Abstract
Mps1 is a conserved protein kinase that during mitosis corrects improper kinetochore–microtubule attachments, thereby ensuring chromosome biorientation and balanced chromosome segregation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation in metaphase until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways.During my PhD thesis, I characterized a novel temperature sensitive mutant, mps1-3, that is defective in chromosome biorientation and SAC signaling. The mps1-3 mutation changes a conserved serine in the kinase domain to phenylalanine, and, surprisingly, the Mps1-3 protein has enhanced catalytic activity compared to the wild type protein. Conversely, the protein does not localize at kinetochores, suggesting that the mps1-3 mitotic defects stem from the lack of phosphorylation of critical kinetochore substrates.Through an unbiased screen for spontaneous suppressors of the temperature-sensitivity of the mps1-3 mutant, I found suppressing mutations that affected the interaction between the kinetochore protein Spc105/KNL1 and the phosphatase PP1, suggesting that reduced levels of PP1 at kinetochores underlie the suppression of the temperature-sensitivity of mps1-3 cells. Importantly, the suppressors restored both SAC signaling and proper chromosome bi-orientation, suggesting that the same molecular mechanism underlies the role of Mps1 in both processes. We have proposed that phosphorylation of Spc105/KNL1, which leads to Bub1 kinetochore recruitment and is antagonized by the phosphatase PP1, is a crucial function of Mps1 in the correction of improper kinetochore-microtubule attachments as it is for SAC activation. Consistently, Spc105 phosphorylation and Bub1 kinetochore localization were affected in mps1-3 cells and restored in the aforementioned suppressors. Moreover, artificial recruitment of Bub1 to Spc105 suppressed the chromosome segregation defects of mps1-3 mutant cells.Thus, a main conclusion of my thesis work is that SAC and error correction are triggered by a single sensory device involving Mps1 and antagonized by PP1.Through the same genetic screen, I also found suppressors carrying mutations in the F-box protein Grr1 that is part of the SCF ubiquitin-ligase complex. My preliminary data indicate that Grr1 might localize at kinetochores, suggesting that the SCFGrr1 complex could be a novel player in the control of chromosome bi-orientation at kinetochores. Finally, I could isolate intragenic suppressors, carrying a second mutation in MPS1 that restored the mitotic functions of the Mps1-3 protein. The data gathered so far indicate that this class of suppressing mutations, unlike the extragenic suppressors above, re-establish Mps1 kinetochore localization. Since Mps1 regulates its own turnover at kinetochores by autophosphorylation, we postulated that the elevated kinase activity of the Mps1-3 mutant protein might be responsible for its displacement from kinetochores and the suppressors might re-establish kinetochore localization of Mps1-3 by decreasing its kinase activity. Surprisingly, although most of the suppressing mutations reduced the ability of Mps1-3 to phosphorylate an exogenous substrate, they did not significantly affect autophosphorylation, suggesting that Mps1-dependent phosphorylation of an unidentified kinetochore protein could influence Mps1 residence time.