Identification of cell cycle-dependent phosphorylation sites on the anaphase-promoting complex/cyclosome by mass spectrometry

Phosphorylation has an almost universal role in controlling the properties of proteins that govern progression through mitosis and meiosis. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) and its cofactors are no exception to this rule. However, it is poorly understood how APC/C pathway components are regulated by phosphorylation, i.e., little is known about which amino acid residues on subunits and regulators of the APC/C are phosphorylated by which kinase, when during the cell cycle, where in the cell, and with which functional consequence. As a first step toward answering these questions we have established a procedure for the sensitive and relatively rapid identification of phosphorylation sites on small microgram amounts of the APC/C and on associated regulatory proteins. This procedure will enable studies on the dynamic changes of APC/C phosphorylation during the cell cycle and, in conjunction with chemical biology approaches, will allow one to determine which phosphorylation sites depend on the presence of which kinase activity in living cells.