Laboratory of Molecular Biophysics Laboratory Journal 2001 Prof. L. N. Johnson

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Nick R. Brown and John Sinclair

CDK Activating Kinase (CAK)

CAK is a positive regulator of CDK1, CDK2 and CDK4. The kinase phosphorylates CDKs on the threonine residue in the activation segment (Thr160 in CDK2). CAK is itself a member of the CDK family and is composed of CDK7, cyclin H and an assembly protein Mat1. In the presence of Mat1, activation of the CDK7/cyclin H complex does not require phosphorylation of the activation segment for activity. In the absence of Mat1, phosphorylation of two residues in the activation segment (Ser170 and Thr176 in Xenopus CDK7) is required for activity. CDK7 raises intriguing questions concerning substrate recognition. CDK7/cyclin H does not promote its own phosphorylation but is able to phosphorylate CDK2/cyclin A. Likewise CDK2/cyclin A is able to phosphorylate CDK7/cyclin H but does not phosphorylate itself. Garrett et al. (Garrett et al. 2001) have produced a hybrid CDK in which the activation segment of CDK2 was replaced with the activation segment from CDK7. It was found that the CDK2 (CDK7activation segment) hybrid was a substrate for CDK7/cyclin H but not for CDK2/cyclin A and the CDK2 (CDK7 activation segment) hybrid could phosphorylate CDK7 but not CDK2. The results demonstrate that sites elsewhere from the catalytic site on the CDK appear to be important for substrate recognition.

Last year we reported the production in baculoviral infected insect cells of quantities of Xenopus CDK7/cyclin H for crystallisation trials. We were able to show by mass spectrometry that in the CDK7/cyclin H complex, CDK7 was fully phosphorylated on its two sites in the activation segment presumably by kinases present in the insect cells (Lawrie, A. M. et al. 2001). This year we have extended the studies to expression of human CDK7/cyclin H by co-infection of insect cells with the viruses encoding the respective proteins and with the third protein Mat1. We are grateful to Dr David O. Morgan (UCSF) for the generous gift of the viruses. Further purification, characterisation and crystallisation trials are in progress.

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