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.

[1]  H. Piwnica-Worms,et al.  Reversible tyrosine phosphorylation and cell cycle control. , 1993, Seminars in cell biology.

[2]  J. Maller,et al.  Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions , 1991, Molecular and cellular biology.

[3]  J. Zheng,et al.  Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[4]  U. Strausfeld,et al.  Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein , 1991, Nature.

[5]  Margaret S. Lee,et al.  Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner. , 1991, The EMBO journal.

[6]  A. Kumagai,et al.  The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system , 1991, Cell.

[7]  M. Kirschner,et al.  Control of p34cdc2 activation. , 1991, Cold Spring Harbor symposia on quantitative biology.

[8]  Marc W. Kirschner,et al.  Cyclin activation of p34 cdc2 , 1990, Cell.

[9]  M. Kirschner,et al.  Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C , 1990, Cell.

[10]  Kathleen L. Gould,et al.  Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis , 1989, Nature.

[11]  D. Beach,et al.  The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis , 1988, Cell.

[12]  T. Hunter,et al.  The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. , 1988, Science.

[13]  M. Wigler,et al.  Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method , 1988, Molecular and cellular biology.

[14]  M. Kirschner,et al.  Regulation of MPF activity in vitro , 1988, Cell.

[15]  D. Beach,et al.  p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. , 1987, The EMBO journal.

[16]  D. Beach,et al.  Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe , 1986, Molecular and cellular biology.

[17]  Andrew R. Cherenson,et al.  The structure of an antigenic determinant in a protein , 1984, Cell.

[18]  K. Kim,et al.  Dephosphorylation and activation of acetyl-CoA carboxylase by phosphorylase phosphatase. , 1980, Biochemical and biophysical research communications.