Figure 3.

Cdk6 is activated irrespective of the presence or absence of oncogenic stimulation. (A) Oncogenic stimulation does not influence Cdk6 activity. NRK cells were arrested in G₁ by serum starvation and contact inhibition and stimulated to start cell cycling by the addition of FCS or FCS containing EGF plus TGF-β (E+T). After incubation for various times, 5 × 10⁶ cells were harvested at each time point and lysates were prepared. Cdk4 and Cdk6 were immunoprecipitated from the lysates and assayed for kinase activity. (B) The Cdk6–cyclin D1 overexpressor K6D1 expresses Cdk6 10-fold more. K6D1 cells in a log phase were lysed, and immunoblot analysis was performed as in Fig. 2. (C) Cdk6 kinase activity in K6D1 cells. K6D1 cells were arrested in G₁ by serum starvation and contact inhibition and stimulated with FCS or FCS containing EGF plus TGF-β for 12 hr. At 0 and 12 hr, 5 × 10⁵ cells were harvested and lysed. Cdk6 then was immunoprecipitated and assayed for kinase activity. (D) Immunodepletion of Cdk4 still blocks the S phase onset of the Cdk6 overexpressor. K6D1 cells were arrested by serum starvation, released to start cell cycling in the presence of FCS alone or FCS containing EGF plus TGF-β, microinjected with αCdk4 and/or αCdk6 antibodies, and examined for S phase entry as in Fig. 1. The results are expressed as the relative percentage of BrdUrd-positive cells, with the data of IgG-injected control cells as 100%, and as average values ± SDs (error bars) in two separate experiments.