REGULATION OF CYCLIN-DEPENDENT KINASE-4 DURING ADIPOGENESIS INVOLVES SWITCHING OF CYCLIN-D SUBUNITS AND CONCURRENT BINDING OF P18(INK4C) AND P27(KIP1)

Terminal differentiation of many cell lineages involves an exit from t he mitotic cycle and entry into, and maintenance of, a permanent state of G(1) arrest. We found that during terminal differentiation of mous e 3T3-L1 preadipocytes, the level of cyclin-dependent kinase 4 (CDK4) remained constant, but the subunit composition of the CDK4 complex und erwent a dynamic rearrangement. As 3T3-L1 cells differentiated, the le vels of cyclin D1 and cyclin D1-CDK4 complexes declined to negligible levels. Meanwhile, cyclins D2 and D3 levels and their associations wit h CDK4 increased transiently and persistently, respectively, with cycl in D3 becoming the predominant cyclin partner of CDK4 in mature adipoc ytes, At least five CDK inhibitors are expressed during the differenti ation program of 3T3-L1 cells. Both p15(INK4b) and p16(INK4a) continuo usly declined to undetectable levels immediately after differentiation induction. p21 was transiently expressed during the exit of 3T3-L1 ce lls from mitotic clonal expansion and then decreased to undetectable l evels in mature adipocytes, The level of p27(Kip1) and p27-CDK4 comple xes remain high during differentiation and in mature adipocytes. Disti nctly, there is a remarkable induction of p18(INK4c) mRNA and protein that was not seen in the closely related nondifferentiating 3T3-C2 cel l line, suggesting that p18 induction in 3T3-L1 cells is related to ce ll differentiation, not cell cycle arrest. The pRb kinase activity of cyclin D3 and CDK4 was not detected in quiescent 3T3-L1 cells and was then induced as the cells entered the mitotic clonal expansion phase. Unexpectedly, cyclin D3 and CDK4 pRb kinase activity remained high aft er 3T3-L1 cells completed their mitotic division and was still readily detectable in mature adipocytes, Our study reveals an active regulati on, rather than passive inhibition, of CDK4 activity during adipocyte differentiation. Two central features of this complex regulation are s witching of activating cyclin D subunits and concurrent binding by the p18 and p27 CDK inhibitors.