The cyclin-dependent kinase inhibitor p27^Kip1 is stabilized in G₀ by Mirk/dyrk1B Kinase

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G₀/G₁ until mitogenic signals activate G ₁ cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G₀ in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G₀-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G₀, but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNA_i to Mirk increased cell entry from G₀ into G₁ as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G₀ when Mirk is most abundant. Mitogen stimulation then causes cells to enter G₁, reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNA_i in postmitotic C2C12 myoblasts decreased protein but not MRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.