Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.     

Induced expression of p16(INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes

To investigate the mode of action of the p16(INK4a) tumor suppressor protein, we have established U2-OS cells in which the expression of p16(INK4a) can be regulated by addition or removal of isopropyl-beta-D-thiogalactopyranoside. As expected, induction of p16(INK4a) results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16(INK4a) also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27(KIP1). Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16(INK4a), this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16(INK4a). Sequestration of CDK4 by p16(INK4a) allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16(INK4a), p27(KIP1) appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27(KIP1) and p21(CIP1). Significantly, p16(INK4a) itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.     

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes

CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols.     

Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.     

Cell cycle and biochemical effects of PD 0183812. A potent inhibitor of the cyclin D-dependent kinases CDK4 and CDK6

Progression through the G1 phase of the cell cycle requires phosphorylation of the retinoblastoma gene product (pRb) by the cyclin D-dependent kinases CDK4 and CDK6, whose activity can specifically be blocked by the CDK inhibitor p16(INK4A). Misregulation of the pRb/cyclin D/p16(INK4A) pathway is one of the most common events in human cancer and has lead to the suggestion that inhibition of cyclin D-dependent kinase activity may have therapeutic value as an anticancer treatment. Through screening of a chemical library, we initially identified the [2,3-d]pyridopyrimidines as inhibitors of CDK4. Chemical modification resulted in the identification of PD 0183812 as a potent and highly selective inhibitor of both CDK4 and CDK6 kinase activity, which is competitive with ATP. Flow cytometry experiments showed that of the cell lines tested, only those expressing pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a loss of pRb phosphorylation and a block in proliferation, which was reversible. These results suggest a potential use of this chemical class of compounds as therapeutic agents in the treatment of tumors with functional pRb, possessing cell cycle aberrations in other members of the pRb/cyclin D/p16(INK4A) pathway.     

Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.

Cyclin-dependent kinases 4 and 6 are complexed with many small cellular proteins in vivo. We have isolated cDNA sequences, INK4d, encoding a 19-kDa protein that is associated with CDK6 in several hematopoietic cell lines. p19 shares equal similarity and a common ancestor with other identified inhibitors of the p16/INK4 family. p19 interacts with and inhibits the activity of both CDK4 and CDK6 and exhibits no detectable interaction with the other known CDKs. p19 protein is present in both cell nuclei and cytoplasm. The p19 gene has been mapped to chromosome 19p13.2, and the level of its mRNA expression varies widely between different tissues. In contrast to p21 and p27 whose interaction with CDK subunits is dependent on or stimulated by the cyclin subunit, the interaction of p19 and p18 with CDK6 is hindered by the cyclin protein. Binary cyclin D1-p18/p19 or cyclin D1-CDK6 complexes are highly stable and cannot be dissociated by excess amounts of cyclin D1 or p19/p18 proteins, suggesting that p16 inhibitors and D cyclins may interact with CDKs 4 and 6 in a competing or potentially mutually exclusive manner.     

The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts

The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D- CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity was well tolerated and had no overt effects on the cell cycle.     

Loss of cyclin-dependent kinase 2 (CDK2) inhibitory phosphorylation in a CDK2AF knock-in mouse causes misregulation of DNA replication and centrosome duplication

Cyclin-dependent kinase 1 (CDK1) inhibitory phosphorylation controls the onset of mitosis and is essential for the checkpoint pathways that prevent the G(2)- to M-phase transition in cells with unreplicated or damaged DNA. To address whether CDK2 inhibitory phosphorylation plays a similar role in cell cycle regulation and checkpoint responses at the start of the S phase, we constructed a mouse strain in which the two CDK2 inhibitory phosphorylation sites, threonine 14 and tyrosine 15, were changed to alanine and phenylalanine, respectively (CDK2AF). This approach showed that inhibitory phosphorylation of CDK2 had a major role in controlling cyclin E-associated kinase activity and thus both determined the timing of DNA replication in a normal cell cycle and regulated centrosome duplication. Further, DNA damage in G(1) CDK2AF cells did not downregulate cyclin E-CDK2 activity when the CDK inhibitor p21 was also knocked down. We were surprised to find that this was insufficient to cause cells to bypass the checkpoint and enter the S phase. This led to the discovery of two previously unrecognized pathways that control the activity of cyclin A at the G(1) DNA damage checkpoint and may thereby prevent S-phase entry even when cyclin E-CDK2 activity is deregulated.     

The fate of pancreatic tumor cell lines following p16 overexpression depends on the modulation of CDK2 activity

Restitution of lost tumor-suppressor activities may be a promising strategy to target specifically cancer cells. However, the action of ectopically expressed tumor-suppressor genes depends on genetic background of tumoral cells. Ectopic expression of p16(INK4a) induces either cell cycle arrest or apoptosis in different pancreatic cancer cell lines. We examined the molecular mechanisms mediating these two different cellular responses to p16 overexpression. Ectopic expression of p16 leads to G1 arrest in NP-9 cells by redistributing p21/p27 CKIs and inhibiting cyclin-dependent kinase CDK2 activity. In contrast, in NP-18 cells cyclin E (CycE)/CDK2 activity is significantly higher and is not downregulated by p16-mediated redistribution of p21/p27. Moreover, inhibition of CDK4 activity with fascaplysine, which does not affect CycE/CDK2 activity, reduces pocket protein phosphorylation in both cell lines, but fails to induce growth arrest. Like overexpression of p16, fascaplysine induces apoptosis in NP-18 cells, suggesting that inhibition of D-type cyclin/CDK activity in cells with high levels of CycE/CDK2 activity activates an apoptotic pathway. Inhibition of CycE/CDK2 activity via ectopic expression of p21 in NP-18 cells overexpressing p16 induces growth arrest and prevents p16-mediated apoptosis. Accordingly, silencing of p21 expression by using small interfering RNA switches the fate of p16-expressing NP-9 cells from cell cycle arrest to apoptosis. Our data suggest that, after CDK4/6 inactivation, the fate of pancreatic tumor cells depends on the ability to modulate CDK2 activity.     

Critical reanalysis of the methods that discriminate the activity of CDK2 from CDK1

Cyclin dependent kinases 1 and 2 (CDK1 and CDK2) play crucial roles in regulating cell cycle progression from G1 to S, through S, and G2 to M phase. Both inhibition and aberrant activation of CDK1/2 can be detrimental to cancer cell growth. However, the tools routinely employed to discriminate between the activities of these 2 kinases do not have the selectivity commonly attributed to them. Activation of these kinases is often assayed as a decrease of the inhibitory tyrosine-15 phosphorylation, yet the antibodies used cannot discriminate between phosphorylated CDK1 and CDK2. Inhibitors of these kinases, while partially selective against purified kinases, may lack selectivity when applied to intact cells. High levels of cyclin E are often considered a marker of increased CDK2 activity, yet active CDK2 targets cyclin E for degradation, hence high levels usually reflect inactive CDK2. Finally, inhibition of CDK2 does not arrest cells in S phase suggesting CDK2 is not required for S phase progression. Furthermore, activation of CDK2 in S phase can rapidly induce DNA double-strand breaks in some cell lines. The misunderstandings associated with the use of these tools has led to misinterpretation of results. In this review, we highlight these challenges in the field.     

Cooperation of p27(Kip1) and p18(INK4c) in progestin-mediated cell cycle arrest in T-47D breast cancer cells

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.     

CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis

Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.     

Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27.

The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic activity. p27(Kip1) is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non-covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27(c-)), CDKs (p27(k-)) or both (p27(ck-)), and demonstrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27(k-) associated with active cyclin E-CDK2 and, unlike wild type p27, p27(c-) or p27(ck-), was efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site (TPKK). Retrovirally expressed p27(k-) was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild-type p27 formed inactive ternary complexes with cellular cyclin E-CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation. p27(ck-), which was not recruited to cyclin E-CDK2, also remained stable in vivo. Thus, selective degradation of p27(k-) depended upon association with active cyclin E-CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin-bound non-inhibitory conformation in vivo.     

Lineage specific composition of cyclin D–CDK4/CDK6–p27 complexes reveals distinct functions of CDK4, CDK6 and individual D‐type cyclins in differentiating cells of embryonic origin

Abstract. Objectives: This article is to study the role of G₁/S regulators in differentiation of pluripotent embryonic cells. Materials and methods: We established a P19 embryonal carcinoma cell‐based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G₁/S regulators were analysed with respect to growth and differentiation parameters of the cells. Results and Conclusions: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E–CDK2 but not of cyclin D–CDK4/6–p27 complexes. In an exponentially growing P19 cell population, the cyclin D1–CDK4 complex is detected, which is replaced by cyclin D2/3–CDK4/6–p27 complex following density arrest. During endodermal differentiation kinase‐inactive cyclin D2/D3–CDK4–p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2–CDK4–p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D–CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G₁/S transition‐regulating machinery in early embryonic cells.     

Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation.

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. Here we report an analysis of the behavior and partners of p27 in Swiss 3T3 mouse fibroblasts during normal mitotic cell cycle progression, as well as in cells arrested at different stages in the cycle by growth factor deprivation, lovastatin treatment, or ultraviolet (UV) irradiation. We found that the level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 was predominantly monomeric, although some portion was associated with residual cyclin A.Cdk2. During G1, all of p27 was associated with cyclin D1.Cdk4 and was then redistributed to cyclin A.Cdk2 as cells entered S phase. The loss of the monomeric p27 pool as cyclins accumulate in G1 is consistent with the in vivo and in vitro data showing that p27 binds better to cyclin.CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 was associated with cyclin D1 and the level of p27 was significantly lower than the level of cyclin D1. In cells arrested in G1 with lovastatin, cyclin D1 was degraded and p27 was redistributed to cyclin A.Cdk2. In contrast to p21 (which is a p27-related CDK inhibitor and is induced by UV irradiation), the level of p27 was reduced after UV irradiation, but because cyclin D1 was degraded more rapidly than p27, there was a transient increase in binding of p27 to cyclin A.Cdk2. These data suggest that cyclin D1.Cdk4 acts as a reservoir for p27, and p27 is redistributed from cyclin D1.Cdk4 to cyclin A.Cdk2 complexes during S phase, or when cells are arrested by growth factor deprivation, lovastatin treatment, or UV irradiation. It is likely that a similar principle of redistribution of p27 is used by the cell in other instances of cell cycle arrest.     

RNA沉默抑制子p19调控细胞周期相关蛋白表达

番茄丛矮病毒的P19蛋白不仅是一个重要的病毒致病因子,而且还可作为RNA干扰(RNAi)的抑制子．这种作用是通过限制细胞内的小RNA,比如小干扰RNA(siRNAs)和微RNA(miRNAs)来实现．但是目前对P19蛋白在哺乳动物细胞上的作用还未见报道．构建了一株p19稳定表达的293细胞系,即293-p19．流式细胞仪分析发现在293细胞中过量表达P19蛋白可显著引发细胞周期的G2/M阻滞．细胞增殖实验显示,293-p19细胞的DNA复制及细胞生长均受到显著的抑制． 此外,研究还发现p19可使人胚肾293细胞内的细胞周期调控子的表达谱发生改变． 其中包括上调cyclin A1,CDK2,CDK4,CDK6,p18,cyclin D2,p19INK4d和E2F1,及下调p15,cyclin A,cyclin B1和cyclin E1的表达．上述研究结果提示,p19有可能靶向多个G2/M调控蛋白从而引发细胞的G2/M阻滞．     

G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.     

Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Here we show that although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules.