Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.     

Kinetochore localization and microtubule interaction of the human spindle checkpoint kinase Mps1

Members of the Mps1 protein kinase family have been implicated in the regulation of the kinetochore-mediated spindle assembly checkpoint in species ranging from yeast to man. However, conflicting data have been reported on the subcellular localization of vertebrate Mps1 kinases and their possible roles in centrosome duplication. Moreover, little is presently known about the regulation of Mps1 kinases during the cell cycle. Here, we have used immunofluorescence microscopy, immunoblotting and siRNA-mediated depletion of hMps1 to re-investigate the subcellular localization of this kinase. Our data confirm the kinetochore association of hMps1 but suggest that the centrosome staining produced by some anti-hMps1 antibodies could be due to cross-reactivity with other proteins. We also show that the kinetochore association of hMps1 is mediated by the amino-terminal, non-catalytic domain and specifically requires the presence of the Hec1/Ndc80-Nuf2 complex at the kinetochore. Finally, we have combined in vitro binding studies and kinase assays to explore the influence of microtubules on hMps1 activity. Our data indicate that the catalytic domain of hMps1 displays affinity for microtubules and that microtubule binding could contribute to the regulation of kinase activity.Electronic Supplementary Material Supplementary material is available for this article at .Abbreviations DAPI 4,6-Diamidino-2-phenylindole - EGFP Enhanced green fluorescent protein - Mab Monoclonal antibody - MBP Myelin basic protein - PBS Phosphate-buffered saline - RT Room temperature     

Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1

Background

Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.

Methodology/Principal Findings

Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.

Conclusions/Significance

hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.     

Mps1 kinase promotes sister-kinetochore bi-orientation by a tension-dependent mechanism

Segregation of sister chromatids to opposite spindle poles during anaphase is dependent on the prior capture of sister kinetochores by microtubules extending from opposite spindle poles (bi-orientation). If sister kinetochores attach to microtubules from the same pole (syntelic attachment), the kinetochore-spindle pole connections must be re-oriented to be converted to proper bi-orientation. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension. Mps1 is another evolutionarily conserved protein kinase, required for spindle-assembly checkpoint and, in some organisms, for duplication of microtubule-organizing centers. Separately from these functions, however, Mps1 has an important role in chromosome segregation. Here we show that, in budding yeast, Mps1 has a crucial role in establishing sister-kinetochore bi-orientation on the mitotic spindle. Failure in bi-orientation with inactive Mps1 is not due to a lack of kinetochore-spindle pole connections by microtubules, but due to a defect in properly orienting the connections. Mps1 promotes re-orientation of kinetochore-spindle pole connections and eliminates those that do not generate tension between sister kinetochores. We did not find evidence that Ipl1 regulates Mps1 or vice versa; therefore, they play similar, but possibly independent, roles in facilitating bi-orientation.     

The mouse Mps1p-like kinase regulates centrosome duplication.

The yeast Mps1p protein kinase acts in centrosome duplication and the spindle assembly checkpoint. We demonstrate here that a mouse Mps1p ortholog (esk, which we designate mMps1p) regulates centrosome duplication. Endogenous mMps1p and overexpressed GFP-mMps1p localize to centrosomes and kinetochores in mouse cells. Overexpression of GFP-mMps1p causes reduplication of centrosomes during S phase arrest. In contrast, a kinase-deficient mutant blocks centrosome duplication altogether. Control of centrosome duplication by mMps1p requires a known regulator of the process, Cdk2. Inhibition of Cdk2 prevents centrosome reduplication and destabilizes mMps1p, causing its subsequent loss from centrosomes, suggesting that Cdk2 promotes mMps1p's centrosome duplication function by regulating its stability during S phase. Thus, mMps1p, an in vitro Cdk2 substrate, regulates centrosome duplication jointly with Cdk2.     

Phosphoregulation of mixed-lineage kinase 1 activity by multiple phosphorylation in the activation loop

Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.     

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop

Monopolar spindle 1 (Mps1) is a dual-specificity protein kinase, orchestrating faithful chromosome segregation during mitosis. All reported structures of the Mps1 kinase adopt the hallmarks of an inactive conformation, which includes a mostly disordered activation loop. Here, we present a 2.4 Å resolution crystal structure of an “extended” version of the Mps1 kinase domain, which shows an ordered activation loop. However, the other structural characteristics of an active kinase are not present. Our structure shows that the Mps1 activation loop can fit to the ATP binding pocket and interferes with ATP, but less so with inhibitors binding, partly explain the potency of various Mps1 inhibitors.     

Metaphase arrest by cyclin E-Cdk2 requires the spindle-checkpoint kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.     

Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.     

Novel Mps1 kinase inhibitors: From purine to pyrrolopyrimidine and quinazoline leads

Mps1, also known as TTK, is a mitotic checkpoint protein kinase that has become a promising new target of cancer research. In an effort to improve the lead-likeness of our recent Mps1 purine lead compounds, a scaffold hopping exercise has been undertaken. Structure-based design, principles of conformational restriction, and subsequent scaffold hopping has led to novel pyrrolopyrimidine and quinazoline Mps1 inhibitors. These new single-digit nanomolar leads provide the basis for developing potent, novel Mps1 inhibitors with improved drug-like properties.     

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.Massive chromosome missegregation induces cell death as observed by Theodor Boveri in the early 1900s.¹ However, the underlying mechanism remains elusive. The spindle assembly checkpoint (SAC) is a dominant machine monitoring chromosomal segregation during mitosis by delaying the onset of anaphase until all chromosomes are properly captured by microtubules. The SAC consists of kinetochore association sensors, including Mps1 (monopolar spindle 1), Bub1 (budding uninhibited by benzimidazole 1 homolog) and Aurora B; a signaling transducer termed the mitotic checkpoint complex (MCC), including CDC20 (cell division cycle 20), BubR1 (Bub1-related kinase), Bub3 (budding uninhibited by benzimidazole 3 homolog) and Mad2 (mitotic arrest deficient-like 2); and an effector APC/C (anaphase-promoting complex/cyclosome) that is inhibited by MCC in response to an active SAC.² Loss of SAC by inactivation of checkpoint sensors or signaling transducers elicits massive chromosome missegregation, induces severe gain or loss of chromosomes and eventually causes cell death.^{3, 4, 5, 6} Meanwhile, a weakened SAC due to the haploinsufficiency of the checkpoint proteins Mad1, Mad2, Bub1, BubR1 and CENP-E (centromere protein E) does not cause cell death but facilitates tumorigenesis.^{7, 8, 9, 10, 11} These studies suggest that the fate of these cells is dependent on their respective degree of SAC deficiency. Notably, in these studies SAC proteins were constitutively disturbed, raising the possibility that other signaling pathways could be affected as SAC proteins have functions beyond SAC regulation.^{12, 13, 14}Mps1 is an essential component of SAC that senses SAC signal by promoting MCC formation via kinetochore recruitment of Mad2, CENP-E and Knl1 (kinetochore-null protein 1).^{15, 16, 17, 18, 19} Recent studies show that Mps1 can discriminate between on or off SAC signaling by binding to NDC80c via the motif that associates microtubules.^{20, 21} Following SAC, Mps1 is involved in regulating chromosome alignment by phosphorylating Borealin, a component of chromosomal passenger complex (CPC).^{22, 23} In addition, Mps1 plays multiple roles beyond mitosis, including centrosome duplication, cytokinesis, ciliogenesis and DNA damage response.^{18, 24, 25, 26, 27, 28} Mps1 is indispensable for cell survival as loss of Mps1 function by specific siRNA or Mps1 kinase inhibitors causes significant cell death; it has been proposed that Mps1 regulates this process through its roles in SAC.^{29, 30, 31}Mps1 kinase is overexpressed in a variety of tumor types.^{32, 33, 34, 35} In breast cancer, high levels of Mps1 correlate with tumor grades; reducing Mps1 level induces massive apoptosis but allows a selective survival of tumor cells with less aneuploidy.³² Our recent results in colon cancer cells showed that overexpression of Mps1 facilitate the survival of tumor cells with higher aneuploidy by decreasing SAC threshold.³⁵ To further uncover the roles of high levels of Mps1 in tumorigenesis, we examined Mps1 levels in various stages of colon cancer tissues and found that Mps1 level peaks in tissues at stage II, at which stage tumor cells encounter various survival stresses, including genome instability. Aneuploid colon cancer cell lines bear higher levels of Mps1 than diploid cell lines and the amount of Mps1 required for cell survival is far more than that of maintaining SAC, suggesting that other functions of Mps1 are also employed to maintain cell viability. Short-term inhibition of Mps1 activity in mitosis with inhibitors at a dose of more than SAC depletion is sufficient to cause dividing cell death and increase mitochondrial fragmentation simultaneously. Finally, we found that Mps1 can regulate the release of cytochrome c by associating with mitochondrial protein VDAC1 (voltage-dependent anion channel 1). Based on these findings, we postulated that high levels of Mps1 contribute to survival of aneuploid cancer cells via its roles in SAC and mitochondria.     

Mps1 phosphorylation by MAP kinase is required for kinetochore localization of spindle-checkpoint proteins

The spindle checkpoint delays anaphase onset until all chromosomes have achieved bipolar attachment to the spindle microtubules. Unattached kinetochores activate the spindle checkpoint by recruiting several spindle-checkpoint proteins, including Mps1, Mad1, Mad2, Bub1, Bub3, and BubR1 (Mad3 in yeast). In vertebrate cells, active MAP kinase (MAPK) is also enriched at unattached kinetochores and is required for the spindle checkpoint. It has been shown that the kinase activity of Mps1 is required for the spindle checkpoint and for kinetochore localization of Bub1, Bub3, Mad1, and Mad2 . We herein demonstrate that MAPK phosphorylates Mps1 at S844 in Xenopus egg extracts. Interestingly, changing S844 to unphosphorylatable alanine (S844A) has no effect on the kinase activity of Mps1, although it abolishes the checkpoint function of Mps1. Biochemical and immunofluorescence studies show that S844A mutation perturbs kinetochore localization of Mps1 and other spindle-checkpoint proteins, whereas the phosphorylation-mimicking S844D mutant restores their functions. Our studies suggest that Mps1 phosphorylation by MAPK at S844 might create a phosphoepitope that allows Mps1 to interact with kinetochores. In addition, our results indicate that active Mps1 must localize to kinetochores in order to execute its checkpoint function.     

Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.     

Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase

Monopolar spindle 1 (Mps1, also known as TTK) is a protein kinase crucial for ensuring that cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules. Incomplete chromosomal attachment leads to abnormal chromosome counts in the daughter cells (aneuploidy), a condition common in many solid cancers. Therefore Mps1 is an established target in cancer therapy. Mps1 kinase inhibitors include reversine (2‐(4‐morpholinoanilino)‐6‐cyclohexylaminopurine), a promiscuous compound first recognized as an inhibitor of the Aurora B mitotic kinase. Here, we present the 3.0‐Å resolution crystal structure of the Mps1 kinase domain bound to reversine. Structural comparison of reversine bound to Mps1 and Aurora B, indicates a similar binding pose for the purine moiety of reversine making three conserved hydrogen bonds to the protein main chain, explaining the observed promiscuity of this inhibitor. The cyclohexyl and morpholinoaniline moieties of reversine however, have more extensive contacts with the protein in Mps1 than in Aurora B. This is reflected both in structure‐based docking energy calculations, and in new experimental data we present here, that both confirm that the affinity of reversine towards Mps1 is about two orders of magnitude higher than towards Aurora B. Thus, our data provides detailed structural understanding of the existing literature that argues reversine inhibits Mps1 more efficiently than Aurora B based on biochemical and in‐cell assays. Proteins 2016; 84:1761–1766. © 2016 Wiley Periodicals, Inc.     

Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores

Mps1 is an upstream component of the spindle assembly checkpoint, which, in human cells, is required for checkpoint activation in response to spindle damage but not apparently during an unperturbed mitosis. Mps1 also recruits Mad1 and Mad2 to kinetochores. However, whether the enzymatic activity of Mps1 is required for these processes is unclear. To address this question, we established an RNA interference (RNAi) complementation assay. Repression of Mps1 triggers premature anaphase, often with unaligned or maloriented chromosomes. This phenotype is rescued by an RNAi-resistant wild-type Mps1 transgene but not by a catalytically inactive mutant. An analogue-sensitive allele, Mps1(M602A), also rescues the RNAi-induced defect, but not when inhibited by the adenosine triphosphate analogue 1-NM-PP1. Thus, Mps1 activity does restrain anaphase during an unperturbed mitosis. Furthermore, although catalytically inactive Mps1 can restore kinetochore localization of Mad1, only the active kinase restores Mad2 localization. Thus, in human cells, Mps1 catalytic activity is required for spindle checkpoint function and recruitment of Mad2.     

Human Mps1 kinase is required for the spindle assembly checkpoint but not for centrosome duplication

Budding yeast Mps1p kinase has been implicated in both the duplication of microtubule-organizing centers and the spindle assembly checkpoint. Here we show that hMps1, the human homolog of yeast Mps1p, is a cell cycle-regulated kinase with maximal activity during M phase. hMps1 localizes to kinetochores and its activity and phosphorylation state increase upon activation of the mitotic checkpoint. By antibody microinjection and siRNA, we demonstrate that hMps1 is required for human cells to undergo checkpoint arrest in response to microtubule depolymerization. In contrast, centrosome (re-)duplication as well as cell division occur in the absence of hMps1. We conclude that hMps1 is required for the spindle assembly checkpoint but not for centrosome duplication.     

Crystal structure of the catalytic domain of the mitotic checkpoint kinase Mps1 in complex with SP600125

Chromosomal instability can result from defective control of checkpoints and is associated with malignant cell growth. Monopolar spindle 1 (Mps1) is a dual-specificity protein kinase that has important roles in the prevention of aneuploidy during the cell cycle and might therefore be a potential target for new therapeutic agents in the treatment of cancer. To gain insights into the molecular mechanism of Mps1 inhibition by small molecules, we determined the x-ray structure of Mps1, both alone and in complex with the ATP-competitive inhibitor SP600125. Mps1 adopts a classic protein kinase fold, with the inhibitor sitting in the ATP-binding site where it is stabilized by hydrophobic interactions. We identified a secondary pocket, not utilized by SP600125, which might be exploited for the rational design of specific Mps1 inhibitors. These structures provide important insights into the interaction of this protein kinase with small molecules and suggest potential mechanisms for Mps1 regulation.