Centrosomes are the organelles at mitotic spindle poles that organize microtubules essential for chromosome segregation. Assembly of nascent centrosomes is a tightly regulated process that occurs exactly once each cell cycle. Defects in this process can lead to centrosomal abnormalities, which are frequently observed in tumor cells and are thought to contribute to genetic instability, a hallmark of cellular transformation. Protein phosphorylation is a critical signal involved in centrosome assembly and function, though the molecular details are not well understood. Using the S. cerevisiae centrosome (the Spindle Pole Body, SPB) as a model system, we will identify phosphorylation events that are important to the controlled assembly of a functional organelle. Key events will be tested for conservation in vertebrate cells. The first specific aim of this proposal focuses on the effects phosphorylation has on an important regulator of centrosome duplication, the protein kinase Mps1. Mps1 is tightly regulated through the cell cycle;it is subject to ubiquitin mediated proteolysis by the APC, and stabilized by cyclin dependant kinase phosphorylation. We will test whether phosphorylation by Cdk and autophosphorylation directly prevents ubiquitination in vitro, and assay the in vivo consequences of Mps1 mutated at these sites. The second specific aim is designed to obtain a comprehensive map of the phosphorylation state of the SPB. Phosphorylation sites will be identified on purified SPBs by mass spectrometry. By isolating SPBs from synchronized cells, we will monitor changes in phosphorylation that occur at particular points in the cell cycle. In addition, we will phosphorylate isolated SPBs with candidate kinases to gain an understanding of their contribution to the overall phosphorylation state of the SPB. The final specific aim focuses on the conserved gamma-tubulin complex. Although vertebrate centrosomes and yeast SPBs are structurally distinct, components involved in the nucleation of microtubules are conserved. Once in vivo and in vitro gamma-tubulin complex phosphorylation sites are mapped, we will determine the effects of mutations in these sites on cells, and on the ability of the complex to nucleate microtubules in vitro. By understanding the role phosphorylation plays at centrosomes, we will gain important insights into how centrosome assembly is coordinated with the cell cycle, and how the fidelity of mitosis is maintained.
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