ASSEMBLY OF CYCLIN D-DEPENDENT KINASE AND TITRATION OF P27(KIP1) REGULATED BY MITOGEN-ACTIVATED PROTEIN-KINASE KINASE (MEK1)

A constitutively active form of mitogen-activated protein kinase kinas e (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expr ession of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphory lated the retinoblastoma protein inefficiently, Activation of the MEK1 /ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27(Kip1)) nor led to activation of cyclin E- a nd A-dependent CDK2, and such cells did not enter the DNA synthetic (S ) phase of the cell division cycle. In contrast, zinc induction of act ive MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblasto ma protein kinase activity approximating those achieved in cells stimu lated by serum. In this setting, p27(Kip1) was mobilized into complexe s containing cyclin D1; cyclin E-and A-dependent CDK2 complexes were a ctivated; and serum-starved cells entered S phase, Thus, although the activity of p27(Kip1) normally is canceled through a serum-dependent d egradative process, overexpressed cyclin D1-CDK complexes sequestered p27(Kip1) and reduced the effective inhibitory threshold through a sto ichiometric mechanism, A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting f or cell cycle progression, Therefore, the MEK/ERK pathway not only act s transcriptionally to induce the cyclin D1 gene but functions posttra nslationally to regulate cyclin D1 assembly with CDK4 and to thereby h elp cancel p27(Kip1)-mediated inhibition.