Abstract
Activation of cyclin-dependent kinase 2 (CDK2)-cyclin E in the late G(1) phase of the cell cycle is important for transit into S phase. In Chinese hamster embryonic fibroblasts (IIC9) phosphatidylinositol 3-kinase and ERK regulate alpha-thrombin-induced G(1) transit by their effects on cyclin D1 protein accumulation (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053). Here, we show that ERK also affects CDK2-cyclin E activation by regulating the subcellular localization of CDK2. Ectopic expression of cyclin E rescues the inhibition of alpha-thrombin-induced activation of CDK2-cyclin E and transit into S phase brought about by treatment of IIC9 cells with LY29004, a selective inhibitor of mitogen stimulation of phosphatidylinositol 3-kinase activity. However, cyclin E expression is ineffectual in rescuing these effects when ERK activation is blocked by treatment with PD98059, a selective inhibitor of MEK activation of ERK. Investigation into the mechanistic reasons for this difference found the following. 1) Although treatment with LY29004 inhibits alpha-thrombin-stimulated nuclear localization, ectopic expression of cyclin E rescues CDK2 translocation. 2) In contrast to treatment with LY29004, ectopic expression of cyclin E fails to restore alpha-thrombin-stimulated nuclear CDK2 translocation in IIC9 cells treated with PD98059. 3) CDK2-cyclin E complexes are not affected by treatment with either inhibitor. These data indicate that, in addition to its effects on cyclin D1 expression, ERK activity is an important controller of the translocation of CDK2 into the nucleus where it is activated.
Highlights
Cyclin-dependent kinase 2 (CDK2)1 is a member of a family of serine/threonine kinases essential for ordered progression through the cell cycle [2]
Because phosphorylates retinoblastoma (pRb), which is the only known substrate of CDK4-cyclin D, binds to and represses the E2F family of transcription factors [9], and the cyclin E gene promoter is under E2F regulation, we expect that cyclin E expression and CDK2-cyclin E activity will be blocked in cells treated with either PD98059, an inhibitor of MEK activation of ERK, or LY294002, a selective inhibitor of PI 3-kinase (Fig. 1)
We initially examined by Western blot analysis the time course of cyclin E protein expression (Fig. 1A)
Summary
Cyclin-dependent kinase 2 (CDK2)1 is a member of a family of serine/threonine kinases essential for ordered progression through the cell cycle [2]. Ectopic expression of cyclin E rescues the inhibition of ␣-thrombin-induced activation of CDK2cyclin E and transit into S phase brought about by treatment of IIC9 cells with LY29004, a selective inhibitor of mitogen stimulation of phosphatidylinositol 3-kinase activity.
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