Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.

We have established a one-hybrid screen that allows the in vivo localization of proteins at a functional Saccharomyces cerevisiae centromere. Applying this screen we have identified three proteins-Ctf19, Mcm21, and the product of an unspecified open reading frame that we named Okp1-as components of the budding yeast centromere. Ctf19, Mcm21, and Okp1 most likely form a protein complex that links CBF3, a protein complex directly associated with the CDE III element of the centromere DNA, with further components of the budding yeast centromere, Cbf1, Mif2, and Cse4. We demonstrate that the CDE III element is essential and sufficient to localize the established protein network to the centromere and propose that the interaction of the CDE II element with the CDE III localized protein complex facilitates a protein-DNA conformation that evokes the active centromere.

Stable maintenance of genetic information requires chromosome segregation to occur with high accuracy. Anaphase is triggered when ring-shaped cohesin is cleaved by separase, a protease regulated by association with its inhibitor securin. Dispensability of vertebrate securin strongly suggests additional means of separase regulation. Indeed, sister chromatid separation but not securin degradation is inhibited by constitutively active cyclin-dependent kinase 1 (Cdk1) and can be rescued solely by preventing phosphorylation of separase. We demonstrate that Cdk1-dependent phosphorylation of separase is not sufficient for inhibition. In a second step, Cdk1 stably binds phosphorylated separase via its regulatory cyclin B1 subunit. Complex formation results in inhibition of both protease and kinase, and we show that vertebrate separase is a direct inhibitor of Cdk1. This unanticipated function of separase is negatively regulated by securin but independent of separase's proteolytic activity.

Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.