Figure 4.

Cdk6 phosphorylation and activation by CAK. (A) Wild-type and mutant forms of GST-cdk6 expressed in bacteria were incubated in the presence of radiolabeled ATP with GST-Cak1p (upper panel) or p40^MO15/cyclin H (lower panel). T177A contains a mutation of the activating phosphorylation site (lanes 6, 7, and 8). Purified His₆-cyclin D1, D2, or D3 was present in the indicated lanes. (B) Activation of cdk6 by CAKs. Wild-type and mutant forms of GST-cdk6 expressed in bacteria were incubated with the indicated D-type cyclins and GST-Cak1p (upper panel) or p40^MO15/cyclin H (lower panel) in the presence of unlabeled ATP. The reaction was then incubated with GST-Rb_605–928 as a substrate in the presence of radiolabeled ATP. Samples were analyzed by 10% SDS-PAGE and autoradiography. His₆-cyclin D1, D2, and D3 proteins were expressed in insect cells and purified via their His₆-tags, whereas GST-cdk6 was expressed in E. coli.