Human MPS1 Kinase Is Required for Mitotic Arrest Induced by the Loss of CENP-E from Kinetochores

Abstract

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore. INTRODUCTIONThe mitotic checkpoint is a fail-safe mechanism that ensures accurate chromosome segregation by preventing cells from prematurely exiting mitosis in the presence of unaligned chromosomes (Nicklas, 1997;Rieder and Salmon, 1998;Amon, 1999). This checkpoint system is highly sensitive, because even a single unaligned chromosome is sufficient to block cells from entering anaphase (Rieder et al., 1994;Li and Nicklas, 1997). The mitotic checkpoint has been shown to monitor both microtubule attachment and tension generated across sister kinetochores by poleward forces (Rieder et al., 1994;Li and Nicklas, 1997;Waters et al., 1998). Failure of the mitotic checkpoint causes cells to exit mitosis in the presence of unaligned chromosomes and is a major mechanism responsible for aneuploidy (Jallepalli and Lengauer, 2001). Seven mitotic checkpoint genes, BUB1, BUB2, BUB3, MAD1, MAD2, MAD3, and MPS1, were originally identified via genetic screens in Saccharomyces cerevisiae (Hoyt et al., 1991;Li and Murray, 1991;Weiss and Winey, 1996). These genes act along two separate mitotic checkpoint pathways (Clarke and Gimenez-Abian, 2000;Daum et al., 2000;Gardner and Burke, 2000). MPS1, BUB1, BUB3, MAD1, MAD2, and MAD3 monitor kinetochore microtubule attachments and prevent premature chromosome segregation by inhibiting degradation of securin/Pds1 and mitotic cyclins (Wassmann and Benezra, 2001;Peters, 2002). BUB2 acts along a different pathway that monitors spindle integrity and orientation and prevents premature cytokinesis by inhibiting the degradation of the mitotic cyclin Clb2 (Alexandru et al., 1999;Fesquet et al., 1999;Fraschini et al., 1999;Li, 1999;Bardin et al., 2000;Bloecher et al., 2000;Pereira et al., 2000).Many of the mitotic checkpoint genes in yeast are evolutionarily conserved, because orthologues of M...

Dynamic distribution of TTK in HeLa cells: insights from an ultrastructural study

“…of mitotic cells, which is consistent with the outcome from an immunoflourescence study recently reported by Liu et al [26].…”

“…The centrosomal TTK may regulate spindle integrity and dynamics via phosphorylation of MAPs or other proteins involved in centrosomal duplication. During the preparation of this study, Liu et al (2003) have reported that human MPS1 is located at nuclear pore of interphase nucleus and to kinetochore and centrosome of mitotic cells [26]. Given the fact that the temporal order of dissociation of TTK from the kinetochore is correlated with the dephosphorylation profile of TTK reported by Liu et al [26], it is then possible that the phosphorylation of TTK may account for its function at the kinetochore.…”

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…It remains unclear whether the SAC component, Mps1p, also plays a role in centrosome duplication in higher eukaryotes [Fischer et al, 2004;Fisk et al, 2003;Liu et al, 2003;Stucke et al, 2004;Stucke et al, 2002] as it does in budding [Winey et al, 1991] but not in fission yeast [He et al, 1998]. The mammalian Ndc80p complex [McCleland et al, 2003], the ROD and ZW10 kinetochore proteins [Basto et al, 2000;Chan et al, 2000], the outer kinetochore protein Zwint-1 [Obuse et al, 2004;Wang et al, 2004a], and the kinetochore-associated NEK2A protein [Lou et al, 2004] are also required for the spindle checkpoint, indicating that the kinetochore's role in SAC signaling is evolutionarily conserved, although no ROD, ZW10, or Zwint-1 homologues have been identified in yeast.…”

Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint

“…Overexpression of Mps1 in yeast is sufficient to activate the spindle checkpoint and arrests cells in mitosis in the absence of spindle damage (8). Subsequent studies then identified orthologs of Mps1 in various organisms, revealing that the function of Mps1 in the spindle checkpoint is conserved from yeast to man (9)(10)(11)(12). In addition, Mps1 has meiotic and developmental functions in multicellular organisms, such as fly and zebra fish (13)(14)(15)(16)(17).…”