The Plk1-dependent phosphoproteome of the early mitotic spindle

Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. Inearly mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindleassembly and microtubule attachment to kinetochores. However, despite the essential and diversefunctions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate thePlk1-dependent phosphoproteome of the human mitotic spindle, we combine SILAC with Plk1inactivation or depletion, followed by spindle isolation and mass spectrometry. Our studyidentifies 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novelsubstrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 siteswere validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1consensus motif. Collectively our data provides a rich source of information on Plk1-dependentphosphorylation, Plk1 docking to substrates, the influence of phosphorylation on proteinlocalization, and the functional interaction between Plk1 and Aurora A on the early mitoticspindle.