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 G 1 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 G 1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G 1 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.Both positive and negative extracellular growth factors exert their influence upon proliferation during the G 1 phase of the cell cycle. At a point in late G 1 , termed the restriction point (R), cells become largely refractory to these factors and once past R are committed to completing the mitotic cycle (62,87,97). The action of serum growth factors is mediated, in part, through their activation of receptor tyrosine kinases. Ras, an inner plasma membrane-bound GTPase, plays a significant role in receiving and transducing extracellular signals by functioning as a downstream mediator of several membrane-bound receptor and nonreceptor tyrosine kinases (5,45,46,69). Ras influences, and is required for, both the G 0 /G 1 transition and passage through G 1 to a point temporally coincident with the restriction point. In certain situations, Ras has been shown to be sufficient for progression from G 0 to S phase, as demonstrated by the ability of microinjected activated or wild-type Ras to induce DNA synthesis from quiescence in the absence of serum factors (19,84). Furthermore, microinjection of murine fibroblasts with anti-Ras neutralizing antibodies prevents efficient serum-induced S-phase entry from a G 0 state (16,52), and expression of a dominant-negative Ras protein (Ras N17 ) inhibits the proliferation of NIH 3T3 and 32D myeloid cells, resulting in a G 1 arrest (8,18,59).In terms of nuclear events influencing progression through the G 1 phase of the cell cycle, a key role is played by G 1 cyclins and their catalytic subunits, the cyclin-dependent kinases (CDKs). The three D-type cyclins associate with and activate CDK4 and CDK6. Cyclin E is required for the activation of CDK2. The synthesis of D-type cyclins, whose expression is rate limiting for G 1 progression, is ...
