Systematic Phosphorylation Analysis of Human Mitotic Protein Complexes

Analysis of the phosphorylation of mitotic protein complexes suggests that specific members of the complexes relay regulatory signals to these molecular machines. Regulating Mitotic Machines Most proteins do not function in isolation; they are part of large macromolecular complexes. Hegemann et al. used information available about the protein complexes involved in mitosis and then performed mass spectrometry to determine the phosphoproteome of these mitotic machines. Certain proteins in each complex were phosphorylated at many more sites than other proteins in the complex and thus may represent master regulators of the activities of these mitotic machines. Experiments with specific inhibitors of Polo-like kinase 1 and Aurora kinase B enabled the identification of specific targets of these mitotic kinases, providing insight into the mechanism by which these two kinases regulate the activity of mitotic complexes to control progression through the complicated process of cell division. Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as “switchboards” to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.