Subcellular localization of the mitotic checkpoint kinase MPS1: Mechanisms and regulation

Faithful segregation of chromosomes during mitosis is required for the maintenance of genomic stability. The missegregation of chromosomes during mitosis causes aneuploidy and may be an initial step in the development of cancer. To ensure that both daughter cells are endowed with a complete set of chromosomes, a mitotic checkpoint has evolved. This checkpoint ensures that anaphase onset is delayed until all kinetochores have been attached by the mitotic spindle. A separate pathway, the error correction machinery, functions to destabilize incorrect kinetochore-microtubule interactions and creates unattached kinetochores in the process. Together, these separate, but intertwined pathways prevent the onset of anaphase until all kinetochores have been bioriented. The mitotic checkpoint is dominantly regulated by recruitment of the kinase MPS1 to unattached kinetochores. Before the work presented in this thesis, it was however largely unknown which mechanisms govern this process. Here, we report that the MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a TPR domain, for which we have determined the structure. Although the module was necessary for kinetochore localization of MPS1, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required the kinetochore protein HEC1/Ndc80 and Aurora B activity. We show that MPS1 localization to kinetochores depends on the calponin homology domain of HEC1 but not of Aurora B-dependent phosphorylation of the HEC1 tail. Rather, Aurora B impinged on MPS1 localization via the TPR domain, as deletion of the TPR rendered MPS1 localization insensitive to Aurora B inhibition. We suggest that NTE-dependent kinetochore localization can be inhibited by the TPR domain and that this inhibition is relieved by Aurora B activity. We show that Aurora B ensures MPS1 localization by excluding the phosphatase PP1 from kinetochores via phosphorylation of the PP1-docking motifs in KNL1. Reducing kinetochore PP1 renders MPS1 kinetochore levels as well as mitotic checkpoint activity largely resistant to Aurora B inhibition. Conversely, allowing premature PP1 kinetochore binding by mutating the Aurora B phosphorylation site on KNL1 weakens the mitotic checkpoint. This can be overcome by artificially maintaining high levels of MPS1 at kinetochores. This Aurora B-PP1 pathway may impinge on MPS1 localization via the TPR domain, to antagonize the activity of an unknown kinase. These data show that excluding PP1 from kinetochores is a prerequisite for establishing a robust mitotic checkpoint in human cells. We further addressed the role of kinetochore phosphatases in mitotic checkpoint regulation, and we show that PP1 suppresses kinetochore localization of PP2A-B56 by dephosphorylation of its docking site in BUBR1. In turn, PP2A-B56 dephosphorylates the PP1-binding motifs in KNL1, causing PP1 localization and subsequent removal of mitotic checkpoint kinases from kinetochores. Efficient silencing of the mitotic checkpoint thus requires kinetochore localization of both PP1 and PP2A-B56, and we show that tethering the PP2A-B56-binding motif of BUBR1 to kinetochores can force mitotic exit by permitting kinetochore binding of PP1. We thus propose that PP1 and PP2A-B56 are in a localized negative feedback that ensures low levels of mitotic checkpoint-silencing phosphatase activity at kinetochores in prometaphase