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.     

Antizyme Restrains Centrosome Amplification by Regulating the Accumulation of Mps1 at Centrosomes

Extra centrosomes are found in many tumors, and their appearance is an early event that can generate aberrant mitotic spindles and aneuploidy. Because the failure to appropriately degrade the Mps1 protein kinase correlates with centrosome overproduction in tumor-derived cells, defects in the factors that promote Mps1 degradation may contribute to extra centrosomes in tumors. However, while we have recently characterized an Mps1 degradation signal, the factors that regulate Mps1 centrosomal Mps1 are unknown. Antizyme (OAZ), a mediator of ubiquitin-independent degradation and a suspected tumor suppressor, was recently shown to localize to centrosomes and modulate centrosome overproduction, but the known OAZ substrates were not responsible for its effect on centrosomes. We have found that OAZ exerts its effect on centrosomes via Mps1. OAZ promotes the removal of Mps1 from centrosomes, and centrosome overproduction caused by reducing OAZ activity requires Mps1. OAZ binds to Mps1 via the Mps1 degradation signal and modulates the function of Mps1 in centrosome overproduction. Moreover, OAZ regulates the canonical centrosome duplication cycle, and reveals a function for Mps1 in procentriole assembly. Together, our data suggest that OAZ restrains the assembly of centrioles by controlling the levels of centrosomal Mps1 through the Cdk2-regulated Mps1 degradation signal.     

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.     

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.     

A Highlights from MBoC Selection: Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function

Centrins are a family of small, calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified centrin 2 and centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, although Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole overproduction, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, whereas depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr-676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes.     

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.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein we have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis, and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking, and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.     

Centriole assembly and the role of Mps1: defensible or dispensable?

The Mps1 protein kinase is an intriguing and controversial player in centriole assembly. Originally shown to control duplication of the budding yeast spindle pole body, Mps1 is present in eukaryotes from yeast to humans, the nematode C. elegans being a notable exception, and has also been shown to regulate the spindle checkpoint and an increasing number of cellular functions relating to genomic stability. While its function in the spindle checkpoint appears to be both universally conserved and essential in most organisms, conservation of its originally described function in spindle pole duplication has proven controversial, and it is less clear whether Mps1 is essential for centrosome duplication outside of budding yeast. Recent studies of Mps1 have identified at least two distinct functions for Mps1 in centriole assembly, while simultaneously supporting the notion that Mps1 is dispensable for the process. However, the fact that at least one centrosomal substrate of Mps1 is conserved from yeast to humans down to the phosphorylation site, combined with evidence demonstrating the exquisite control exerted over centrosomal Mps1 levels suggest that the notion of being essential may not be the most important of distinctions.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein and have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.Key words: Mps1 kinase, actin, Mip1, cytokinesis     

The spindle pole body assembly component mps3p/nep98p functions in sister chromatid cohesion

For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister chromatids are paired together, identifying the products of replication as sister chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.     

The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.     

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.     

Drosophila Polo regulates the spindle assembly checkpoint through Mps1‐dependent BubR1 phosphorylation

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase‐promoting complex/cyclosome‐inhibitory complexes to levels that ensure long‐term SAC activity. We propose a model in which Polo controls Mps1‐dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.     

Ablation of the spindle assembly checkpoint by a compound targeting Mps1

The spindle assembly checkpoint ensures accurate chromosome segregation by delaying anaphase initiation until all chromosomes are properly attached to the mitotic spindle. Here, we show that the previously reported c-Jun amino-terminal kinase (JNK) inhibitor SP600125 effectively disrupts spindle checkpoint function in a JNK-independent fashion. SP600125 potently inhibits activity of the mitotic checkpoint kinase monopolar spindle 1 (Mps1) in vitro and triggers efficient progression through a mitotic arrest imposed by spindle poisons. Importantly, expression of an Mps1 mutant protein refractory to SP600125-mediated inhibition restores spindle checkpoint function in the presence of SP600125, showing that its mitotic phenotype is induced by Mps1 inhibition in vivo. Remarkably, primary human cells are largely resistant to the checkpoint-inactivating action of SP600125, suggesting the existence of Mps1-independent checkpoint pathways that are compromised in tumour cells.     

Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.     

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.     

Cellular abundance of Mps1 and the role of its carboxyl terminal tail in substrate recruitment

Mps1 is a protein kinase that regulates normal mitotic progression and the spindle checkpoint in response to spindle damage. The levels of Mps1 are relatively low in cells during interphase but elevated in mitosis or upon activation of the spindle checkpoint, although the dynamic range of Mps1 expression and the Mps1 catalytic mechanism have not been carefully characterized. Our recent structural studies of the Mps1 kinase domain revealed that the carboxyl-terminal tail region of Mps1 is unstructured, raising the question of whether this region has any functional role in Mps1 catalysis. Here we first determined the cellular abundance of Mps1 during cell cycle progression and found that Mps1 levels vary between 60,000 per cell in early G(1) and 110,000 per cell during mitosis. We studied phosphorylation of a number of Mps1 substrates in vitro and in culture cells. Unexpectedly, we found that the unstructured carboxyl-terminal region of Mps1 plays an essential role in substrate recruitment. Kinetics studies using the purified recombinant wild type and mutant kinases indicate that the carboxyl-terminal tail is largely dispensable for autophosphorylation of Mps1 but critical for trans-phosphorylation of substrates in vitro and in cultured cells. Mps1 mutant without the unstructured tail region is defective in mediating spindle assembly checkpoint activation. Our results underscore the importance of the unstructured tail region of Mps1 in kinase activation.     

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.     

Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G2/M transition and sustained spindle checkpoint responses

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G₂/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses.