Figure 2.

Defective activation of G₁ CDKs in fibroblasts lacking c-Jun. (A) Induction of cyclin D1 associated pRb kinase activity and cyclin E-associated histone H1 kinase activity in 3T3 fibroblasts. Quiescent wild-type (+/+) and c-jun^−/− 3T3 fibroblasts (−/−) were stimulated to reenter the cell cycle (see Materials and Methods). At the indicated time points after serum stimulation, whole-cell extracts were prepared, anti-cyclin D1 and anti-cyclin E immunoprecipitates were isolated, and their GST–Rb and histone H1 phosphorylating activities were determined. The levels of coimmunoprecipitated CDK2 in the cyclin E/CDK2 immune complexes were determined by Western blotting. (B) Serum induction of expression of the indicated G₁ cyclins and CDKs in G₀ synchronized and restimulated wild-type (+/+) and c-jun^−/− 3T3 fibroblasts (−/−) was determined by Western blotting. (C) Expression levels of G₁ cyclins and CDKs in two independent wild-type (+/+, lanes 1,2) and two independent c-jun^−/− (−/−, lanes 3,4) exponentially growing 3T3 fibroblast lines. Only the 33-kD splice variant of CDK2 is shown in B and C.