DUAL PHOSPHORYLATION OF THE T-LOOP IN CDK7 - ITS ROLE IN CONTROLLING CYCLIN-H BINDING AND CAK ACTIVITY

A cyclin-dependent kinase (cdk)-activating kinase (CAK) has been shown previously to catalyze T-loop phosphorylation of cdks in most eukaryo tic cells, This enzyme exists in either of two forms: the major one co ntains cdk7, cyclin H and an assembly factor called MAT-1, whilst the minor one lacks MAT-1, Cdk7 is unusual among cdks because it contains not one but two residues (S170 and T176 in Xenopus cdk7) in its T-loop that are phosphorylated in vivo, We have investigated the role of S17 0 and T176 phosphorylation in the assembly and activity of cyclin H-cd k7 dimers, In the absence of MAT-1, phosphorylation of the T-loop appe ars to be required for cdk7 to bind cyclin H. Phosphorylation of both residues does not require cyclin H binding in vitro, Phosphorylation o f S170 is sufficient for cdk7 to bind cyclin H with low affinity, but high affinity binding requires T176 phosphorylation, By mutational ana lysis, we demonstrate that in addition to its role in promotion of cyc lin H binding, S170 phosphorylation plays a direct role in the control of CAK activity, Finally, we show that dual phosphorylation of S170 a nd T176, or substitution of both phosphorylatable residues by aspartic residues, is sufficient to generate CAK activity to one-third of its maximal value in vitro, even in the absence of cyclin H and MAT-1, and may thus provide further clues as to how cyclins activate cdk subunit s.