Regulation of the Mps1 Protein Kinase and Centrosome Duplication

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DESCRIPTION (provided by applicant): The mammalian Mps1 protein kinase is a key regulator of centrosome duplication and is the focus of our proposed project. Centrosomes are critical for mitotic spindle assembly, and excess centrosomes lead to abnormal spindles that cannot faithfully segregate chromosomes. Errors in chromosome segregation are the leading cause of miscarriage and are observed in the majority of human tumors. The number of centrosomes must be tightly controlled, because as observed in many human tumors excess centrosomes generate aberrant spindles that cause chromosome segregation errors. As with many important cell cycle events, the duplication of centrosomes is temporally restricted by cyclin-dependent kinase activity (Cdk2 in vertebrate cells). However, the mechanisms of temporal regulation and of centrosome duplication itself remain unknown. Our work suggests that Mps1 might be the key to both mechanisms. In the absence of Cdk2 activity Mps1 is removed from centrosomes. This removal requires proteasome activity and is regulated by a single Cdk2 site within Mps1, T468. Mutating T468 to alanine prevents Mps1 from accumulating at centrosomes, but has no effect on whole-cell Mps1 levels. Moreover, Mps1T468A cannot substitute for the function of endogenous Mps1 in centrosome duplication. In contrast, preventing the removal of Mps1 from centrosomes causes the production of extra centrosomes. We identified a centrosome localization domain in the Mps1 N-terminus and a degradation signal surrounding T468, as well as centrosomal proteins that bind to these domains. We also identified an Mps1 phosphorylation site within a centrosomal protein, and mimicking phosphorylation at this site causes centrosome overproduction. Accordingly, we hypothesize that both the targeting of Mps1 to centrosomes and its subsequent degradation at centrosomes are critical for the temporal control of centrosome duplication, and that phosphorylation of Mps1 substrates regulates the assembly of new centrosomes. We have three aims, and we propose to; 1) define specific residues responsible for binding of Mps1 to centrosomes, and to test the role of these residues in the interaction between the Mps1 centrosome localization domain and its binding partners; 2) define specific residues responsible for degradation of Mps1 at centrosomes, and determine how these residues cooperate with T468 to regulate interactions between the Mps1 degradation signal and its binding partners; and 3) determine the contribution of Mps1 phosphorylation to assembly of its substrates into centrosomes. Our experiments will not only elucidate the regulation and function of Mps1, a key centrosome regulator, but will also address two major unanswered biological problems, the temporal control and molecular mechanisms of centrosome duplication. PUBLIC HEALTH RELEVANCE: Chromosome segregation errors are the most common cause of miscarriage and are also observed early in tumorigenesis. This project seeks to characterize the regulation and function of the Mps1 protein kinase, whose misregulation causes the production of extra centrosomes that, like those found in many human tumors, perturb mitotic spindle assembly and cause errors in chromosome segregation. We propose to exploit domains within Mps1 that control its targeting and degradation, centrosomal proteins that bind to these domains, and Mps1 substrates to elucidate the mechanisms that control the production of centrosomes.