Stimulation of the Raf/MEK/ERK cascade is necessary and sufficient for activation and Thr-160 phosphorylation of a nuclear-targeted CDK2.
The activity of cyclin-dependent kinase 2 is required for G(1)-S-phase progression of the eukaryotic cell cycle. In this study, we examine the activation of CDK2-cyclin E by constructing a CDK2 that is constitutively targeted to the nucleus. Activation of CDK2 requires the removal of two inhibitory phosphates (Thr-14 and Tyr-15) and the addition of one activating phosphate (Thr-160) by a nuclear localized CDK-activating kinase, which is thought to be constitutively active. Surprisingly, nuclear localized CDK2-NLS and CDK2-NLS(A14,F15), which lacks the inhibitory phosphorylation sites, require serum to become active, despite complexing with expressed cyclin E. We show that inhibition of mitogen-mediated ERK activation by treatment with U0126, a selective MEK inhibitor, or expression of dominant-negative ERK markedly reduces the phosphorylation of Thr-160 and enzymatic activity of both CDK2-NLS constructs. Consistent with a role for ERK in Thr-160 phosphorylation, expression of constitutively active Raf-1 induces Thr-160 phosphorylation of CDK2-NLS in serum-arrested cells, an effect that is blocked by treatment with U0126. Taken together, these data show a new role for ERK in G1 cell cycle progression: In addition to its role in stimulating cyclin D1 expression and nuclear translocation of CDK2, ERK regulates Thr-160 phosphorylation of CDK2-cyclin E.
D021581:Active Transport, Cell Nucleus; D000818:Animals; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D008957:Models, Genetic; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D015854:Up-Regulation
D002070:Butadienes; D018797:Cell Cycle Proteins; D019927:Cyclin E; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D009570:Nitriles; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; C113580:U 0126; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases