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

Progress through the G1 phase of the mammalian cell cycle is regulated by the ordered synthesis, assembly, and activation of distinct cyclin-CDK holoenzymes (45, 46). Cyclins D1, D2, and D3 are up-regulated as cells exit from quiescence and associate with their major kinase partners CDK4 and CDK6 (3, 29, 32, 53). These two kinase molecules are highly homologous and associate exclusively with the D-type cyclins (3). Numerous studies have implicated cyclin D-CDK4-CDK6 complexes as key regulators of the cell cycle up to a hypothetical point during late G1 (24, 25), the restriction point, when hyperphosphorylation and inactivation of the retinoblastoma tumor suppressor gene product, pRB, occur (37, 44). In contrast to mitotic cyclin-CDK complexes, the D-type cyclins do not automatically assemble into complexes with either CDK4 or CDK6. For example, when overexpressed in NIH 3T3 cells in the absence of serum, D-type cyclins and CDK4 do not interact efficiently (30). Hence, assembly of D-type cyclins and CDK4 and CDK6 into functional complexes in vivo is likely to depend on numerous factors, in particular, synthesis rates and stability of the various components. Indeed, the D-type cyclins possess canonical PEST sequences near their C termini and have short half-lives in vivo (4, 31). Association of the D-type cyclins with CDK4 and CDK6 is also influenced by the INK4 family of CDK inhibitors (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) (9, 10, 12, 18, 42). INK4 polypeptides bind to the catalytic subunits and inhibit the association of D-type cyclins (10, 38). Typically, human cell lines that lack functional pRB express very high levels of p16INK4a (1, 35, 38). In such cells, CDK4 and CDK6 do not interact with D-type cyclins but are sequestered into long-half-life, binary complexes with p16INK4a (38). These observations have led to a simple model whereby INK4 family members compete with the D-type cyclins for binding to their target CDKs, and uncomplexed D-type cyclins are rapidly degraded. Members of a second family of CDK-regulatory molecules (p21CIP1, p27KIP1, and p57KIP2) (8, 15, 22, 28, 40, 48) comprise a class of polypeptides thought to be broad-spectrum inhibitors of different cyclin-CDK complexes. The prototypic member is p21CIP1, a molecule independently identified by several laboratories. Variously described as a p53-regulated cell cycle inhibitor and a marker induced during cellular senesence (7, 34), p21CIP1 was also cloned biochemically by virtue of copurification with cyclin D1 from mammalian cell extracts (52). In vitro, p21 family members bind to and inhibit the kinase activities of many mammalian cyclin-CDK complexes (16). Recently, evidence has emerged that in addition to simply inhibiting kinase activity, members of the p21 family of molecules may have additional roles. For instance, in overexpression studies, p21CIP1 seemed to play a role during assembly of cyclin D-CDK complexes (20). Aberrant accumulation of active cyclin D-CDK complexes and the inappropriate phosphorylation of pRB are common events in a variety of human tumors (11). Cyclin D1 becomes amplified or overexpressed in many different tumor types (5, 21). Similarly, CDK4 is subject to amplification (17, 26, 41), as well as to point mutations that render it insensitive to INK4 inhibition (50). However, of all the CDK inhibitors, only p16INK4a has been shown convincingly to be a tumor suppressor (19, 39). Consistent with this, p16INK4a levels rise dramatically during cellular senescence (2, 13, 36). In this work, we address the mechanism of cyclin D-CDK assembly by examining specific biochemical properties of individual CDK inhibitors and their associated target molecules. The data suggest that the INK4 and p21 families of CDK-regulatory polypeptides play antagonistic roles during the formation of cyclin D-CDK4-CDK6 holoenzymes. Moreover, we find that the p16INK4a tumor suppressor gene product is unique among the INK4 group, as it alone forms stable complexes with both CDK4 and CDK6 under proliferative conditions, suggesting that this molecule is a specialized member of the INK4 family.