Multi-kinase phosphorylation of the APC/C activator Cdh1 revealed by mass spectrometry.

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Cdh1 contributes to proper exit from mitosis and maintenance of G(1) phase in eukaryotic cells by activating a large ubiquitin ligase called the anaphase-promoting complex, or cyclosome (APC/C). At the end of G(1), APC/C(Cdh1) is inhibited by cyclin-dependent kinase (CDK) phosphorylation of Cdh1. The specific Cdh1 phosphorylation sites used to regulate APC/C(Cdh1) activity have not been directly identified. Here, we used a mass spectrometric approach to identify the in vivo phosphorylation sites on yeast Cdh1. Surprisingly, in addition to several expected CDK phosphorylation sites, we discovered numerous nonCDK phosphorylation sites. In total, at least 19 serine and threonine residues on Cdh1 are phosphorylated in vivo. Seventeen of these sites are located in the N-terminal half of Cdh1, outside the highly conserved WD40 repeats. The pattern of phosphorylation was the same when Cdh1 was purified from yeast cultures arrested in S, early M and late M. Mutation of CDK consensus sequences eliminated detectable phosphorylation at many of the nonCDK sites. In contrast, mutation of nonCDK sites had no significant effect on CDK phosphorylation. We conclude that phosphorylation of CDK sites promotes the subsequent recognition of Cdh1 by at least one additional kinase. The function of nonCDK phosphorylation may differ from CDK phosphorylation because mutation of nonCDK sites did not result in constitutive activation of APC and consequent cell cycle arrest. These results suggest that phosphoregulation of APC/C(Cdh1) activity is much more complex than previously thought.
Mesh Terms:
Amino Acid Sequence, Cell Cycle, Consensus Sequence, Cyclin-Dependent Kinases, Gene Expression, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Phosphorylation, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Ubiquitin-Protein Ligase Complexes
Cell Cycle Oct. 01, 2004; 3(10);1278-84 [PUBMED:15467459]
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