Investigation of the cell cycle regulation of cdk3-associated kinase activity and the role of cdk3 in proliferation and transformation

Abstract

The G1-S transition in mammalian cells has been demonstrated to require the cyclin-dependent kinases cdk2, cdk3 and cdk4/6. Here we show that a novel kinase activity associated with cdk3 fluctuates throughout the cell cycle differently from the expression of cyclin D1-, E- and A-associated kinase activities. Cdk3 kinase activity is neither affected by p16 (in contrast to cdk4/6) nor by E2F-1 (in contrast to cdk2), but is downregulated upon transient p27 expression. We found cdk3 to bind to p21 and p27. We provide evidence that p27 could be involved in the regulation of the cell cycle fluctuation of cdk3 activity: cdk3 protein does not fluctuate and interaction of cdk3 with p27, but not with p21, is lost when cdk3 kinase becomes active during the cell cycle. In Myc-overexpressing cells, but not in normal Ratl cells, constitutive ectopic expression of cdk3 induces specific upregulation of cdk3-associated kinase activity that is still cell cycle phase dependent. Ectopic cdk3, but not cdk2, enhances Myc-induced proliferation and anchorage-independent growth associated with Myc activation, without effects on cyclin D1, E and A protein expression or kinase activities. High levels of cdk3 in Myc-overexpressing cells trigger up- and deregulation of E2F-dependent transcription without inducing the E2F-DNA binding capacity. In contrast to all other studied positive G regulators, cdk3 is unable to cooperate with ras in fibroblast transformation suggesting a function of cdk3 in G1 progression that is different from cyclin D- or E-associated kinase activities. Our data provide first insights into the regulation of cdk3-associated kinase activity and suggest a model how cdk3 participates in the regulation of the G1-S transition.