Role of CDK-mediated phosphorylation in the regulation of the spindle pole body duplication in budding yeast

Younghoon Oh, Purdue University

Abstract

The spindle pole body (SPB) of the budding yeast Saccharomyces cerevisiae is functionally equivalent to the metazoan centrosome and is the sole microtubule organizing center for the cell. The SPB is necessary for bipolar spindle formation and sister chromatid segregation. To form a functional bipolar spindle, the SPB that each daughter cell inherits must be duplicated only once during each round of cell division. Thus, to ensure proper chromosome segregation and genomic stability, both SPB duplication and SPB-mediated spindle formation must be tightly coordinated with cell cycle progression. Bbp1 and Spc29 are essential components of the yeast SPB that are required for duplication of this organelle. As part of an effort to examine the role of phosphorylation in coordinating SPB function with cell cycle events, we employed mass spectrometry to identify residues of Bbp1 and Spc29 that are phosphorylated in vivo. This analysis revealed that Bbp1 was phosphorylated at four proline-directed sites (Ser/Thr-Pro). Phosphorylation at one of these sites, Ser 29, is essential for cell viability. We demonstrate that Cdk1-dependent phosphorylation of Ser 29 is necessary for normal SPB duplication and formation of a bipolar spindle. Mutations in Bbp1 that prevent phosphorylation at the three other potential Cdk1 sites do not affect cell viability raising the possibility that Cdk1 has multiple roles in controlling SPB function(s). Analyses using a phosphosite-specific antibody show that Ser 29 of Bbp1 is phosphorylated during a period extending from G1 to late mitosis. We identified that Spc29 was phosphorylated at three Cdk-consensus sites and seven non-Cdk phosphorylation sites in vivo. Phosphorylation of Spc29 occurs in both Cdk1- and Mps1 kinase-dependent manner. Several lines of evidence indicate that the protein phosphatase Cdc14 dephosphorylates Ser 29 in Bbp1 during exit from mitosis. We propose a model in which Ser 29 of Bbp1 is phosphorylated by Cdk1 to promote SPB duplication in G1 and is dephosphorylated in late mitosis by Cdc14 to reset the SPB so that its duplication can be properly coordinated with crucial events of the subsequent cell cycle.

Degree

Ph.D.

Advisors

Charbonneau, Purdue University.

Subject Area

Biochemistry

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