the same three INK4 family members, although in relatively low level of cyclin D1 in p21/p27 double-null comparison with wild-type MEFs, less CDK4 was bound INK4c INK4b MEFs also reflects a 3-fold reduced rate of D1 synthesis to p18 and more was bound to p15 (Figure 4B). versus that in wild-type MEFs (data not shown). Cyclin In both MEF strains, more CDK4 was complexed with INK4a D1 synthesis was only modestly increased following acute p16 than with other INK4 family members, and no INK4d infection of the cells with the p27 retrovirus (Figure 3B, association with p19 was detected. compare lane 6 with lane 2), so the restoration of D1 To determine the relative pools of total INK4-bound levels following reintroduction of p21 or p27 (Figure 3) and -unbound CDK4, MEF lysates were depleted with primarily reflects increased D1 stability. mixtures of antibodies directed to all four INK4 family members, and the above analysis was repeated (Figure Association of CDK4 with Cdc37 and INK4 proteins 4C). From several such experiments, we estimated that in in p21/p27 double-null cells wild-type cells, ~40% of the total CDK4 pool stably Although CDK4 in mouse fibroblasts has a half-life of associated with INK4 proteins. As expected, cyclin D1 ~4 h (Matsushime et al., 1992), unassembled CDK4 co-precipitated only with those CDK4 molecules that subunits are unstable, and the levels of monomeric CDK4 were not bound to INK4 proteins (/ cells, lanes 3). 1574 CDK inhibitors promote cyclin D–CDK assembly Fig. 4. Association of CDK4 with INK4 proteins and Cdc37. Cell lysates from wild-type (A) or p21/p27 double-null cells (B) were sequentially depleted by two rounds of precipitation with non-immune rabbit serum (NRS) or with antibodies to the designated INK4 proteins (lanes 1 and 2) and then precipitated with anti-CDK4 (lanes 3). All recovered proteins were immunoblotted with anti-CDK4. (C) Experiments were performed as above, except that immunodepletion was carried out with a mixture of antibodies to all four INK4 family members prior to blotting of precipitated proteins with anti-CDK4 and anti-D1. (D) Cells of the indicated genotype were lysed and equal quantities of protein (100 μg) were resolved on denaturing gels and immunoblotted directly with anti-CDK4 (lanes 1 and 2) or rabbit anti-Cdc37 (lanes 5 and 6; produced by J.A.D. and C.J.S. to recombinant mouse protein synthesized in bacteria, unpublished). The arrows indicate the position of authentic CDK4 (34 kDa, left) and Cdc37 (50 kDa, right). The faster-migrating band detected with commercial polyclonal antibodies to mouse CDK4 used in this experiment (Santa Cruz Biotechnology) is not observed using antisera raised in our laboratory (R or R ; Matsushime et al., 1994). To quantitate complex formation, 5-fold Y Z more lysate protein (500 μg/lane) precipitated with anti-Cdc37 was separated on denaturing gels and blotted with anti-CDK4 (lanes 3 and 4). Importantly, all enzymatically active CDK4 is bound to Table I. Subcellular localization of cyclin D1 in cells lacking CKIs D-type cyclins, and this fraction also contained associated p21 and p27 molecules (Figure 2). In p21/p27 double- Genotype % Nuclear % Nuclear and % Cytoplasmic cytoplasmic null cells, the INK4-bound CDK4 fraction was increased to ~50–60%; D1–CDK4 complexes were again not p21 p27 detected even though a substantial pool of non-INK4- 61 421 318  3 bound CDK4 remained (–/– cells, lanes 3). Therefore, in 49 423 328  4 wild-type MEFs, ~40% of CDK4 is associated with cyclin 46 426 428  4 D1 and 10–15% is associated with D2 (Figure 1), ~40% 38 328 334  4 is associated with INK4 proteins (Figure 4C), and much Flag-tagged D1 retrovirus of the remainder is bound to Cdc37 (Figure 4D; see figure 59 523 218  2 legend for amounts of protein loading per lane). In p21/ 7 364 529  4 p27 double-null cells, little CDK4 binding to D cyclins was detected (Figure 1), ~60% was bound to INK4 Flag-tagged D1 (T286A) retrovirus proteins (Figure 4C) and 10% was complexed to Cdc37. 82 514 24  1 Therefore, a substantial fraction of CDK4 must either 77 717 56  2 remain monomeric or is associated with as yet unidentified Proliferating wild-type MEFs and those lacking p21, p27 or both were molecules. This indicates that p21 and p27 do not simply stained with monoclonal antibody to cyclin D1 and scored by compete with INK4 proteins in directing cyclin D–CDK immunofluorescence for the presence of nuclear and/or cytoplasmic assembly. D1. Cells infected with retroviruses encoding Flag-tagged D1 or the D1 (T286A) mutant that is stable and remains in the nucleus throughout interphase were studied similarly. Because ectopic p21 or p27 can facilitate nuclear accumulation of expression of D1 greatly exceeded that of the endogenous protein, no cyclin D1 background signals were detected at the exposures used. At least 500 Cyclin D1 normally accumulates in the nuclei of cells cells were counted per experiment and the results show mean  SD during G phase but relocalizes to the cytoplasm during 1 from three such experiments. S phase (Baldin et al., 1993). Although cyclin D1 has no obvious nuclear import signal, p21 family members can direct the nuclear localization of cyclin D1–CDK4 com- stained with antibody to cyclin D1, 61% of the cells plexes (Diehl and Sherr, 1997; LaBaer et al., 1997), exhibited strong nuclear fluorescence (Table I). Fluores- raising the possibility that cyclin D1 might not be able to cence-activated cell sorter (FACS) analysis of DNA con- enter the nucleus in MEFs lacking both p21 and p27. tent indicated that 52% of the total asynchronously When asynchronously proliferating wild-type MEFs were proliferating population were in G phase, and in agree- 1575 M.Cheng et al. CDK4 in cells lacking both p21 and p27 (Figure 5, lane 4, CDK4 blot; also see Figure 1C). These experiments left open the possibility that the ability of p21 and p27 to promote assembly of cyclin D1– CDK4 complexes was an indirect effect of their directing cyclin D1 to the nucleus. Accordingly, we took advantage of a mutant form of cyclin D1 (T286A) that contains an alanine for threonine-286 substitution, is remarkably stable (t 3 h) (Diehl et al., 1997), and remains in the nucleus 1/2 throughout the cell cycle (Diehl et al., 1998). When infected with a vector encoding D1 (T286A), both wild- type MEFs (82%) and p21/p27 double-null MEFs (77%) displayed an exclusively nuclear staining pattern of the D1 mutant (Table I), reinforcing the concept that D1 can enter the nucleus in the absence of both p21 and p27. Moreover, the abundance of cyclin D1 (T286A) was similar in both wild-type and double-null MEFs (Figure Fig. 5. Assembly of ectopically expressed cyclin D1 and D1 (T286A) 5, lanes 3 and 5). Even under these conditions, D1 (T286A) with CDK4. Wild-type and double-null MEFs were infected with retrovirus encoding Flag-tagged cyclin D1 or D1 (T286A) for 48 h. could not assemble with CDK4 (Figure 5, lane 5, CDK4 Lysates were then precipitated with a control monoclonal antibody (C) blot). This argues that CKIs do not ensure assembly by or with the monoclonal antibody to the flag epitope (M2), and the simply contributing a nuclear import signal, in agreement resulting precipitates were resolved on a denaturing gel and transferred with results obtained with mutants of p21 (LaBaer et al., to nitrocellulose. Proteins were visualized by enhanced chemiluminescence using a monoclonal antibody to D1 or antibodies 1997) and p27 (not shown) that lack the signal sequences. to CDK4. These results also confirm that decreased assembly of cyclin D1–CDK4 complexes is not simply secondary to ment with others’ results (Baldin et al., 1993), double- the decreased abundance of cyclin D1 in p21/p27 double- labeling with BrdU for 2 h prior to staining indicated null MEFs. that ~90% of cells exhibiting bright nuclear cyclin D1 fluorescence were not in S phase (data not shown). Rb phosphorylation in p21/p27 double-null cells Although a smaller fraction of cells lacking p21, p27 or In normal MEFs, phosphorylation of Rb is triggered by both exhibited exclusively nuclear staining, the lower cyclin D-dependent kinases and is probably completed by levels of cyclin D1 expressed were still able to enter the cyclin E–CDK2 (and/or cyclin A–CDK2) as cells enter S nucleus (Table I). In agreement with immunoblotting phase. Since active cyclin D-dependent kinase activity results (Figure 1), the intensity of cyclin D1 staining in was not detected in the p21/p27 double-null MEFs (Figure p21/p27 double-null MEFs was much lower than that of 2), we studied the kinetics of Rb phosphorylation in these wild-type MEFs (as judged by the need for a 6-fold cells. MEFs arrested by contact inhibition and serum increase in exposure time to obtain an almost comparable starvation were trypsinized and reseeded at lower density signal). Hence, the CDK inhibitors are not strictly required in complete medium containing 10% fetal bovine serum for cyclin D1 nuclear import. Moreover, the fact that a (FBS). Cell lysates prepared at different times thereafter significant fraction of cyclin D1 was detected in the were precipitated with antibody to Rb, and the resulting nucleus of double-null cells (Table I) whereas 95% immunoprecipitates were resolved on a denaturing gel, failed to assemble with CDK4 (Figure 1) suggests that transferred to nitrocellulose and blotted with the cognate stable association with catalytic subunits is also not antibodies. The percentage of cells in S phase was estim- essential for D1 nuclear import. ated by flow cytometric analysis of DNA content. As cells We next used infection with a retrovirus encoding Flag- approached S phase, Rb appeared to undergo phosphoryl- tagged D1 to increase cyclin D levels in double-null cells. ation, as manifested by its characteristic retardation in After infection for 36 h, similar amounts of Flag-D1 were electrophoretic mobility on denaturing gels (Figure 6A). expressed in both wild-type and the double-null MEFs, The kinetics of Rb phosphorylation and the rate of cell as demonstrated by immunoprecipitation with M2 anti- cycle progression were quite similar in p21/p27 double- bodies to the tag followed by immunoblotting with anti- null and wild-type MEFs. Hence, in the absence of the bodies to D1 (Figure 5, lanes 2 versus 4, D1 blot). These CKIs, resident cyclin-dependent kinases are sufficient levels of ectopically expressed cyclin D1 exceeded the to phosphorylate Rb, canceling its growth suppressive endogenous level of cyclin D1 in double-null MEFs by function as cells exit G phase. 10-fold (data not shown). Most infected wild-type MEFs Although the levels of cyclin D-dependent kinase (59%) displayed an exclusively nuclear cyclin D1 staining activity expressed in p21/p27 double-null cells were pattern (Table I). In marked contrast, 7% of p21/p27 reduced below the limits of detection (Figure 2), several double-null MEFs exhibited exclusively nuclear cyclin D1 lines of evidence suggest that there may be some residual staining, and instead, the cells displayed both nuclear and kinase activity. First, using antibodies that detect Rb cytoplasmic staining or exclusively cytoplasmic staining phosphorylated on Ser780, a site reported to be specifically (Table I). Therefore, under conditions in which cyclin D1 phosphorylated by cyclin D-dependent kinases (Kitagawa was restored, an absence of p21 and p27 limited D1 et al., 1996), forms of Rb phosphorylated on this residue nuclear accumulation. Importantly, ectopically expressed could be detected in cycling (lanes 4 and 6) but not in Flag-tagged cyclin D1 was still unable to assemble with quiescent (lane 5) p21/p27 double-null cells (Figure 6B). 1576 CDK inhibitors promote cyclin D–CDK assembly Second, when we infected double-null cells with a retro- INK4a virus encoding p16 , we observed inhibition of S phase entry, albeit not nearly to the same extent as that observed with the wild-type MEFs (Figure 6C). As expected, both MEF strains remained highly sensitive to growth arrest by a retrovirus encoding p27. Since, unlike p27, INK4 proteins appear to specifically target CDK4 and CDK6, these data imply that p21/p27 double-null cells are not entirely devoid of cyclin D-dependent kinase activity. We also measured total CDK2 or cyclin E-dependent kinase activity in lysates of the various MEF strains, using either histone H1 or GST-Rb as the substrate (Figure 7). Although the levels of CDK2 were comparable in the different cell strains (Figure 7D), both CDK2 and cyclin E-dependent kinase activity were enhanced in p21/p27 double-null MEFs, as compared with those in wild-type MEFs (Figure 7A–C). Therefore, in the absence of p21 and p27, unopposed CDK2 activity may compensate for the severe reduction in CDK4 function. In agreement, p21- and p27-null MEFs are not significantly perturbed in cell cycle progression and exhibit generation times and S phase fractions similar to those of wild-type cells (Figure 6A and data not shown). Assembly of cyclin D–CDK complexes in tissues from double-null mice Although the above studies were performed with primary MEFs, a clear prediction is that assembly of different cyclin D-dependent kinases would be perturbed in many other cell types. Extracts of whole liver from wild-type and double-null mice expressed all three D-type cyclins together with CDK6 (Figure 8, lanes 1 and 2). When these were immunoprecipitated with a mixture of antibodies to cyclins D1, D2 and D3 and then immunoblotted with antibodies to CDK6, significantly fewer cyclin D–CDK6 complexes were observed in liver from double-null animals. Similar results were obtained with CDK4 in complexes with D1 (data not shown). T lymphocytes primarily express cyclins D2 and D3 in conjunction with CDK6 (Meyerson and Harlow, 1994), and thymic extracts from double-null mice also contained much lower levels of cyclin D–CDK6 complexes than those from wild-type animals (Figure 8, lanes 3 and 4). Assembly of cyclin D– CDK4 complexes was only modestly reduced in extracts Fig. 6. Rb status and p16-induced arrest. (A) Wild-type and double- null MEFs made quiescent by contact inhibition and serum starvation of kidney and heart from double-null mice (data not were trypsinized, reseeded and stimulated to enter the cell cycle in shown). Therefore, despite the combinatorial nature of complete medium containing 10% FBS. Cell lysates prepared at expressed cyclin D–CDK complexes in different tissues different times thereafter were immunoprecipitated with polyclonal and the potential participation of other CKIs such as antibodies to mouse Rb, separated on denaturing gels and blotted with Kip2 monoclonal antibody to Rb. The position of the hypophosphorylated p57 in the assembly process, assembly of cyclin D- form of Rb is indicated by the signal in starved cells, whereas dependent kinases was impaired in vivo. additional phosphorylation is connoted by the retardation in Rb’s mobility as cells progress through the division cycle. The fraction of cells in S phase was determined by flow cytometric analysis of DNA Discussion content. Cells entered S phase by ~12 h and were predominately in Cip1 G /M by the completion of the experiment. (B) Rb precipitated from 2 CDK inhibitors of the Cip/Kip family, including p21 , quiescent (lane 5) or proliferating (all other lanes) MEFs of the Kip1 Kip2 p27 and p57 , negatively regulate cell cycle progres- indicated genotypes was immunoblotted with anti-Rb (lanes 1 and 2) sion and enforce cell cycle arrest when expressed at high or with an antibody that specifically detects an epitope containing levels (Elledge and Harper, 1994; Sherr and Roberts, pSer780 (lanes 3–6) (Kitagawa et al., 1996). The position of the pSer780 form of Rb is indicated by arrows in the right margin. 1995). Our results provide a different perspective in (C) MEFs infected for 24 h with a control CD8 retrovirus or with showing that p21 and p27 are necessary for certain INK4a vectors encoding CD8 plus either p16 or p27 were scored for processes that positively regulate cell cycle progression: [ H]thymidine incorporation (2 h pulse). Results with the control CD8 cyclin D assembly with CDK4, its stability and its nuclear vector were normalized to 100%. Standard errors (bars) were calculated from several independent experiments. localization. Hence, the generally prevailing view of Cip/ Kip proteins as universal inhibitors of CDKs appears to 1577 M.Cheng et al. Fig. 7. CDK2 activities in MEFs lacking p21 and/or p27. Lysates from proliferating MEFs were precipitated with antibodies to CDK2 (A and B)or to cyclin E (C), and immune complex kinase assays were performed using histone H1 (A and C) or GST-Rb (B) as substrates. All reactions were stopped at the times indicated by heating samples to 85°C for 5 min in gel sample buffer containing SDS. Labeled proteins were resolved on denaturing gels, which were dried and subjected to autoradiography. Excised slices containing substrates were rehydrated and radioactivity was determined by liquid scintillation. Results are plotted in (A–C) for wild-type MEFs (squares), p21-null MEFs (triangles), p27-null MEFs (inverted triangles) and double-null MEFs (diamonds). (D) CDK2 protein in MEFs of the indicated genotype was detected by immunoblotting with the cognate antibody. Equal aliquots of lysate were separated per lane, and sites of antibody binding were detected by enhanced chemiluminescence. portray too simple a picture of their regulatory effects on the cell cycle. A more accurate representation is that these CKIs are activators of CDK4 and inhibitors of CDK2. This reformulation of the role of Cip/Kip proteins places their inhibitory effect on cell proliferation more specifically at the pivotal transition in the cell cycle between the cyclin D-mediated responses to extrinsic mitogenic cues and the CDK2-mediated progression from G to S phase. Stable association of p21 and p27 with active cyclin D–CDK4 complexes in vivo The solved structure of p27 in a complex with cyclin A– CDK2 illustrates that a single p27 molecule can bind to both the cyclin and CDK subunit and can disrupt the CDK to dismantle its ATP binding site (Russo et al., 1996). Although no analogous structure of a cyclin D–CDK–CKI Fig. 8. Cyclin D–CDK assembly in mouse tissues. Tissue extracts from liver and thymus of wild-type or p21/p27 double-null mice were complex is yet available, p21 and p27 were found to be precipitated with a mixture of antibodies to cyclins D1, D2 and D3 potent inhibitors of binary cyclin D–CDK4 complexes (top panel) or anti-CDK6 (middle panel), and precipitated proteins assembled from recombinant protein subunits (Harper separated on denaturing gels were blotted with the same antibodies. In et al., 1993; Polyak et al., 1994; Toyoshima and Hunter, parallel, cyclin D precipitates were blotted with anti-CDK6 (bottom) to score for complex formation. Proteins were detected by enhanced 1994). However, the idea that p21 and p27 differentially chemiluminescence using exposure times of 10 s (top, middle) and regulate various cyclin–CDK complexes is consistent with 60 s (bottom). data that cyclin E–CDK2 and cyclin A–CDK2 are much more susceptible to inhibition by these CKIs than are cyclin D-dependent kinases in vivo (Soos et al., 1996; prepared in this manner retain Rb but not histone H1 Blain et al., 1997; LaBaer et al., 1997). As also observed kinase activity (Soos et al., 1996), a feature of substrate in our studies, enzymatically active cyclin D–CDK4 com- specificity that distinguishes cyclin D-dependent holo- plexes can be depleted from mammalian cell lysates with enzymes from cyclin A-, B- and E-dependent kinases antibodies to these CKIs. In turn, immune complexes (Matsushime et al., 1992). Therefore, p21 and p27 remain 1578 CDK inhibitors promote cyclin D–CDK assembly stably bound to active holoenzyme complexes during the complex formation. Interactions between p21/p27 and cell cycle. CDK4/CDK6 might also prevent INK4 binding to the How can cyclin D–CDK complexes containing p21 and catalytic subunits (Reynisdottir and Massague´, 1997), p27 retain activity? One possibility is that higher order diverting CDK4 into cyclin D-containing complexes and cyclin D–CDK–CKI complexes recovered from mamma- facilitating assembly through a less direct competitive lian cells contain additional components that protect the mechanism. Cells lacking p27 and p21 would be expected core binary enzyme from CKI-mediated inhibition (see, to accumulate more CDK4–INK4 complexes, and under for example, Zhang et al., 1994). Consistent with this these conditions, cyclin D1 should be destabilized (Bates idea, the mass of active cyclin D–CDK6 complexes et al., 1994; Parry et al., 1995). Increased binding of recovered from T cells has been estimated at 150–170 kDa CDK4 to INK4 proteins was indeed observed, but its (Mahony et al., 1998). Post-translational modifications of association with Cdc37 was diminished relative to that in the included subunits might also alter their activities. This wild-type cells. A significant fraction (~30%) of CDK4 is not to say that Cip/Kip proteins cannot, under certain remained unbound to INK4 proteins or to Cdc37 in circumstances, inhibit CDK4 and CDK6. For example, in double-null MEFs, arguing that mechanisms other than response to negative regulators of G progression such as competition between p21/p27 and INK4 proteins for CDK4 cAMP or TGF-β, the accumulation of Cip/Kip proteins binding play a role in the assembly process. can occlude cyclin D–CDK activation by CAK (Kato Both the levels and assembly promoting activities of et al., 1994; Polyak et al., 1994; Aprelikova et al., 1995). p21 and p27 are governed by mitogenic signals. For Clearly, the nature of the active holoenzymes expressed instance, p21 is frequently induced in cells entering the in mammalian cells needs to be better clarified. cycle from a quiescent state, whereas p27 levels are The fact that Cip/Kip proteins enter into stable, generally high in quiescent cells but fall prior to their enzymatically active complexes with cyclin D and CDK4 entry into S phase (Firpo et al., 1994; Kato et al., 1994; subunits highlights a second, non-catalytic function of Nourse et al., 1994). Hence, p27 should not be limiting cyclin D–CDKs: to ‘titrate’ p21 and p27, thereby freeing in promoting the association of cyclin D1 with CDK4 as other CDKs from their constraint. In the face of an cells enter the cycle, but its presence in a quiescent cell inhibitory threshold set by the CKIs, the latter process is still incapable of assisting assembly of cyclin–CDK sets a dependency of CDK2 activity on the mitogen- complexes (Matsushime et al., 1994). While it might be stimulated assembly of cyclin D–CDK complexes, thereby argued that the level of cyclin D1 in quiescent cells is coordinating the sequential activities of these enzymes as simply too low to allow its entry into complexes with quiescent cells enter the cycle and progress toward S phase. CDK4, ectopically expressed D1 subunits are also unable to assemble with CDK4 in serum-starved fibroblasts Cip/Kip regulation of cyclin D–CDK assembly (Matsushime et al., 1994) and require signals via the The loss of both p21 and p27 in MEF strains decreased Ras-Raf1-MEK-ERK kinase cascade to ensure complex the steady-state levels of assembled cyclin D–CDK4 formation (Cheng et al., 1998). One scenario is that p27 complexes 10-fold and lowered cyclin D- and CDK4- or p21 is subject to phosphorylation by ERKs, and that associated Rb kinase activities to undetectable levels. The only the appropriately modified forms of the CKIs are turnover of D-type cyclins was accelerated in cells lacking able to promote cyclin D–CDK complex formation. Both both CKIs and their overall levels were diminished. of these CKIs are phosphoproteins, and p27 can be Nonetheless, the reduction in cyclin D levels per se did phosphorylated on serine by ERK1 (Zhang et al., 1994; not account for their failure to assemble with catalytic Alessandrini et al., 1997). However, induction of subunits because ectopically overexpressed cyclin D1, enzymatically active MEK1 in NIH 3T3 fibroblasts was whether located primarily in the nucleus or cytoplasm, unaccompanied by detectable phosphorylation of p21 or was also unable to associate with CDK4 in this setting. p27. Also, a mutant version of p27 in which the ERK1 Conversely, reintroduction of p21 or p27 into double- phosphorylation site was changed to alanine was still able null cells reconstituted the formation of cyclin D–CDK to promote cyclin D1–CDK4 assembly (data not shown). complexes and increased the stability of cyclin D1. Other possibilities are that cyclin D and CDK4 must be Assembly of D-type cyclins with CDK4 requires several phosphorylated prior to assembly, or that the chaperone steps. Proper folding of CDK4 relies on the chaperone activity of the Cdc37/hsp90 complex can be regulated function of a cytoplasmic complex that includes Hsp90/ by ERKs. Cdc37 (Dai et al., 1996; Stepanova et al., 1996). Once Several factors are likely to contribute to added com- released from this complex, CDK4 can either enter into plexity in living animals. First, different cell types exhibit complexes with a D-type cyclin or can accumulate in varying levels of Cip/Kip proteins in vivo, so the extent enzymatically inactive binary complexes with an INK4 of assembly promoting activity attributed to each of these protein. The fate of CDK4 is largely determined by the CKIs likely varies between tissues. Double-null mice availability of cyclin D subunits, which accumulate in continue to express cyclin D–CDK4 complexes, albeit at Kip2 response to mitogenic signaling, but how p21 and p27 reduced levels, in many tissues. Although p57 seems contribute mechanistically to the assembly process remains not to be required for assembly of cyclin D-dependent unclear. Both of the latter CKIs contain distinct binding kinases in cultured MEFs, it may well promote assembly sites for CDKs and cyclins, which enable them to contact in other tissues. The possibility that new more distantly both subunits simultaneously (Toyoshima and Hunter, related Cip/Kip family members may be identified and be 1994; Chen et al., 1995, 1996; Luo et al., 1995; Nakanishi shown to play a role in cyclin–CDK assembly cannot be et al., 1995; Lin et al., 1996; Russo et al., 1996). Hence, formally excluded. Secondly, while MEFs preferentially their binding to cyclin D and CDK4 might stabilize synthesize cyclin D1 and D2 in complexes with CDK4, 1579 M.Cheng et al. other cell types such as T lymphocytes, for example, cyclin. Phosphorylation of cyclin D1 on Thr286 is medi- express D3 instead of D1 and much more CDK6 than ated by GSK-3β, and although cyclin D1 turnover in CDK4 (Ajchenbaum et al., 1993; Meyerson and Harlow, proliferating cells is relatively rapid (t  25 min), 1/2 1994). Therefore, the six cyclin D-dependent kinases decreased signaling through the Ras-PI3K-Akt pathway (containing D1, D2 or D3 with CDK4 or CDK6) are activates GSK-3β and further shortens the half-life of likely to be differentially regulated by Cip/Kip family cyclin D1 to ~12 min (Diehl et al., 1998). Moreover, members. Thirdly, the levels of cyclin D–CDK4 complexes overexpression of an active, but not kinase-defective form were only modestly reduced in MEFs lacking p21 or p27, of GSK-3β in mouse fibroblasts causes a redistribution of as compared with those in cells lacking both CKIs. cyclin D1 from the cell nucleus to the cytoplasm (Diehl Possibly, the loss of one of the CKIs facilitates compensa- et al., 1998). Therefore, by suppressing Thr286 phos- tion by the other. Finally, CKIs themselves are regulated phorylation, CKIs might affect the stability of cyclin D1 by signals that affect cell proliferation, organismal via two mechanisms: by promoting its nuclear retention development and cell differentiation (Elledge and Harper, and by preventing its targeting to proteasomes. 1994; Sherr and Roberts, 1995). As a singular example, the Cip1 gene is directly regulated by p53 (El-Deiry et al., Consequences of Cip/Kip loss during the cell 1993; Dulic et al., 1994), and in cancer cells containing division cycle mutant p53, the levels of p21 are generally reduced Remarkably, MEFs lacking p21 and p27 did not exhibit making other CKIs more likely to promote cyclin D–CDK overtly aberrant cell cycles. Indeed, our failure to detect assembly under such circumstances. cyclin D-dependent kinase activity in cells lacking both p21 and p27 raised the possibility that these kinases are CKIs affect cyclin D stability and nuclear not required for cell cycle progression. In an attempt to localization test directly whether p21/p27 double-null cells lack all Cyclin D1 accumulates in the nucleus during G phase cyclin D-dependent kinase activity, we infected these cells but redistributes into the cytoplasm during S phase (Baldin with retroviruses encoding the CDK4- and CDK6-specific INK4a et al., 1993). D-type cyclins and CDK4 lack obvious inhibitor, p16 . Proteins of the INK4 family are pre- nuclear localization signals (NLSs), and p21 or p27 can sumed to act specifically as inhibitors of cyclin D-depend- promote the nuclear import of cyclin D1–CDK4 through ent kinases and are only able to arrest cells that retain Rb putative NLSs at their C-termini (LaBaer et al., 1997). function (Koh et al., 1995; Lukas et al., 1995; Medema When cyclin D1 was ectopically overexpressed in wild- et al., 1995). Despite the absence of detectable cyclin D- type MEFs, most of it localized to the nucleus, but in dependent kinase activity, p21/p27 double-null MEFs p21/p27 double-null cells, it was largely confined to the retained some sensitivity to the cell cycle inhibitory effects INK4a cytoplasm. Therefore, under conditions in which cyclin of overexpressed p16 . Even more inhibition was D1 levels are relatively high, the absence of p21 and p27 obtained using adenovirus vectors that programmed higher can limit its nuclear accumulation. However, assembly of levels of ectopic protein expression (data not shown). In cyclin D–CDK complexes per se does not drive their addition, Rb was phosphorylated on at least one site that nuclear uptake, since endogenous cyclin D1 expressed at is preferentially recognized by cyclin D-dependent kinases. much reduced levels in p21/p27 double-null cells was Therefore, cells lacking p21 and p27 might well express able to accumulate in the nucleus, albeit less efficiently. cryptic cyclin D-dependent kinase activity. In short, while Moreover, a cyclin D1 (T156A) mutant that assembles we were unable to resolve whether cyclin D-dependent with CDK4 remains largely cytoplasmic, although its kinases are dispensable for the division cycle in Rb- nuclear entry can be enforced by p21 overexpression positive cells lacking Cip/Kip proteins, it is evident that (Diehl and Sherr, 1997). Conversely, the D1 (T286A) such cells tolerate a significant reduction in enzyme mutant is preferentially retained in the nucleus even in activity. p21/p27 double-null cells where it does not undergo Although mice lacking cyclins D1 or D2 (or both) assembly. Indeed, this underscores the fact that assembly exhibit focal developmental anomalies (Fantl et al., 1995; is inhibited in the absence of CKIs irrespective of whether Sicinski et al., 1995, 1996), two of the three D-type D1 is predominately cytoplasmic or nuclear. We conclude cyclins are dispensable for most cell divisions in the life that cyclin D–CDK assembly and nuclear uptake are of these animals. Indeed, if the key functions of cyclin separable functions to which the CKIs contribute inde- D-dependent kinases are to phosphorylate Rb and to titrate pendently. CDK inhibitors, then their loss in p21/p27 double-null The overall levels of cyclins D1 and D2 were signific- cells should be well tolerated. In the absence of these antly reduced in MEFs lacking both p21 and p27, but CKIs, the titration function of cyclin D-dependent kinases cyclin D1 was stabilized and its levels were restored when would be superfluous, and unopposed cyclin E–CDK2 either CKI was reintroduced into the double-null cells. and cyclin A–CDK2 activities might be sufficient to How might the presence of CKIs affect cyclin D stability? phosphorylate Rb. Based on changes in its electrophoretic Phosphorylation of cyclin D1 on Thr286 targets it for mobility, Rb phosphorylation increased as p21/p27-null degradation, and elimination of this threonine markedly cells approached the G /S boundary, and the kinetics of increases the half-life of cyclin D1 in proliferating cells cell cycle progression were remarkably similar to those (t 3 h) (Diehl et al., 1997). Cyclins D2 and D3 appear of wild-type MEFs. Together, our results argue that p21 1/2 subject to similar controls (our unpublished data). One and p27 positively regulate the assembly, stability and possibility, then, is that binding of p21 or p27 to cyclin nuclear localization of D-type cyclins. In turn, the activities D1–CDK4 complexes can partially suppress D1 phos- of cyclin E–CDK2 and A–CDK2 are normally opposed phorylation on Thr286, thereby helping to stabilize the by these CKIs, but in their absence, the latter enzymes 1580 CDK inhibitors promote cyclin D–CDK assembly 10 mM MgCl , 1 mM DTT) Reaction mixtures (50 μl kinase buffer) are likely to compensate for loss of cyclin D-dependent 2 . contained 30 μM ATP with 10 μCi [γ- P]ATP plus 10 μg histone H1 kinase activity. or 5 μg GST-Rb. All reactions were stopped by adding 1/3 volume 3 gel sample buffer and heating at 85°C for 5 min. Labeled proteins were resolved on denaturing polyacrylamide gels, which were dried and Materials and methods subjected to autoradiography. Cells and culture conditions Virus production and infection Cip1 Mouse embryonic fibroblasts (MEFs) from animals deficient in p21 , Human kidney 293T cells were transfected (Chen and Okayama, 1987) Kip1 p27 or both CKIs were established as described previously (Zindy with 15 μg of ecotropic helper retrovirus plasmid plus 15 μgofSRα et al., 1997) and maintained in Dulbecco’s modified Eagle’s medium INK4a vector DNA encoding p21, p27, p16 or Flag-tagged cyclin D1 (DMEM) supplemented with 10% FBS, 2 mM glutamine, 0.1 mM non- (WT) and cyclin D1 (T286A). Cell supernatants containing infectious essential amino acids, 55 μM 2-mercaptoethanol and 100 U/ml each of retroviral pseudotypes were harvested 24–60 h post-transfection, pooled penicillin and streptomycin. To make them quiescent, confluent MEFs on ice and filtered (0.45 μm membrane). Virus infections of exponentially were washed twice with phosphate-buffered saline (PBS) and cultured growing MEFs were performed in a 9% CO atmosphere with 5 ml of in serum-depleted medium [DMEM with 0.1% FBS, 0.04% bovine virus-containing culture supernatants plus 10 μg/ml polybrene (Sigma, serum albumin (BSA), glutamine, penicillin and streptomycin] for 18 h. St Louis, MO) for each 100 mm diameter culture dish. After 5 h, 10 ml Quiescent cells were trypsinized, re-plated at low density and stimulated fresh medium was added, and medium was changed 24 h later. Cells with complete medium containing 10% FBS to enter the division cycle, were harvested 48 h after infection, and the percentage of cells in S and entry into S phase was monitored by estimating the DNA content phase was determined by flow cytometric analysis of DNA content of propidium iodide-stained nuclei using fluorescence-activated flow (Matsushime et al., 1991) or by incorporation of [ H]thymidine into cytometry (Matsushime et al., 1991). The 293T retrovirus packaging replicating cell DNA (Kamijo et al., 1997). cell line and helper virus plasmid (Pear et al., 1993) were obtained from C.Sawyers (University of California, Los Angeles) with permission from David Baltimore (California Institute of Technology). Acknowledgements Immunoblotting, immunoprecipitation and We thank Dr Yoichi Taya for generously supplying antibodies to the Rb immunofluorescence epitope containing phosphoserine 780, and Carol Bockhold, Erin Randell Immunoblotting of cyclin D1, cyclin D2, CDK4, CDK6, p21, p27 and and Esther Van der Kamp for excellent technical assistance. This work the detection of cyclin D–CDK complexes was performed as described was supported in part by NIH grant CA-56819 to M.F.R., by Cancer previously (Cheng et al., 1998). INK4 proteins were precipitated from Center Core Grant CA-21765 and by the American Lebanese Syrian MEFs or mouse tissues as described (Zindy et al., 1997). For detection Associated Charities (ALSAC) of St Jude Children’s Research Hospital. of Rb protein, cells were disrupted in lysis buffer containing 50 mM M.C. is supported by NIH training grant T32-CA-70089 and J.P.O. by HEPES pH 7.0, 0.5% Nonidet P-40, 250 mM NaCl, 1 mM phenylmethyl- the Human Frontier Science Program. J.M.R. and C.J.S. are Investigators sulfonyl fluoride (PMSF), 5 mM EDTA, 4 μg/ml aprotinin, 4 μg/ml of the Howard Hughes Medical Institute. pepstatin (protease inhibitors from Sigma Chemicals, St Louis, MO), 0.5 mM sodium orthovanadate, 5 mM sodium fluoride, and 50 mM β-glycerophosphate. Lysates were clarified by centrifugation, and protein References concentration was determined using a BCA assay kit (Pierce, Rockford, IL). Protein (2 mg) was precipitated with rabbit antibody to Rb (SC- Ajchenbaum,F., Ando,K., DeCaprio,J.A. and Griffin,J.D. (1993) 050, Santa Cruz, CA) and collected with 30 μl of protein A–Sepharose Independent regulation of human D-type cyclin gene expression during (Pharmacia Biotechnology, Uppsala, Sweden). After three washes in G phase in primary human T lymphocytes. J. Biol. Chem., 268, lysis buffer, immune complexes resuspended in 30 μl gel sample buffer 4113–4119. [50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1 mM dithiothreitol Aktas,H., Cai,H. and Cooper,G.M. (1997) Ras links growth factor (DTT), 0.1% Bromophenol Blue], were electrophoretically resolved on signaling to the cell cycle machinery via regulation of cyclin D1 and Kip1 denaturing polyacrylamide gels, transferred to nitrocellulose and probed the cdk inhibitor p27 . Mol. Cell. Biol., 17, 3850–3857. with the mouse monoclonal antibody to Rb (14001A, Pharmingen, Albanese,C., Johnson,J., Watanabe,G., Eklund,N., Vu,D., Arnold,A. and ras CA). Rb protein phosphorylated on Ser780 was detected by direct Pestell,R.G. (1995) Transforming p21 mutants and c-Ets-2 activate immunoblotting using purified antibodies specifically reactive with a the cyclin D1 promoter through distinguishable regions. J. Biol. phosphorylated epitope (Kitagawa et al., 1996). Metabolic labeling and Chem., 270, 23589–23597. measurements of cyclin D1 turnover were performed as previously Alessandrini,A., Chiaur,D.S. and Pagano,M. (1997) Regulation of the described (Diehl et al., 1997). Indirect immunofluorescence was also cyclin-dependent kinase inhibitor p27 by degradation and carried out according to previous methods (Diehl and Sherr, 1997) phosphorylation. Leukemia, 11, 342–345. except that the cells were fixed with 3.7% paraformaldehyde at room Aprelikova,O., Xiong,Y. and Liu,E.T. (1995) Both p16 and p21 families temperature for 15–20 min and permeabilized with acetone for 5 min of the cyclin-dependent kinase (CDK) inhibitors block the at –20°C. phosphorylation of cyclin-dependent kinases by the CDK-activating kinase. J. Biol. Chem., 270, 18195–18197. Protein kinase assays Baldin,V., Lukas,J., Marcote,M.J., Pagano,M. and Draetta,G. (1993) Immune complex kinase assays using 5 μg recombinant GST-Rb as Cyclin D1 is a nuclear protein required for cell cycle progression in substrate were performed (Matsushime et al., 1994) using 500 μg total G1. 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The p21Cip1 and p27Kip1 CDK ‘inhibitors’ are essential activators of cyclin D‐dependent kinases in murine fibroblasts

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Copyright © European Molecular Biology Organization 1999
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10.1093/emboj/18.6.1571
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Abstract

The EMBO Journal Vol.18 No.6 pp.1571–1583, 1999 Cip1 Kip1 The p21 and p27 CDK ‘inhibitors’ are essential activators of cyclin D-dependent kinases in murine fibroblasts 1 2,3 has not been defined. Finally, proteasomal degradation of Mangeng Cheng , Paul Olivier , 1,3 2,3 cyclin D1 is triggered by its phosphorylation on a single J.Alan Diehl , Matthew Fero , 1 2,3 threonine residue (Thr286) by glycogen synthase kinase- Martine F.Roussel , James M.Roberts and 1,3,4 3β (Diehl et al., 1997, 1998), a process antagonized Charles J.Sherr by signaling through a separate Ras-dependent pathway Department of Tumor Cell Biology, St Jude Children’s Research involving phosphatidylinositol 3-kinase (PI3K) and protein Hospital, 332 N. Lauderdale, Memphis, TN 38105, Division of Basic kinase B (also called Akt) (Boudewijn et al., 1995; Cross Sciences, Fred Hutchinson Cancer Center, Seattle, WA 98104 and et al., 1995; Franke et al., 1995, 1997; Klinghoffer et al., Howard Hughes Medical Institute, USA 1996; Dudek et al., 1997; Vanhaesebroeck et al., 1997). Corresponding author In the continued presence of mitogenic signals, cyclin D1– e-mail: [email protected] CDK4 complexes assemble and accumulate throughout G phase, enter the nucleus and undergo phosphorylation by The widely prevailing view that the cyclin-dependent CDK-activating kinase (CAK) to yield active holoen- kinase inhibitors (CKIs) are solely negative regulators zymes. One key function of the cyclin D-dependent kinases of cyclin-dependent kinases (CDKs) is challenged here is to initiate phosphorylation of the retinoblastoma protein by observations that normal up-regulation of cyclin D– (Rb), thereby helping to cancel its activity as a transcrip- CDK4 in mitogen-stimulated fibroblasts depends tional repressor of a bank of genes, including cyclins E Cip1 Kip1 redundantly upon p21 and p27 . Primary mouse and A, whose activities are required for S phase entry embryonic fibroblasts that lack genes encoding both (Weinberg, 1995; Sherr, 1996). p21 and p27 fail to assemble detectable amounts of A separate, non-catalytic action of cyclin D-dependent cyclin D–CDK complexes, express cyclin D proteins at Kip1 kinases is the sequestration of CKIs, including p27 much reduced levels, and are unable to efficiently Cip1 and p21 (Sherr and Roberts, 1995). The Cip/Kip direct cyclin D proteins to the cell nucleus. Restoration proteins interact with a variety of cyclin–CDK complexes of CKI function reverses all three defects and thereby through a conserved N-terminal domain that contains both restores cyclin D activity to normal physiological levels. cyclin and CDK binding sites (Toyoshima and Hunter, In the absence of both CKIs, the severe reduction in 1994; Chen et al., 1995, 1996; Luo et al., 1995; Nakanishi cyclin D-dependent kinase activity was well tolerated et al., 1995; Lin et al., 1996; Russo et al., 1996). Cyclin and had no overt effects on the cell cycle. Cip1 D-dependent CDKs isolated from mammalian cells appear Keywords: CDK4/cell cycle/D-type cyclins/p21 / Kip1 to be less susceptible to Cip/Kip-mediated inhibition than p27 are other classes of cyclin–CDKs (Soos et al., 1996; Blain et al., 1997; LaBaer et al., 1997), and sequestration of Cip1 Kip1 p21 and p27 into higher order complexes with Introduction cyclin D-dependent kinases during G phase helps to relieve cyclin E–CDK2 from their constraint, thereby Regulation of mammalian cell proliferation by extracellu- facilitating its activation later in G phase. This ability lar mitogens is governed through receptor-mediated sig- 1 to ‘titrate’ CKIs therefore sets a dependency of cyclin naling circuits which ultimately converge on the cell cycle E-dependent kinase on the mitogen-stimulated assembly of machinery driven by cyclin-dependent kinases (CDKs) cyclin D-dependent kinases. Cyclin E–CDK2 collaborates and opposed by CDK inhibitors (CKIs) (Sherr and Roberts, with cyclin D-dependent kinases to phosphorylate Rb 1995). One important example is receptor-activated Ras (Hatakeyama et al., 1994; Mittnacht et al., 1994; Lee signaling, which governs the accumulation of cyclin D1– et al., 1996; Kelly et al., 1998; Lundberg and Weinberg, CDK4 complexes by at least three independent but comple- Kip1 1998), phosphorylates p27 to trigger its degradation mentary pathways: gene transcription, protein association (Sheaff et al., 1997; Vlach et al., 1997), and may target and protein stabilization. First, Ras signaling promotes other proteins whose modifications trigger origin firing transcription of the cyclin D1 gene via a kinase cascade and DNA replication per se (Stillman, 1996; Krude that depends upon the sequential activities of Ras, Raf-1, et al., 1997). mitogen-activated protein kinase kinase (MEK1) and Although it is generally assumed that CKIs act solely mitogen-activated protein kinases (MAPKs), also referred to retard G progression, the fact that they can be found to as extracellular signal-regulated protein kinases (ERKs) in complexes with active cyclin–CDKs (Zhang et al., (Albanese et al., 1995; Lavoie et al., 1996; Winston et al., 1994; Soos et al., 1996; Blain et al., 1997; LaBaer et al., 1996; Aktas et al., 1997; Kerkhoff and Rapp, 1997; Weber 1997) raises the possibility that they may also act as et al., 1997). Signaling through this same pathway is also sufficient to promote assembly of cyclin D1–CDK4 positive regulators. Intriguingly, LaBaer et al. (1997) Cip1 complexes (Cheng et al., 1998), although the physiological demonstrated that p21 could promote the assembly of target of ERK phosphorylation that mediates this process active cyclin D1–CDK4 complexes and, in addition, could © European Molecular Biology Organization 1571 M.Cheng et al. provide a localization signal for their nuclear import. However, the fact that p21 nullizygous mice undergo normal development and do not seem to have a significant deficiency in cyclin D-dependent kinase function (Brugarolas et al., 1995; Deng et al., 1995) leaves open the question of whether the CKIs are normal physiological regulators of cyclin D–CDK assembly. In this study, we have used primary mouse embryo fibroblast (MEF) strains deficient in p21, p27 or both to study their roles in governing the activities of cyclin D–CDK holoenzymes. Results Impaired assembly of cyclin D–CDK4 complexes in MEFs lacking p21 and p27 Cell lysates from asynchronously proliferating MEFs derived from wild-type mice, p21- and p27-null mice and from animals lacking both genes were precipitated with antibodies to cyclin D1 or CDK4. Precipitated proteins were resolved on denaturing polyacrylamide gels, trans- ferred to nitrocellulose membranes and blotted with the cognate antibodies to quantitate cyclin D1 and CDK4 levels, respectively, or with the reciprocal antibodies to score for the presence of cyclin D1–CDK4 complexes (Figure 1A). In early passage (p5) wild-type MEFs, ~40% of the total CDK4 (lane 1, K4 blot) co-precipitated with antibodies to D1 (lane 2, K4 blot). A smaller percentage of the total D1 pool (lane 2, D1 blot) co-precipitated with CDK4 (lane 1, D1 blot). However, antibodies to full-length recombinant CDK4 used in this experiment preferentially detect free versus cyclin D1-bound catalytic subunits, so the amount of D1 detected in CDK4 immunoprecipitates underestimates the extent of complex formation. Generally equivalent levels of CDK4 were expressed in MEF strains lacking one or both CKIs (Figure 1A, lanes 3, 5 and 7). In contrast, the overall levels of cyclin D1 were significantly lowered in cells lacking either p21 or p27 (D1 blot, lanes 6 and 8 versus lane 2) and were decreased at least 10- fold in lysates of cells lacking both CKIs (lane 4). Cyclin Fig. 1. Impaired assembly of cyclin D–CDK4 complexes in MEFs D1–CDK4 complexes were recovered at similarly reduced lacking p21 and p27. (A) Cell lysates (500 μg total protein per lane) levels from lysates of p21-null and p27-null MEFs (lanes from MEFs of the indicated genotypes were immunoprecipitated (IP) 5–8), but at this level of resolution, no cyclin D1–CDK4 with antibodies to CDK4 (K4) or cyclin D1 and the separated proteins were blotted with the cognate or reciprocal antibodies. (B) Cell lysates complexes were detected in immune precipitates from were normalized for cyclin D1 abundance, and D1 immune cells lacking both CKIs (lanes 3 and 4). precipitates were blotted with antibodies to cyclin D1 or CDK4. Two Reduced cyclin D1–CDK4 complex formation in electrophoretic forms of cyclin D1 detected in this experiment can be double-null MEFs may have simply reflected the lower routinely observed when separation conditions are sufficiently stringent (Matsushime et al., 1991); both are phosphoproteins and the nature of levels of cyclin D1 expressed in these cells. However, the differences between them remains unclear (Diehl et al., 1998). several lines of evidence indicate that this is not the (C) Wild-type or p21/p27 double-null MEFs were infected with explanation. First, when cell lysates were normalized so retrovirus encoding Flag-tagged cyclin D1. Cell lysates prepared 48 h that each contained comparable amounts of cyclin D1 post-infection were precipitated with a control monoclonal antibody protein, the levels of CDK4 that co-precipitated with (C) or with antibodies to the Flag epitope (M2), and the separated proteins were blotted with antibodies to cyclin D1 or CDK4. (D) Cell cyclin D1 were again found to be decreased in p21-null lysates from MEFs with the indicated genotypes were precipitated or p27-null cells (Figure 1B, lanes 2 and 3) and were very with antibodies to cyclin D2 and the separated proteins were blotted much reduced in p21/p27 double-null MEFs, whether the with antibodies to cyclin D2 or CDK4. All immunoblots were cells were in early (p7) or late (p15) passage (Figure 1B, visualized using enhanced chemiluminescence. lanes 4 and 6). We estimated that p7 double-null cells contained 10% of the D1–CDK4 complexes detected in age-matched wild-type MEFs. Secondly and more the p21/p27 double-null cells do not account for the defect importantly, infection of p21/p27 double-null MEFs for observed in cyclin D1–CDK4 assembly. 48 h with a retrovirus encoding Flag epitope-tagged cyclin MEFs also express cyclin D2 (Figure 1D, lane 1) but D1 restored high levels of D1 expression but not D1– little detectable cyclin D3 (data not shown). Compared CDK4 complex formation (Figure 1C). This demonstrated with wild-type MEFs, cyclin D2 levels were reduced by directly that the lower levels of cyclin D1 expressed in ~30% in cells lacking either p21 (lane 2) or p27 (lane 3) 1572 CDK inhibitors promote cyclin D–CDK assembly p27 double-null MEFs (lanes 5 and 9), consistent with observations that few cyclin D–CDK4 complexes were formed (Figure 1). Cell lysates from wild-type MEFs were subjected to two rounds of immunodepletion using antisera to p21, p27 or to both, and CDK4 kinase activity was measured using glutathione S-transferase (GST)-Rb as the substrate. Removal of p27 (Figure 2B, lane 3) or p21 (lane 4) from lysates of wild-type MEFs partially reduced CDK4 kinase activity, whereas elimination of both p21 and p27 (lane 5) completely depleted the activity. Similarly, removal of p27 from lysates of p21-null MEFs (lane 7) or vice versa (lane 9) depleted all CDK4 kinase activity from these lysates. Therefore, both p21- and p27-associated cyclin D–CDK complexes retain activity (Zhang et al., 1994; Soos et al., 1996; Blain et al., 1997; LaBaer et al., 1997), consistent with results that either p21 or p27 is required for efficient assembly of active cyclin D–CDK4 complexes. p21 or p27 promotes assembly of stable cyclin D1–CDK4 complexes in double-null MEFs One prediction is that reintroduction of p21 or p27 into double-null MEFs should increase the assembly of cyclin Fig. 2. Cyclin D1 and CDK4-dependent Rb kinase activity in MEFs D1–CDK4 complexes. We infected these cells either with lacking p21 and p27. (A) Lysates from MEFs of the indicated a control retrovirus encoding the T-cell co-receptor CD8 genotypes were precipitated with non-immune rabbit serum (NRS) or or with retroviruses encoding either p21 or p27 (Figure with antibodies to cyclin D1 or CDK4. Resulting complexes were 3A). Lysates prepared from MEFs infected for 48 h were assayed for kinase activity using GST-Rb as the substrate. (B) Lysates from cells of the indicated genotype were depleted of p21, p27 or precipitated with antibodies to cyclin D1 or CDK4, and both, and then precipitated with antibodies to CDK4 or control NRS. assayed for complex formation. Ectopic expression of Washed immune complexes were assayed for Rb kinase activity. either p21 (Figure 3A, lane 3) or p27 (lane 4) but not CD8 (lane 1) increased cyclin D1–CDK4 complex and, like D1, were significantly decreased in cells lacking formation in p21/p27 double-null MEFs. The N-terminal both CKIs (lane 4). Despite the fact that the D2 signal in portion of p27, which contains the cyclin and CDK binding Figure 1D exceeds the D1 signal in Figure 1A and sites, is sufficient to promote the stable association of B, quantitation of the two cyclins by comparison with cyclin D1 and CDK4, whereas the C-terminal half of p27 recombinant protein standards indicated that the level of is inactive in this assembly assay (data not shown). Ectopic INK4a D1 exceeds that of D2 by 2- to 3-fold (data not shown). expression of another CDK inhibitor, p16 , which Cyclin D2–CDK4 complexes were readily detected in binds to CDK4 or CDK6 but not to D cyclins (Serrano wild-type MEFs and in those lacking either CKI but et al., 1993), did not promote assembly of cyclin D1– were significantly reduced in cells lacking both inhibitors CDK4 in these cells (data not shown). Ectopic expression (Figure 1D, K4 blot). In agreement with previous data of p21 or p27 not only increased the assembly of cyclin showing that CDK4 is the predominant partner of D-type D–CDK4 complexes but also increased the overall levels cyclins in rodent fibroblasts (Matsushime et al., 1994), of cyclin D1 in p21/p27 double-null MEFs to levels that virtually no cyclin D1–CDK6 or D2–CDK6 complexes approached those in wild-type MEFs (Figure 3A, lanes 3 were detected in MEFs. and 4 versus lane 2). D-type cyclin binding is essential for activating CDK4 Cyclin D1 is a labile protein (Matsushime et al., kinase activity (Matsushime et al., 1992). Since association 1992), and its rapid proteolytic degradation is triggered of D-type cyclins with CDK4 was significantly compro- by phosphorylation on Thr286 (Diehl et al., 1997). By mised in the p21/p27 double null MEFs, both cyclin D1- interacting with both cyclin D1 and CDK4, p21 and p27 dependent and total CDK4 kinase activity were measured might slow cyclin D1 turnover, possibly by promoting in these cells (Figure 2A). Cell lysates from proliferating nuclear localization of the complexes (see below) and/or MEFs were precipitated with either non-immune rabbit by interfering with cyclin D1 phosphorylation. Double- serum (NRS), antibody to cyclin D1 or antibody to CDK4, null MEFs were infected for 48 h with a retrovirus and the resulting immune complexes were assayed for encoding p27, and cells were metabolically labeled with kinase activity using recombinant GST-Rb as the substrate. [ S]methionine for 30 min. Medium containing labeled Note that CDK4-dependent kinase activity should include methionine was removed, and cells were incubated in contributions from both D1- and D2-containing holo- complete medium containing a 100-fold excess of un- enzymes (Figure 1). Active cyclin D1- and CDK4-depend- labeled methionine. Cell lysates prepared after different ent Rb kinase activities were detected in precipitates from periods of ‘chase’ were precipitated with the monoclonal wild-type MEFs (Figure 2A, lanes 2 and 6), p21-null antibody to cyclin D1, and the labeled proteins were MEFs (lanes 3 and 7) and p27-null MEFs (lanes 4 and resolved on a denaturing gel (Figure 3B). The half-life of 8). In contrast, only background levels of kinase activity cyclin D1 in p21/p27 double-null MEFs in several such were detected in immune complexes recovered from p21/ experiments was calculated to be 15 min (Figure 3B, lanes 1573 M.Cheng et al. are very low (Dai et al., 1996; Stepanova et al., 1996). Similarly, the vast majority of CDK6 subunits are bound to other molecules (Mahony et al., 1998). CDK4 requires association with Hsp90/Cdc37 for stabilization, suggesting that the latter acts as a chaperone for the proper folding of kinase subunits (Dai et al., 1996; Stepanova et al., 1996). High molecular weight complexes containing Hsp90, Cdc37 and CDK4 or CDK6 are cytoplasmic and do not contain D-type cyclins, so assembly of cyclin D with CDK4, the nuclear translocation of these complexes, and their activation by CAK presumably occur as later steps. We considered the possibility that in p21/p27 double- null cells, the lack of complexes between CDK4 and cyclins D1 and D2 might result in a greater association of CDK4 subunits with Cdc37. Instead, the level of Cdc37- bound CDK4 was lower in double-null cells than in wild- type cells (Figure 4D), suggesting that most CDK4 was complexed with other molecules or remained monomeric. Apart from interacting with D-type cyclins, CDK4 and CDK6 can independently associate with INK4 proteins (Serrano et al., 1993; Guan et al., 1994; Hannon and Beach, 1994; Chan et al., 1995; Hirai et al., 1995). CDK– INK4 complexes are stable, lack Cdc37, cannot assemble with cyclins, and therefore appear inaccessible for enzym- atic activation (Parry et al., 1995; Stepanova et al., 1996). It is therefore presumed that CDK4 can achieve alternative fates after release from the chaperone complex, either assembling with D-type cyclins or being inactivated through INK4 binding. Possibly, p21 and p27 might facilitate assembly of CDK4 and D-type cyclins by blocking the ability of INK4 proteins to sequester CDK4 Fig. 3. Reconstitution of cyclin D1–CDK4 complexes in vivo and in an inactive pool. We therefore studied the expression restabilization of cyclin. (A) Wild-type MEFs or those lacking both INK4a p21 and p27 were infected for 48 h with control virus (CD8) or with of the four different INK4 family members (p16 , INK4b INK4c INK4d viruses encoding p21 or p27. Cells were lysed and immunoprecipitated p15 , p18 and p19 ) and compared their with antibodies to cyclin D1 or CDK4. Separated immune complexes associations with CDK4 in wild-type and p21/p27 double- were then blotted with the cognate or reciprocal antibodies, and sites null MEFs. of antibody binding were detected by enhanced chemiluminescence. Individual INK4 proteins were depleted from cell lysates (B) MEFs lacking both p21 and p27 were infected with a control retrovirus encoding CD8 or with a virus encoding p27. Two days post- by two sequential immunoprecipitations, and the levels of infection, cells were pulse-labeled for 30 min with [ S]methionine CDK4 associated with each INK4 family member were and then ‘chased’ in the presence of 100-fold excess of unlabeled determined by blotting the precipitated proteins with methionine for the indicated times. Lysates normalized for protein antibodies to CDK4 (Figure 4A, lanes 1 and 2). Lysates concentration were precipitated with a monoclonal antibody to cyclin depleted of individual INK4 proteins were then precipit- D1, and the labeled proteins were resolved on a denaturing gel, which was dried and subjected to autoradiography. ated and blotted with antibodies to CDK4, in order to estimate the levels of residual CDK4 that remained 2–5), which is shorter than that in wild-type cells (t  unassociated with INK4 proteins (Figure 4A, lanes 3). 1/2 25 min) (Matsushime et al., 1992; Diehl et al., 1997, Wild-type MEFs expressed significant amounts of CDK4 INK4a INK4b INK4c 1998). In contrast, in cells infected with p27-virus, the in complexes with p16 , p15 and p18 (Figure half-life of D1 exceeded 40 min (Figure 3B, lanes 6– 4A). In cells lacking both p21 and p27, CDK4 associated 9). Metabolic labeling experiments indicated that the with the same three INK4 family members, although in relatively low level of cyclin D1 in p21/p27 double-null comparison with wild-type MEFs, less CDK4 was bound INK4c INK4b MEFs also reflects a 3-fold reduced rate of D1 synthesis to p18 and more was bound to p15 (Figure 4B). versus that in wild-type MEFs (data not shown). Cyclin In both MEF strains, more CDK4 was complexed with INK4a D1 synthesis was only modestly increased following acute p16 than with other INK4 family members, and no INK4d infection of the cells with the p27 retrovirus (Figure 3B, association with p19 was detected. compare lane 6 with lane 2), so the restoration of D1 To determine the relative pools of total INK4-bound levels following reintroduction of p21 or p27 (Figure 3) and -unbound CDK4, MEF lysates were depleted with primarily reflects increased D1 stability. mixtures of antibodies directed to all four INK4 family members, and the above analysis was repeated (Figure Association of CDK4 with Cdc37 and INK4 proteins 4C). From several such experiments, we estimated that in in p21/p27 double-null cells wild-type cells, ~40% of the total CDK4 pool stably Although CDK4 in mouse fibroblasts has a half-life of associated with INK4 proteins. As expected, cyclin D1 ~4 h (Matsushime et al., 1992), unassembled CDK4 co-precipitated only with those CDK4 molecules that subunits are unstable, and the levels of monomeric CDK4 were not bound to INK4 proteins (/ cells, lanes 3). 1574 CDK inhibitors promote cyclin D–CDK assembly Fig. 4. Association of CDK4 with INK4 proteins and Cdc37. Cell lysates from wild-type (A) or p21/p27 double-null cells (B) were sequentially depleted by two rounds of precipitation with non-immune rabbit serum (NRS) or with antibodies to the designated INK4 proteins (lanes 1 and 2) and then precipitated with anti-CDK4 (lanes 3). All recovered proteins were immunoblotted with anti-CDK4. (C) Experiments were performed as above, except that immunodepletion was carried out with a mixture of antibodies to all four INK4 family members prior to blotting of precipitated proteins with anti-CDK4 and anti-D1. (D) Cells of the indicated genotype were lysed and equal quantities of protein (100 μg) were resolved on denaturing gels and immunoblotted directly with anti-CDK4 (lanes 1 and 2) or rabbit anti-Cdc37 (lanes 5 and 6; produced by J.A.D. and C.J.S. to recombinant mouse protein synthesized in bacteria, unpublished). The arrows indicate the position of authentic CDK4 (34 kDa, left) and Cdc37 (50 kDa, right). The faster-migrating band detected with commercial polyclonal antibodies to mouse CDK4 used in this experiment (Santa Cruz Biotechnology) is not observed using antisera raised in our laboratory (R or R ; Matsushime et al., 1994). To quantitate complex formation, 5-fold Y Z more lysate protein (500 μg/lane) precipitated with anti-Cdc37 was separated on denaturing gels and blotted with anti-CDK4 (lanes 3 and 4). Importantly, all enzymatically active CDK4 is bound to Table I. Subcellular localization of cyclin D1 in cells lacking CKIs D-type cyclins, and this fraction also contained associated p21 and p27 molecules (Figure 2). In p21/p27 double- Genotype % Nuclear % Nuclear and % Cytoplasmic cytoplasmic null cells, the INK4-bound CDK4 fraction was increased to ~50–60%; D1–CDK4 complexes were again not p21 p27 detected even though a substantial pool of non-INK4- 61 421 318  3 bound CDK4 remained (–/– cells, lanes 3). Therefore, in 49 423 328  4 wild-type MEFs, ~40% of CDK4 is associated with cyclin 46 426 428  4 D1 and 10–15% is associated with D2 (Figure 1), ~40% 38 328 334  4 is associated with INK4 proteins (Figure 4C), and much Flag-tagged D1 retrovirus of the remainder is bound to Cdc37 (Figure 4D; see figure 59 523 218  2 legend for amounts of protein loading per lane). In p21/ 7 364 529  4 p27 double-null cells, little CDK4 binding to D cyclins was detected (Figure 1), ~60% was bound to INK4 Flag-tagged D1 (T286A) retrovirus proteins (Figure 4C) and 10% was complexed to Cdc37. 82 514 24  1 Therefore, a substantial fraction of CDK4 must either 77 717 56  2 remain monomeric or is associated with as yet unidentified Proliferating wild-type MEFs and those lacking p21, p27 or both were molecules. This indicates that p21 and p27 do not simply stained with monoclonal antibody to cyclin D1 and scored by compete with INK4 proteins in directing cyclin D–CDK immunofluorescence for the presence of nuclear and/or cytoplasmic assembly. D1. Cells infected with retroviruses encoding Flag-tagged D1 or the D1 (T286A) mutant that is stable and remains in the nucleus throughout interphase were studied similarly. Because ectopic p21 or p27 can facilitate nuclear accumulation of expression of D1 greatly exceeded that of the endogenous protein, no cyclin D1 background signals were detected at the exposures used. At least 500 Cyclin D1 normally accumulates in the nuclei of cells cells were counted per experiment and the results show mean  SD during G phase but relocalizes to the cytoplasm during 1 from three such experiments. S phase (Baldin et al., 1993). Although cyclin D1 has no obvious nuclear import signal, p21 family members can direct the nuclear localization of cyclin D1–CDK4 com- stained with antibody to cyclin D1, 61% of the cells plexes (Diehl and Sherr, 1997; LaBaer et al., 1997), exhibited strong nuclear fluorescence (Table I). Fluores- raising the possibility that cyclin D1 might not be able to cence-activated cell sorter (FACS) analysis of DNA con- enter the nucleus in MEFs lacking both p21 and p27. tent indicated that 52% of the total asynchronously When asynchronously proliferating wild-type MEFs were proliferating population were in G phase, and in agree- 1575 M.Cheng et al. CDK4 in cells lacking both p21 and p27 (Figure 5, lane 4, CDK4 blot; also see Figure 1C). These experiments left open the possibility that the ability of p21 and p27 to promote assembly of cyclin D1– CDK4 complexes was an indirect effect of their directing cyclin D1 to the nucleus. Accordingly, we took advantage of a mutant form of cyclin D1 (T286A) that contains an alanine for threonine-286 substitution, is remarkably stable (t 3 h) (Diehl et al., 1997), and remains in the nucleus 1/2 throughout the cell cycle (Diehl et al., 1998). When infected with a vector encoding D1 (T286A), both wild- type MEFs (82%) and p21/p27 double-null MEFs (77%) displayed an exclusively nuclear staining pattern of the D1 mutant (Table I), reinforcing the concept that D1 can enter the nucleus in the absence of both p21 and p27. Moreover, the abundance of cyclin D1 (T286A) was similar in both wild-type and double-null MEFs (Figure Fig. 5. Assembly of ectopically expressed cyclin D1 and D1 (T286A) 5, lanes 3 and 5). Even under these conditions, D1 (T286A) with CDK4. Wild-type and double-null MEFs were infected with retrovirus encoding Flag-tagged cyclin D1 or D1 (T286A) for 48 h. could not assemble with CDK4 (Figure 5, lane 5, CDK4 Lysates were then precipitated with a control monoclonal antibody (C) blot). This argues that CKIs do not ensure assembly by or with the monoclonal antibody to the flag epitope (M2), and the simply contributing a nuclear import signal, in agreement resulting precipitates were resolved on a denaturing gel and transferred with results obtained with mutants of p21 (LaBaer et al., to nitrocellulose. Proteins were visualized by enhanced chemiluminescence using a monoclonal antibody to D1 or antibodies 1997) and p27 (not shown) that lack the signal sequences. to CDK4. These results also confirm that decreased assembly of cyclin D1–CDK4 complexes is not simply secondary to ment with others’ results (Baldin et al., 1993), double- the decreased abundance of cyclin D1 in p21/p27 double- labeling with BrdU for 2 h prior to staining indicated null MEFs. that ~90% of cells exhibiting bright nuclear cyclin D1 fluorescence were not in S phase (data not shown). Rb phosphorylation in p21/p27 double-null cells Although a smaller fraction of cells lacking p21, p27 or In normal MEFs, phosphorylation of Rb is triggered by both exhibited exclusively nuclear staining, the lower cyclin D-dependent kinases and is probably completed by levels of cyclin D1 expressed were still able to enter the cyclin E–CDK2 (and/or cyclin A–CDK2) as cells enter S nucleus (Table I). In agreement with immunoblotting phase. Since active cyclin D-dependent kinase activity results (Figure 1), the intensity of cyclin D1 staining in was not detected in the p21/p27 double-null MEFs (Figure p21/p27 double-null MEFs was much lower than that of 2), we studied the kinetics of Rb phosphorylation in these wild-type MEFs (as judged by the need for a 6-fold cells. MEFs arrested by contact inhibition and serum increase in exposure time to obtain an almost comparable starvation were trypsinized and reseeded at lower density signal). Hence, the CDK inhibitors are not strictly required in complete medium containing 10% fetal bovine serum for cyclin D1 nuclear import. Moreover, the fact that a (FBS). Cell lysates prepared at different times thereafter significant fraction of cyclin D1 was detected in the were precipitated with antibody to Rb, and the resulting nucleus of double-null cells (Table I) whereas 95% immunoprecipitates were resolved on a denaturing gel, failed to assemble with CDK4 (Figure 1) suggests that transferred to nitrocellulose and blotted with the cognate stable association with catalytic subunits is also not antibodies. The percentage of cells in S phase was estim- essential for D1 nuclear import. ated by flow cytometric analysis of DNA content. As cells We next used infection with a retrovirus encoding Flag- approached S phase, Rb appeared to undergo phosphoryl- tagged D1 to increase cyclin D levels in double-null cells. ation, as manifested by its characteristic retardation in After infection for 36 h, similar amounts of Flag-D1 were electrophoretic mobility on denaturing gels (Figure 6A). expressed in both wild-type and the double-null MEFs, The kinetics of Rb phosphorylation and the rate of cell as demonstrated by immunoprecipitation with M2 anti- cycle progression were quite similar in p21/p27 double- bodies to the tag followed by immunoblotting with anti- null and wild-type MEFs. Hence, in the absence of the bodies to D1 (Figure 5, lanes 2 versus 4, D1 blot). These CKIs, resident cyclin-dependent kinases are sufficient levels of ectopically expressed cyclin D1 exceeded the to phosphorylate Rb, canceling its growth suppressive endogenous level of cyclin D1 in double-null MEFs by function as cells exit G phase. 10-fold (data not shown). Most infected wild-type MEFs Although the levels of cyclin D-dependent kinase (59%) displayed an exclusively nuclear cyclin D1 staining activity expressed in p21/p27 double-null cells were pattern (Table I). In marked contrast, 7% of p21/p27 reduced below the limits of detection (Figure 2), several double-null MEFs exhibited exclusively nuclear cyclin D1 lines of evidence suggest that there may be some residual staining, and instead, the cells displayed both nuclear and kinase activity. First, using antibodies that detect Rb cytoplasmic staining or exclusively cytoplasmic staining phosphorylated on Ser780, a site reported to be specifically (Table I). Therefore, under conditions in which cyclin D1 phosphorylated by cyclin D-dependent kinases (Kitagawa was restored, an absence of p21 and p27 limited D1 et al., 1996), forms of Rb phosphorylated on this residue nuclear accumulation. Importantly, ectopically expressed could be detected in cycling (lanes 4 and 6) but not in Flag-tagged cyclin D1 was still unable to assemble with quiescent (lane 5) p21/p27 double-null cells (Figure 6B). 1576 CDK inhibitors promote cyclin D–CDK assembly Second, when we infected double-null cells with a retro- INK4a virus encoding p16 , we observed inhibition of S phase entry, albeit not nearly to the same extent as that observed with the wild-type MEFs (Figure 6C). As expected, both MEF strains remained highly sensitive to growth arrest by a retrovirus encoding p27. Since, unlike p27, INK4 proteins appear to specifically target CDK4 and CDK6, these data imply that p21/p27 double-null cells are not entirely devoid of cyclin D-dependent kinase activity. We also measured total CDK2 or cyclin E-dependent kinase activity in lysates of the various MEF strains, using either histone H1 or GST-Rb as the substrate (Figure 7). Although the levels of CDK2 were comparable in the different cell strains (Figure 7D), both CDK2 and cyclin E-dependent kinase activity were enhanced in p21/p27 double-null MEFs, as compared with those in wild-type MEFs (Figure 7A–C). Therefore, in the absence of p21 and p27, unopposed CDK2 activity may compensate for the severe reduction in CDK4 function. In agreement, p21- and p27-null MEFs are not significantly perturbed in cell cycle progression and exhibit generation times and S phase fractions similar to those of wild-type cells (Figure 6A and data not shown). Assembly of cyclin D–CDK complexes in tissues from double-null mice Although the above studies were performed with primary MEFs, a clear prediction is that assembly of different cyclin D-dependent kinases would be perturbed in many other cell types. Extracts of whole liver from wild-type and double-null mice expressed all three D-type cyclins together with CDK6 (Figure 8, lanes 1 and 2). When these were immunoprecipitated with a mixture of antibodies to cyclins D1, D2 and D3 and then immunoblotted with antibodies to CDK6, significantly fewer cyclin D–CDK6 complexes were observed in liver from double-null animals. Similar results were obtained with CDK4 in complexes with D1 (data not shown). T lymphocytes primarily express cyclins D2 and D3 in conjunction with CDK6 (Meyerson and Harlow, 1994), and thymic extracts from double-null mice also contained much lower levels of cyclin D–CDK6 complexes than those from wild-type animals (Figure 8, lanes 3 and 4). Assembly of cyclin D– CDK4 complexes was only modestly reduced in extracts Fig. 6. Rb status and p16-induced arrest. (A) Wild-type and double- null MEFs made quiescent by contact inhibition and serum starvation of kidney and heart from double-null mice (data not were trypsinized, reseeded and stimulated to enter the cell cycle in shown). Therefore, despite the combinatorial nature of complete medium containing 10% FBS. Cell lysates prepared at expressed cyclin D–CDK complexes in different tissues different times thereafter were immunoprecipitated with polyclonal and the potential participation of other CKIs such as antibodies to mouse Rb, separated on denaturing gels and blotted with Kip2 monoclonal antibody to Rb. The position of the hypophosphorylated p57 in the assembly process, assembly of cyclin D- form of Rb is indicated by the signal in starved cells, whereas dependent kinases was impaired in vivo. additional phosphorylation is connoted by the retardation in Rb’s mobility as cells progress through the division cycle. The fraction of cells in S phase was determined by flow cytometric analysis of DNA Discussion content. Cells entered S phase by ~12 h and were predominately in Cip1 G /M by the completion of the experiment. (B) Rb precipitated from 2 CDK inhibitors of the Cip/Kip family, including p21 , quiescent (lane 5) or proliferating (all other lanes) MEFs of the Kip1 Kip2 p27 and p57 , negatively regulate cell cycle progres- indicated genotypes was immunoblotted with anti-Rb (lanes 1 and 2) sion and enforce cell cycle arrest when expressed at high or with an antibody that specifically detects an epitope containing levels (Elledge and Harper, 1994; Sherr and Roberts, pSer780 (lanes 3–6) (Kitagawa et al., 1996). The position of the pSer780 form of Rb is indicated by arrows in the right margin. 1995). Our results provide a different perspective in (C) MEFs infected for 24 h with a control CD8 retrovirus or with showing that p21 and p27 are necessary for certain INK4a vectors encoding CD8 plus either p16 or p27 were scored for processes that positively regulate cell cycle progression: [ H]thymidine incorporation (2 h pulse). Results with the control CD8 cyclin D assembly with CDK4, its stability and its nuclear vector were normalized to 100%. Standard errors (bars) were calculated from several independent experiments. localization. Hence, the generally prevailing view of Cip/ Kip proteins as universal inhibitors of CDKs appears to 1577 M.Cheng et al. Fig. 7. CDK2 activities in MEFs lacking p21 and/or p27. Lysates from proliferating MEFs were precipitated with antibodies to CDK2 (A and B)or to cyclin E (C), and immune complex kinase assays were performed using histone H1 (A and C) or GST-Rb (B) as substrates. All reactions were stopped at the times indicated by heating samples to 85°C for 5 min in gel sample buffer containing SDS. Labeled proteins were resolved on denaturing gels, which were dried and subjected to autoradiography. Excised slices containing substrates were rehydrated and radioactivity was determined by liquid scintillation. Results are plotted in (A–C) for wild-type MEFs (squares), p21-null MEFs (triangles), p27-null MEFs (inverted triangles) and double-null MEFs (diamonds). (D) CDK2 protein in MEFs of the indicated genotype was detected by immunoblotting with the cognate antibody. Equal aliquots of lysate were separated per lane, and sites of antibody binding were detected by enhanced chemiluminescence. portray too simple a picture of their regulatory effects on the cell cycle. A more accurate representation is that these CKIs are activators of CDK4 and inhibitors of CDK2. This reformulation of the role of Cip/Kip proteins places their inhibitory effect on cell proliferation more specifically at the pivotal transition in the cell cycle between the cyclin D-mediated responses to extrinsic mitogenic cues and the CDK2-mediated progression from G to S phase. Stable association of p21 and p27 with active cyclin D–CDK4 complexes in vivo The solved structure of p27 in a complex with cyclin A– CDK2 illustrates that a single p27 molecule can bind to both the cyclin and CDK subunit and can disrupt the CDK to dismantle its ATP binding site (Russo et al., 1996). Although no analogous structure of a cyclin D–CDK–CKI Fig. 8. Cyclin D–CDK assembly in mouse tissues. Tissue extracts from liver and thymus of wild-type or p21/p27 double-null mice were complex is yet available, p21 and p27 were found to be precipitated with a mixture of antibodies to cyclins D1, D2 and D3 potent inhibitors of binary cyclin D–CDK4 complexes (top panel) or anti-CDK6 (middle panel), and precipitated proteins assembled from recombinant protein subunits (Harper separated on denaturing gels were blotted with the same antibodies. In et al., 1993; Polyak et al., 1994; Toyoshima and Hunter, parallel, cyclin D precipitates were blotted with anti-CDK6 (bottom) to score for complex formation. Proteins were detected by enhanced 1994). However, the idea that p21 and p27 differentially chemiluminescence using exposure times of 10 s (top, middle) and regulate various cyclin–CDK complexes is consistent with 60 s (bottom). data that cyclin E–CDK2 and cyclin A–CDK2 are much more susceptible to inhibition by these CKIs than are cyclin D-dependent kinases in vivo (Soos et al., 1996; prepared in this manner retain Rb but not histone H1 Blain et al., 1997; LaBaer et al., 1997). As also observed kinase activity (Soos et al., 1996), a feature of substrate in our studies, enzymatically active cyclin D–CDK4 com- specificity that distinguishes cyclin D-dependent holo- plexes can be depleted from mammalian cell lysates with enzymes from cyclin A-, B- and E-dependent kinases antibodies to these CKIs. In turn, immune complexes (Matsushime et al., 1992). Therefore, p21 and p27 remain 1578 CDK inhibitors promote cyclin D–CDK assembly stably bound to active holoenzyme complexes during the complex formation. Interactions between p21/p27 and cell cycle. CDK4/CDK6 might also prevent INK4 binding to the How can cyclin D–CDK complexes containing p21 and catalytic subunits (Reynisdottir and Massague´, 1997), p27 retain activity? One possibility is that higher order diverting CDK4 into cyclin D-containing complexes and cyclin D–CDK–CKI complexes recovered from mamma- facilitating assembly through a less direct competitive lian cells contain additional components that protect the mechanism. Cells lacking p27 and p21 would be expected core binary enzyme from CKI-mediated inhibition (see, to accumulate more CDK4–INK4 complexes, and under for example, Zhang et al., 1994). Consistent with this these conditions, cyclin D1 should be destabilized (Bates idea, the mass of active cyclin D–CDK6 complexes et al., 1994; Parry et al., 1995). Increased binding of recovered from T cells has been estimated at 150–170 kDa CDK4 to INK4 proteins was indeed observed, but its (Mahony et al., 1998). Post-translational modifications of association with Cdc37 was diminished relative to that in the included subunits might also alter their activities. This wild-type cells. A significant fraction (~30%) of CDK4 is not to say that Cip/Kip proteins cannot, under certain remained unbound to INK4 proteins or to Cdc37 in circumstances, inhibit CDK4 and CDK6. For example, in double-null MEFs, arguing that mechanisms other than response to negative regulators of G progression such as competition between p21/p27 and INK4 proteins for CDK4 cAMP or TGF-β, the accumulation of Cip/Kip proteins binding play a role in the assembly process. can occlude cyclin D–CDK activation by CAK (Kato Both the levels and assembly promoting activities of et al., 1994; Polyak et al., 1994; Aprelikova et al., 1995). p21 and p27 are governed by mitogenic signals. For Clearly, the nature of the active holoenzymes expressed instance, p21 is frequently induced in cells entering the in mammalian cells needs to be better clarified. cycle from a quiescent state, whereas p27 levels are The fact that Cip/Kip proteins enter into stable, generally high in quiescent cells but fall prior to their enzymatically active complexes with cyclin D and CDK4 entry into S phase (Firpo et al., 1994; Kato et al., 1994; subunits highlights a second, non-catalytic function of Nourse et al., 1994). Hence, p27 should not be limiting cyclin D–CDKs: to ‘titrate’ p21 and p27, thereby freeing in promoting the association of cyclin D1 with CDK4 as other CDKs from their constraint. In the face of an cells enter the cycle, but its presence in a quiescent cell inhibitory threshold set by the CKIs, the latter process is still incapable of assisting assembly of cyclin–CDK sets a dependency of CDK2 activity on the mitogen- complexes (Matsushime et al., 1994). While it might be stimulated assembly of cyclin D–CDK complexes, thereby argued that the level of cyclin D1 in quiescent cells is coordinating the sequential activities of these enzymes as simply too low to allow its entry into complexes with quiescent cells enter the cycle and progress toward S phase. CDK4, ectopically expressed D1 subunits are also unable to assemble with CDK4 in serum-starved fibroblasts Cip/Kip regulation of cyclin D–CDK assembly (Matsushime et al., 1994) and require signals via the The loss of both p21 and p27 in MEF strains decreased Ras-Raf1-MEK-ERK kinase cascade to ensure complex the steady-state levels of assembled cyclin D–CDK4 formation (Cheng et al., 1998). One scenario is that p27 complexes 10-fold and lowered cyclin D- and CDK4- or p21 is subject to phosphorylation by ERKs, and that associated Rb kinase activities to undetectable levels. The only the appropriately modified forms of the CKIs are turnover of D-type cyclins was accelerated in cells lacking able to promote cyclin D–CDK complex formation. Both both CKIs and their overall levels were diminished. of these CKIs are phosphoproteins, and p27 can be Nonetheless, the reduction in cyclin D levels per se did phosphorylated on serine by ERK1 (Zhang et al., 1994; not account for their failure to assemble with catalytic Alessandrini et al., 1997). However, induction of subunits because ectopically overexpressed cyclin D1, enzymatically active MEK1 in NIH 3T3 fibroblasts was whether located primarily in the nucleus or cytoplasm, unaccompanied by detectable phosphorylation of p21 or was also unable to associate with CDK4 in this setting. p27. Also, a mutant version of p27 in which the ERK1 Conversely, reintroduction of p21 or p27 into double- phosphorylation site was changed to alanine was still able null cells reconstituted the formation of cyclin D–CDK to promote cyclin D1–CDK4 assembly (data not shown). complexes and increased the stability of cyclin D1. Other possibilities are that cyclin D and CDK4 must be Assembly of D-type cyclins with CDK4 requires several phosphorylated prior to assembly, or that the chaperone steps. Proper folding of CDK4 relies on the chaperone activity of the Cdc37/hsp90 complex can be regulated function of a cytoplasmic complex that includes Hsp90/ by ERKs. Cdc37 (Dai et al., 1996; Stepanova et al., 1996). Once Several factors are likely to contribute to added com- released from this complex, CDK4 can either enter into plexity in living animals. First, different cell types exhibit complexes with a D-type cyclin or can accumulate in varying levels of Cip/Kip proteins in vivo, so the extent enzymatically inactive binary complexes with an INK4 of assembly promoting activity attributed to each of these protein. The fate of CDK4 is largely determined by the CKIs likely varies between tissues. Double-null mice availability of cyclin D subunits, which accumulate in continue to express cyclin D–CDK4 complexes, albeit at Kip2 response to mitogenic signaling, but how p21 and p27 reduced levels, in many tissues. Although p57 seems contribute mechanistically to the assembly process remains not to be required for assembly of cyclin D-dependent unclear. Both of the latter CKIs contain distinct binding kinases in cultured MEFs, it may well promote assembly sites for CDKs and cyclins, which enable them to contact in other tissues. The possibility that new more distantly both subunits simultaneously (Toyoshima and Hunter, related Cip/Kip family members may be identified and be 1994; Chen et al., 1995, 1996; Luo et al., 1995; Nakanishi shown to play a role in cyclin–CDK assembly cannot be et al., 1995; Lin et al., 1996; Russo et al., 1996). Hence, formally excluded. Secondly, while MEFs preferentially their binding to cyclin D and CDK4 might stabilize synthesize cyclin D1 and D2 in complexes with CDK4, 1579 M.Cheng et al. other cell types such as T lymphocytes, for example, cyclin. Phosphorylation of cyclin D1 on Thr286 is medi- express D3 instead of D1 and much more CDK6 than ated by GSK-3β, and although cyclin D1 turnover in CDK4 (Ajchenbaum et al., 1993; Meyerson and Harlow, proliferating cells is relatively rapid (t  25 min), 1/2 1994). Therefore, the six cyclin D-dependent kinases decreased signaling through the Ras-PI3K-Akt pathway (containing D1, D2 or D3 with CDK4 or CDK6) are activates GSK-3β and further shortens the half-life of likely to be differentially regulated by Cip/Kip family cyclin D1 to ~12 min (Diehl et al., 1998). Moreover, members. Thirdly, the levels of cyclin D–CDK4 complexes overexpression of an active, but not kinase-defective form were only modestly reduced in MEFs lacking p21 or p27, of GSK-3β in mouse fibroblasts causes a redistribution of as compared with those in cells lacking both CKIs. cyclin D1 from the cell nucleus to the cytoplasm (Diehl Possibly, the loss of one of the CKIs facilitates compensa- et al., 1998). Therefore, by suppressing Thr286 phos- tion by the other. Finally, CKIs themselves are regulated phorylation, CKIs might affect the stability of cyclin D1 by signals that affect cell proliferation, organismal via two mechanisms: by promoting its nuclear retention development and cell differentiation (Elledge and Harper, and by preventing its targeting to proteasomes. 1994; Sherr and Roberts, 1995). As a singular example, the Cip1 gene is directly regulated by p53 (El-Deiry et al., Consequences of Cip/Kip loss during the cell 1993; Dulic et al., 1994), and in cancer cells containing division cycle mutant p53, the levels of p21 are generally reduced Remarkably, MEFs lacking p21 and p27 did not exhibit making other CKIs more likely to promote cyclin D–CDK overtly aberrant cell cycles. Indeed, our failure to detect assembly under such circumstances. cyclin D-dependent kinase activity in cells lacking both p21 and p27 raised the possibility that these kinases are CKIs affect cyclin D stability and nuclear not required for cell cycle progression. In an attempt to localization test directly whether p21/p27 double-null cells lack all Cyclin D1 accumulates in the nucleus during G phase cyclin D-dependent kinase activity, we infected these cells but redistributes into the cytoplasm during S phase (Baldin with retroviruses encoding the CDK4- and CDK6-specific INK4a et al., 1993). D-type cyclins and CDK4 lack obvious inhibitor, p16 . Proteins of the INK4 family are pre- nuclear localization signals (NLSs), and p21 or p27 can sumed to act specifically as inhibitors of cyclin D-depend- promote the nuclear import of cyclin D1–CDK4 through ent kinases and are only able to arrest cells that retain Rb putative NLSs at their C-termini (LaBaer et al., 1997). function (Koh et al., 1995; Lukas et al., 1995; Medema When cyclin D1 was ectopically overexpressed in wild- et al., 1995). Despite the absence of detectable cyclin D- type MEFs, most of it localized to the nucleus, but in dependent kinase activity, p21/p27 double-null MEFs p21/p27 double-null cells, it was largely confined to the retained some sensitivity to the cell cycle inhibitory effects INK4a cytoplasm. Therefore, under conditions in which cyclin of overexpressed p16 . Even more inhibition was D1 levels are relatively high, the absence of p21 and p27 obtained using adenovirus vectors that programmed higher can limit its nuclear accumulation. However, assembly of levels of ectopic protein expression (data not shown). In cyclin D–CDK complexes per se does not drive their addition, Rb was phosphorylated on at least one site that nuclear uptake, since endogenous cyclin D1 expressed at is preferentially recognized by cyclin D-dependent kinases. much reduced levels in p21/p27 double-null cells was Therefore, cells lacking p21 and p27 might well express able to accumulate in the nucleus, albeit less efficiently. cryptic cyclin D-dependent kinase activity. In short, while Moreover, a cyclin D1 (T156A) mutant that assembles we were unable to resolve whether cyclin D-dependent with CDK4 remains largely cytoplasmic, although its kinases are dispensable for the division cycle in Rb- nuclear entry can be enforced by p21 overexpression positive cells lacking Cip/Kip proteins, it is evident that (Diehl and Sherr, 1997). Conversely, the D1 (T286A) such cells tolerate a significant reduction in enzyme mutant is preferentially retained in the nucleus even in activity. p21/p27 double-null cells where it does not undergo Although mice lacking cyclins D1 or D2 (or both) assembly. Indeed, this underscores the fact that assembly exhibit focal developmental anomalies (Fantl et al., 1995; is inhibited in the absence of CKIs irrespective of whether Sicinski et al., 1995, 1996), two of the three D-type D1 is predominately cytoplasmic or nuclear. We conclude cyclins are dispensable for most cell divisions in the life that cyclin D–CDK assembly and nuclear uptake are of these animals. Indeed, if the key functions of cyclin separable functions to which the CKIs contribute inde- D-dependent kinases are to phosphorylate Rb and to titrate pendently. CDK inhibitors, then their loss in p21/p27 double-null The overall levels of cyclins D1 and D2 were signific- cells should be well tolerated. In the absence of these antly reduced in MEFs lacking both p21 and p27, but CKIs, the titration function of cyclin D-dependent kinases cyclin D1 was stabilized and its levels were restored when would be superfluous, and unopposed cyclin E–CDK2 either CKI was reintroduced into the double-null cells. and cyclin A–CDK2 activities might be sufficient to How might the presence of CKIs affect cyclin D stability? phosphorylate Rb. Based on changes in its electrophoretic Phosphorylation of cyclin D1 on Thr286 targets it for mobility, Rb phosphorylation increased as p21/p27-null degradation, and elimination of this threonine markedly cells approached the G /S boundary, and the kinetics of increases the half-life of cyclin D1 in proliferating cells cell cycle progression were remarkably similar to those (t 3 h) (Diehl et al., 1997). Cyclins D2 and D3 appear of wild-type MEFs. Together, our results argue that p21 1/2 subject to similar controls (our unpublished data). One and p27 positively regulate the assembly, stability and possibility, then, is that binding of p21 or p27 to cyclin nuclear localization of D-type cyclins. In turn, the activities D1–CDK4 complexes can partially suppress D1 phos- of cyclin E–CDK2 and A–CDK2 are normally opposed phorylation on Thr286, thereby helping to stabilize the by these CKIs, but in their absence, the latter enzymes 1580 CDK inhibitors promote cyclin D–CDK assembly 10 mM MgCl , 1 mM DTT) Reaction mixtures (50 μl kinase buffer) are likely to compensate for loss of cyclin D-dependent 2 . contained 30 μM ATP with 10 μCi [γ- P]ATP plus 10 μg histone H1 kinase activity. or 5 μg GST-Rb. All reactions were stopped by adding 1/3 volume 3 gel sample buffer and heating at 85°C for 5 min. Labeled proteins were resolved on denaturing polyacrylamide gels, which were dried and Materials and methods subjected to autoradiography. Cells and culture conditions Virus production and infection Cip1 Mouse embryonic fibroblasts (MEFs) from animals deficient in p21 , Human kidney 293T cells were transfected (Chen and Okayama, 1987) Kip1 p27 or both CKIs were established as described previously (Zindy with 15 μg of ecotropic helper retrovirus plasmid plus 15 μgofSRα et al., 1997) and maintained in Dulbecco’s modified Eagle’s medium INK4a vector DNA encoding p21, p27, p16 or Flag-tagged cyclin D1 (DMEM) supplemented with 10% FBS, 2 mM glutamine, 0.1 mM non- (WT) and cyclin D1 (T286A). Cell supernatants containing infectious essential amino acids, 55 μM 2-mercaptoethanol and 100 U/ml each of retroviral pseudotypes were harvested 24–60 h post-transfection, pooled penicillin and streptomycin. To make them quiescent, confluent MEFs on ice and filtered (0.45 μm membrane). Virus infections of exponentially were washed twice with phosphate-buffered saline (PBS) and cultured growing MEFs were performed in a 9% CO atmosphere with 5 ml of in serum-depleted medium [DMEM with 0.1% FBS, 0.04% bovine virus-containing culture supernatants plus 10 μg/ml polybrene (Sigma, serum albumin (BSA), glutamine, penicillin and streptomycin] for 18 h. St Louis, MO) for each 100 mm diameter culture dish. After 5 h, 10 ml Quiescent cells were trypsinized, re-plated at low density and stimulated fresh medium was added, and medium was changed 24 h later. Cells with complete medium containing 10% FBS to enter the division cycle, were harvested 48 h after infection, and the percentage of cells in S and entry into S phase was monitored by estimating the DNA content phase was determined by flow cytometric analysis of DNA content of propidium iodide-stained nuclei using fluorescence-activated flow (Matsushime et al., 1991) or by incorporation of [ H]thymidine into cytometry (Matsushime et al., 1991). The 293T retrovirus packaging replicating cell DNA (Kamijo et al., 1997). cell line and helper virus plasmid (Pear et al., 1993) were obtained from C.Sawyers (University of California, Los Angeles) with permission from David Baltimore (California Institute of Technology). Acknowledgements Immunoblotting, immunoprecipitation and We thank Dr Yoichi Taya for generously supplying antibodies to the Rb immunofluorescence epitope containing phosphoserine 780, and Carol Bockhold, Erin Randell Immunoblotting of cyclin D1, cyclin D2, CDK4, CDK6, p21, p27 and and Esther Van der Kamp for excellent technical assistance. This work the detection of cyclin D–CDK complexes was performed as described was supported in part by NIH grant CA-56819 to M.F.R., by Cancer previously (Cheng et al., 1998). INK4 proteins were precipitated from Center Core Grant CA-21765 and by the American Lebanese Syrian MEFs or mouse tissues as described (Zindy et al., 1997). For detection Associated Charities (ALSAC) of St Jude Children’s Research Hospital. of Rb protein, cells were disrupted in lysis buffer containing 50 mM M.C. is supported by NIH training grant T32-CA-70089 and J.P.O. by HEPES pH 7.0, 0.5% Nonidet P-40, 250 mM NaCl, 1 mM phenylmethyl- the Human Frontier Science Program. J.M.R. and C.J.S. are Investigators sulfonyl fluoride (PMSF), 5 mM EDTA, 4 μg/ml aprotinin, 4 μg/ml of the Howard Hughes Medical Institute. pepstatin (protease inhibitors from Sigma Chemicals, St Louis, MO), 0.5 mM sodium orthovanadate, 5 mM sodium fluoride, and 50 mM β-glycerophosphate. Lysates were clarified by centrifugation, and protein References concentration was determined using a BCA assay kit (Pierce, Rockford, IL). Protein (2 mg) was precipitated with rabbit antibody to Rb (SC- Ajchenbaum,F., Ando,K., DeCaprio,J.A. and Griffin,J.D. (1993) 050, Santa Cruz, CA) and collected with 30 μl of protein A–Sepharose Independent regulation of human D-type cyclin gene expression during (Pharmacia Biotechnology, Uppsala, Sweden). After three washes in G phase in primary human T lymphocytes. J. Biol. Chem., 268, lysis buffer, immune complexes resuspended in 30 μl gel sample buffer 4113–4119. [50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1 mM dithiothreitol Aktas,H., Cai,H. and Cooper,G.M. (1997) Ras links growth factor (DTT), 0.1% Bromophenol Blue], were electrophoretically resolved on signaling to the cell cycle machinery via regulation of cyclin D1 and Kip1 denaturing polyacrylamide gels, transferred to nitrocellulose and probed the cdk inhibitor p27 . Mol. Cell. Biol., 17, 3850–3857. with the mouse monoclonal antibody to Rb (14001A, Pharmingen, Albanese,C., Johnson,J., Watanabe,G., Eklund,N., Vu,D., Arnold,A. and ras CA). Rb protein phosphorylated on Ser780 was detected by direct Pestell,R.G. (1995) Transforming p21 mutants and c-Ets-2 activate immunoblotting using purified antibodies specifically reactive with a the cyclin D1 promoter through distinguishable regions. J. Biol. phosphorylated epitope (Kitagawa et al., 1996). Metabolic labeling and Chem., 270, 23589–23597. measurements of cyclin D1 turnover were performed as previously Alessandrini,A., Chiaur,D.S. and Pagano,M. (1997) Regulation of the described (Diehl et al., 1997). 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Journal

The EMBO Journal – Springer Journals

Published: Mar 15, 1999

Keywords: CDK4; cell cycle; D‐type cyclins; p21Cip1; p27Kip1

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