Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

ABSTRACT

The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G₁ phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G₁ arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G₁ arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.

ACKNOWLEDGMENTS

We thank Martine Roussel and Charles Sherr for the cyclin D1 lines, Christopher Marshall for the activated MEK1 retroviral vector, Jacques Pouysségur and Natalie Rivard for the antisense p27 plasmid, Geoffrey Cooper for the Ras^N17 plasmid, Pamela Silver for the GFP plasmid, Warren Pear and David Baltimore for the Bosc23 packaging line, and Edward Harlow for the CDK4 plasmid. We thank Othon Iliopoulos, Christine McMahon, Justin Lamb, Martine Roussel, and Peter Adams for critical review of the manuscript.

This work was supported by National Institutes of Health grant CA65842 to M.E.E. M.E.E. is a Scholar for the Leukemia Society of America.