Role of phosphorylation in p34cdc2 activation: identification of an activating kinase.
Phosphorylation of p34cdc2 can both positively and negatively regulate its kinase activity. We have mapped two phosphorylation sites in Xenopus p34cdc2 to Thr-14 and Tyr-15 within the putative ATP-binding region of p34cdc2. Mutation of these sites to Ala-14 and Phe-15 has no effect on the final histone H1 kinase activity of the cyclin/p34cdc2 complex. Phosphopeptide analysis shows that there is at least one more site of phosphorylation on p34cdc2. When Thr-161 is changed to Ala, two phosphopeptide spots disappear and it is no longer possible to activate the H1 kinase activity of p34cdc2. We suggest that Thr-161 is a third site of phosphorylation, which is required for kinase activity. All three phosphorylations are induced by cyclin. None of the phosphorylations appears to be required for binding to cyclin, as indicated by the ability of the triple mutant, Ala-14, Phe-15, Ala-161, to bind cyclin. The activating phosphorylation that requires Thr- or Ser-161 occurs even in a catalytically inactive K33R mutant of p34cdc2 and hence does not appear to be the result of intramolecular autophosphorylation. We have detected an activity in Xenopus extracts required for activation of p34cdc2 and present evidence that this is a p34cdc2 activating kinase which, in a cyclin-dependent manner, probably directly phosphorylates Thr-161.
Solomon
M J
MJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Lee
T
T
Kirschner
M W
MW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000302:isolation & purification / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004789:Enzyme Activation; D015151:Immunoblotting; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014638:Vanadates / Q000494:pharmacology; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D016213:Cyclins; D014638:Vanadates; D000255:Adenosine Triphosphate; D016203:CDC2 Protein Kinase