FIGURE 1.
Enforced expression of Cdk6 and cyclin D3 activates Cdk2 in the absence of anchorage. A, Rb remains phosphorylated at Ser807/811, a Cdk2-specific phosphorylation site in anchorage-deprived REF-K6D3 cells. Rapidly proliferating REF and REF-K6D3 were cultured in MC for 48 h and analyzed for the indicated factors by immunoblotting as described (14). B, Cdk2 activation is prolonged in anchorage-deprived REF-K6D3. Cdk2 and Cdk6 in A were assayed for their amounts and activities as described (14). C, E2F-dependent genes driving S phase onset are up-regulated in anchorage-deprived REF-K6D3. RNA was prepared from the cells harvested in A, and cyclin A, E2F1, and Cdc6 mRNAs were quantified by reverse transcription-coupled real time PCR as described (14). The data shown are averaged values with S.D obtained from three independently isolated samples.