The proposed project's goals are to understand the regulation and function of the conserved MPS1 protein kinase in centrosome duplication using both cultured mammalian cells and yeast. Mitotic spindles are responsible for chromosome transmission and their formation depends upon centrosome function. Understanding centrosome duplication is pertinent to dissecting chromosome missegregation that can lead to aneuploidy and cancer in humans.Mps1p is a relatively unstable protein whose levels fluctuate during the cell cycle. The hypothesis that stability of mouse and yeast Mps 1 p is under the control of cyclin dependent kinase (Cdk) phosphorylation will be tested using structure/function analyses. A specific destruction motif (KEN box) will be assayed for its role in yeast Mpslp stability. Domains affecting centrosomal localization also will be identified.An early and critical step in SPB (the yeast centrosome) duplication is formation of the satellite, an SPB precursor. Analysis of a new MPS1 allele, mps1-8, revealed interactions with known SPB components, Spc42p and Cdc31p. To understand the mechanism of satellite formation, we will analyze the molecular interactions of Mpslp with these and other satellite components, and with a newly identified SPB component, Mps3p. A genetic screen using mps1-8 will be used to uncover new factors required for satellite formation.The mouse mMps1p has been shown to play a role in centrosome duplication using dominant negative and over-expression alleles. The step at which mMps1p acts in centrosome duplication will be determined using RNA interference techniques to inactivate mMps1p The timing of mMpslp function will be examined with respect to other known duplication regulators with the goal of constructing a centrosome assembly pathway. Centrosomal substrates of mMps1p will be identified to elucidate how it affects centrosome duplication.As well as its roles in SPB duplication and the spindle checkpoint in yeast, it is likely that Mps1p has an additional role in chromosome segregation. High resolution imaging analysis and a chemically inhibitable allele of MPS1 will be used to verify the additional mitotic role for Mps1p after SPB duplication. Interactions between Mpslp and a kinetochore protein, Dam1p, will be examined, and a genetic screen using a novel MPS1 mutation will be employed to uncover other genes involved in this function of Mps1p.Overall, we will explore further how Mps1p kinase acts in centrosome duplication and in spindle function.
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