Protein Kinase Mps1 Research Articles

Mps1-Targeted Molecular Design of Melatonin for Broad-Spectrum Antifungal Agent Discovery.

Melatonin, a multifunctional class of natural products, has demonstrated antifungal activity, making it a promising candidate for developing antifungal agents. The mitogen-activated protein kinase (Mps1) within fungal pathogens has a target inhibitory effect of melatonin in fungi. We use a virtual screening strategy to design melatonin derivatives based on the melatonin-Mps1 targeting model. Of these, a multiflorane-substitution compound M-12 emerges as a potent antifungal agent, exhibiting broad-spectrum efficacy against eight phytopathogenic fungal species, and effectively reduces the severity of tomato gray mold, Fusarium head blight in wheat, Sclerotinia stem rot in rape, and peach brown rot. M-12 half-maximal effective concentration values (5.50 μM against Botrytis cinerea, 5.21 μM against Fusarium graminearum, 10.6 μM against Rhizoctonia solani, and 9.02 μM against Sclerotinia sclerotiorum) are better than those of commercial broad-spectrum fungicide azoxystrobin (55.0, 23.2, 46.5, and 17.7 μM, respectively). Antifungal activity of enantiomer (S)-M-12 (5.02 μM) is significantly greater than its (R)-enantiomer (23.6 μM) against B. cinerea. Molecular docking and transcriptome analysis reveal that M-12 achieves its antifungal effects by inhibiting Mps1 kinase, thereby suppressing fungal growth and virulence.

Rational modification of melatonin for broad-spectrum antifungal agents discovery.

Natural products, known for their environmental safety, are regarded as a significant basis for the modification and advancement of fungicides. Melatonin, as a low-cost natural indole, exhibits diverse biological functions, including antifungal activity. However, its potential as an antifungal agent has not been fully explored. In this study, a series of melatonin derivatives targeting the mitogen-activated protein kinase (Mps1) protein of fungal pathogens were synthesized based on properties of melatonin, among which the trifluoromethyl-substituted derivative Mt-23 exhibited antifungal activity against seven plant pathogenic fungi, and effectively reduced the severity of crop diseases, including rice blast, Fusarium head blight of wheat and gray mold of tomato. In particular, its EC50 (5.4 µM) against the rice blast fungus Magnaporthe oryzae is only one-fourth that of isoprothiolane (22 µM), a commercial fungicide. Comparative analyzes revealed that Mt-23 simultaneously targets the conserved protein kinase Mps1 and lipid protein Cap20. Surface plasmon resonance assays showed that Mt-23 directly binds to Mps1 and Cap20. In this study, we provide a strategy for developing antifungal agents by modifying melatonin, and the resultant melatonin derivative Mt-23 is a commercially valuable, eco-friendly and broad-spectrum antifungal agent to combat crop disease.

Spindle checkpoint activation by fungal orthologs of the S. cerevisiae Mps1 kinase.

There is an ongoing need for antifungal agents to treat humans. Identification of new antifungal agents can be based on screening compounds using whole cell assays. Screening compounds that target a particular molecule is possible in budding yeast wherein sophisticated strain engineering allows for controlled expression of endogenous or heterologous genes. We have considered the yeast Mps1 protein kinase as a reasonable target for antifungal agents because mutant or druggable forms of the protein, upon inactivation, cause rapid loss of cell viability. Furthermore, extensive analysis of the Mps1 in budding yeast has offered potential tactics for identifying inhibitors of its enzymatic activity. One such tactic is based on the finding that overexpression of Mps1 leads to cell cycle arrest via activation of the spindle assembly checkpoint. We have endeavored to adapt this assay to be based on the overexpression of Mps1 orthologs from pathogenic yeast in hopes of having a whole-cell assay system to test the activity of these orthologs. Mps1 orthologous genes from seven pathogenic yeast or other pathogenic fungal species were isolated and expressed in budding yeast. Two orthologs clearly produced phenotypes similar to those produced by the overexpression of budding yeast Mps1, indicating that this system for heterologous Mps1 expression has potential as a platform for identifying prospective antifungal agents.

Computational Biology Dynamics of Mps1 Kinase Molecular Interactions with Isoflavones Reveals a Chemical Scaffold with Potential to Develop New Therapeutics for the Treatment of Cancer.

The protein kinase Mps1 (monopolar spindle 1) is an important regulator of the Spindle Assembly Checkpoint (SAC), the evolutionary conserved checkpoint system of higher organisms that monitors the proper bipolar attachment of all chromosomes to the mitotic spindle during cell division. Defects in the catalytic activity and the transcription regulation of Mps1 are associated with genome instability, aneuploidy, and cancer. Moreover, multiple Mps1 missense and frameshift mutations have been reported in a wide range of types of cancer of different tissue origin. Due to these features, Mps1 arises as one promising drug target for cancer therapy. In this contribution, we developed a computational biology approach to study the dynamics of human Mps1 kinase interaction with isoflavones, a class of natural flavonoids, and compared their predicted mode of binding with that observed in the crystal structure of Mps1 in complex with reversine, a small-sized inhibitor of Mps1 and Aurora B kinases. We concluded that isoflavones define a chemical scaffold that can be used to develop new Mps1 inhibitors for the treatment of cancer associated with Mps1 amplification and aberrant chromosome segregation. In a broader context, the present report illustrates how modern chemoinformatics approaches can accelerate drug development in oncology.

Monopolar Spindle 1 Kinase (MPS1/TTK) mRNA Expression is Associated with Earlier Development of Clinical Symptoms, Tumor Aggressiveness and Survival of Glioma Patients.

Inhibition of the protein kinase MPS1, a mitotic spindle-checkpoint regulator, reinforces the effects of multiple therapies against glioblastoma multiforme (GBM) in experimental settings. We analyzed MPS1 mRNA-expression in gliomas WHO grade II, III and in clinical subgroups of GBM. Data were obtained by qPCR analysis of tumor and healthy brain specimens and correlated with the patients’ clinical data. MPS1 was overexpressed in all gliomas on an mRNA level (ANOVA, p < 0.01) and correlated with tumor aggressiveness. We explain previously published conflicting results on survival: high MPS1 was associated with poorer long term survival when all gliomas were analyzed combined in one group (Cox regression: t < 24 months, p = 0.009, Hazard ratio: 8.0, 95% CI: 1.7–38.4), with poorer survival solely in low-grade gliomas (LogRank: p = 0.02, Cox regression: p = 0.06, Hazard-Ratio: 8.0, 95% CI: 0.9–66.7), but not in GBM (LogRank: p > 0.05). This might be due to their lower tumor volume at the therapy start. GBM patients with high MPS1 mRNA-expression developed clinical symptoms at an earlier stage. This, however, did not benefit their overall survival, most likely due to the more aggressive tumor growth. Since MPS1 mRNA-expression in gliomas was enhanced with increasing tumor aggressiveness, patients with the worst outcome might benefit best from a treatment directed against MPS1.

Autophosphorylation is sufficient to release Mps1 kinase from native kinetochores

Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible "wait" signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.

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.

Cytotoxic and Apoptotic Effects of Novel Pyrrolo[2,3-d]Pyrimidine Derivatives Containing Urea Moieties on Cancer Cell Lines.

Pyrrolo[2,3-d]pyrimidines have been recently reported to have anticancer activities through inhibition of different targets such as, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, Janus Kinase (JAK), mitotic checkpoint protein kinase (Mps1), carbonic anhydrase, MDM-2. On the other hand, aryl urea moieties which are found in some tyrosine kinase inhibitors such as Sorafenib and Linifanib have aroused recent attention as responsible for anticancer activities. The aims of this paper are to synthesize pyrrolo[ 2,3-d]pyrimidine derivatives containing urea moiety and evaluate their anti-cancer activity against human lung cancer cell line (A549), prostate cancer cell line (PC3), human colon cancer cell line (SW480) and human breast cancer cell line (MCF-7). A series of new pyrrolo[2,3-d]pyrimidines containing urea moieties have been synthesized as Scheme 1. In vitro cytotoxicity of target compounds were evaluated against, SW480, PC3, A549 and MCF-7 human cancer cell lines using a MTT assay. In order to evaluate the mechanism of cytotoxic activity of compounds 9e, 10a and 10b, having the best cytotoxic activity, Annexin V binding assay, cell cycle analysis and western blot analysis were performed. Among the target compounds, 10a (IC50 = 0.19 µM) was found to be the most potent derivative against PC3 cells. Compound 10b and 9e showed the strong cytotoxic activity against MCF-7 and A549 cells with IC50 value of 1.66 µM and 4.55 µM, respectively. Flow cytometry data suggest that the cytotoxic activity of the compounds on cancer cells might be mediated by apoptosis revealing a significant increase in the percentage of late apoptotic cells and causing a cell cycle arrest at different stages. Western blot analysis of apoptosis marker demonstrated that these compounds induce apoptosis through the intrinsic pathway. Compound 9e displayed the strongest cytotoxicity against A549 cancer cell line, and induced late apoptosis in A549, as confirmed by cell cycle arrest in G0/G1 phase. In addition, compound 9e reduced expression of the anti-apoptotic protein Bcl-2 and enhanced expression of the pro-apoptotic protein Bax, besides increased caspase-9 and caspase-3, as well as cleavage of PARP levels. These results suggest that compound 9e showed a cytotoxic effect in A549 cells through activation of the mitochondrial apoptotic pathway. Further studies will be undertaken in our laboratory to improve cytotoxic activity of compound 9e and to identify the biological targets of 9e which are responsible for anticancer activity.

Direct interactions of mitotic arrest deficient 1 (MAD1) domains with each other and MAD2 conformers are required for mitotic checkpoint signaling

As a sensitive signaling system, the mitotic checkpoint ensures faithful chromosome segregation by delaying anaphase onset even when a single kinetochore is unattached to mitotic spindle microtubules. The key signal amplification reaction for the checkpoint is the conformational conversion of "open" mitotic arrest deficient 2 (O-MAD2) into "closed" MAD2 (C-MAD2). The reaction has been suggested to be catalyzed by an unusual catalyst, a MAD1:C-MAD2 tetramer, but how the catalysis is executed and regulated remains elusive. Here, we report that in addition to the well-characterized middle region of MAD1 containing the MAD2-interaction motif (MIM), both N- and C-terminal domains (NTD and CTD) of MAD1 also contribute to mitotic checkpoint signaling. Unlike the MIM, which stably associated only with C-MAD2, the NTD and CTD in MAD1 surprisingly bound both O- and C-MAD2, suggesting that these two domains interact with both substrates and products of the O-to-C conversion. MAD1NTD and MAD1CTD also interacted with each other and with the MPS1 protein kinase, which phosphorylated both NTD and CTD. This phosphorylation decreased the NTD:CTD interaction and also CTD's interaction with MPS1. Of note, mutating the phosphorylation sites in the MAD1CTD, including Thr-716, compromised MAD2 binding and the checkpoint responses. We further noted that Ser-610 and Tyr-634 also contribute to the mitotic checkpoint signaling. Our results have uncovered that the MAD1NTD and MAD1CTD directly interact with each other and with MAD2 conformers and are regulated by MPS1 kinase, providing critical insights into mitotic checkpoint signaling.

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.

How cells sense connected chromosomes

Cells have a “checkpoint” that pauses cell division until all chromosomes are properly arranged on the mitotic spindle to allow precise distribution of one copy of each chromosome to each daughter cell. Hiruma et al . and Ji et al . explain the molecular mechanism by which cells sense that they are ready to divide. The protein kinase MPS1 associates with a protein complex at the kinetochore of the chromosome. Its activity produces signals that pause the cell cycle. When the chromosome becomes properly attached to the mitotic spindle, microtubules of the spindle physically compete for binding to the same site on the kinetochore where MPS1 is bound. Thus, once the kinetochore is properly attached, MPS1 dissociates, the inhibitory signal is lost, and cell division is allowed to proceed. Y. Hiruma, C. Sacristan, S. T. Pachis, A. Adamopoulos, T. Kuijt, M. Ubbink, E. von Castelmur, A. Perrakis, G. J. P. L. Kops, Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling. Science 348 , 1264–1267 (2015). [Abstract][Full Text] Z. Ji, H. Gao, H. Yu, Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80C. Science 348 , 1260–1264 (2015). [Summary][Full Text]

MP39-04 IL-6 RECEPTOR ANTIBODY ENHANCES THE EFFECT OF TKI AGAINST RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Abnormal chromosome segregation during mitosis causes aneuploidy, a hallmark of cancers associated with high risk for tumorigenesis. Mitotic checkpoint, or spindle assembly checkpoint prevents missegregation of chromosomes by arresting cells in metaphase until all the chromosomes are properly aligned. Mps1 kinase activity is essential for spindle checkpoint signaling. High levels of Mps1 serine/threonine kinase are found in colon cancer tissues and several cancer cell line. We found that Mps1 is over-expressed in ccRCC. We have further shown that Mps1 requires the molecular chaperone Hsp90 for its activity. Hsp90 protects an array of mutated, over-expressed and/or chimeric kinases that are vital for tumor growth and survival. The Hsp90 inhibitors are currently undergoing evaluation in human trials. We have previously shown that Hsp90 is a post-translationally modified protein and this regulates its chaperone activity. We hypothesize that Mps1 kinase targets and phosphorylates Hsp90 and therefore regulates its chaperone activity. This in turn impacts the tumor selectivity of Hsp90 inhibitors. METHODS: Functional genomics and quantitative proteomic (stable isotope labeling by amino acids in cell culture) was used to identify the Mps1 kinase that targets and phosphorylates Hsp90. ccRCC and adjacent normal tissue was obtained with written informed consent through the Upstate Department of Urology. 8-mm core of tissue was dissected into 3mm pieces and cultured in 24-well plates containing 1 mL RPMI-1640 with 10% FBS, antibiotic/antimycotic solution. Tissues were cultured at 37 C for 24 hours with or without Mps1 inhibitor NMS-P715 and for further 24 hours in the presence of fluorescently labeled Hsp90 inhibitor ganetespib (FL-ganetespib), then formalin-fixed and paraffin embedded. The tissues were immunostained for Hsp90. RESULTS: Mps1 kinase is over-expressed in ccRCC compared to normal adjacent tissue. Mps1 targets and phosphorylates T115 in the molecular chaperone Hsp90 and this regulates the chaperone activity. Ex vivo data showed that FL-ganetespib selectively accumulates in ccRCC and not in the normal tissue. Pharmacologic inhibition of Mps1 reverts this drug uptake by ccRCC. CONCLUSIONS: Mps1 protein kinase is an Hsp90 client and it is involved in post-translational regulation of the chaperone function. Over-expression of Mps1 in the ccRCC hyperphosphorylates Hsp90 and this is the molecular basis of the tumor selectivity of Hsp90 inhibitors.

MP39-02 OVEREXPRESSION OF MPS1 IN CLEAR CELL RENAL CELL CARCINOMA (CCRCC) CONFERS TUMOR SELECTIVITY ON HEAT SHOCK PROTEIN-90 (HSP90) INHIBITORS

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2015MP39-02 OVEREXPRESSION OF MPS1 IN CLEAR CELL RENAL CELL CARCINOMA (CCRCC) CONFERS TUMOR SELECTIVITY ON HEAT SHOCK PROTEIN-90 (HSP90) INHIBITORS Mourad Abouelleil, Diana Dunn, Mark Woodford, Sandra Jensen, Mahmoud Chehab, Alosh Madala, Tiffany Caza, Steve Landas, William G. Stetler-Stevenson, Len Neckers, Dimitra Bourboulia, Gennady Bratslavsky, and Mehdi Mollapour Mourad AbouelleilMourad Abouelleil More articles by this author , Diana DunnDiana Dunn More articles by this author , Mark WoodfordMark Woodford More articles by this author , Sandra JensenSandra Jensen More articles by this author , Mahmoud ChehabMahmoud Chehab More articles by this author , Alosh MadalaAlosh Madala More articles by this author , Tiffany CazaTiffany Caza More articles by this author , Steve LandasSteve Landas More articles by this author , William G. Stetler-StevensonWilliam G. Stetler-Stevenson More articles by this author , Len NeckersLen Neckers More articles by this author , Dimitra BourbouliaDimitra Bourboulia More articles by this author , Gennady BratslavskyGennady Bratslavsky More articles by this author , and Mehdi MollapourMehdi Mollapour More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.752AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Abnormal chromosome segregation during mitosis causes aneuploidy, a hallmark of cancers associated with high risk for tumorigenesis. Mitotic checkpoint, or spindle assembly checkpoint prevents missegregation of chromosomes by arresting cells in metaphase until all the chromosomes are properly aligned. Mps1 kinase activity is essential for spindle checkpoint signaling. High levels of Mps1 serine/threonine kinase are found in colon cancer tissues and several cancer cell line. We found that Mps1 is over-expressed in ccRCC. We have further shown that Mps1 requires the molecular chaperone Hsp90 for its activity. Hsp90 protects an array of mutated, over-expressed and/or chimeric kinases that are vital for tumor growth and survival. The Hsp90 inhibitors are currently undergoing evaluation in human trials. We have previously shown that Hsp90 is a post-translationally modified protein and this regulates its chaperone activity. We hypothesize that Mps1 kinase targets and phosphorylates Hsp90 and therefore regulates its chaperone activity. This in turn impacts the tumor selectivity of Hsp90 inhibitors. METHODS Functional genomics and quantitative proteomic (stable isotope labeling by amino acids in cell culture) was used to identify the Mps1 kinase that targets and phosphorylates Hsp90. ccRCC and adjacent normal tissue was obtained with written informed consent through the Upstate Department of Urology. 8-mm core of tissue was dissected into 3mm pieces and cultured in 24-well plates containing 1 mL RPMI-1640 with 10% FBS, antibiotic/antimycotic solution. Tissues were cultured at 37°C for 24 hours with or without Mps1 inhibitor NMS-P715 and for further 24 hours in the presence of fluorescently labeled Hsp90 inhibitor ganetespib (FL-ganetespib), then formalin-fixed and paraffin embedded. The tissues were immunostained for Hsp90. RESULTS Mps1 kinase is over-expressed in ccRCC compared to normal adjacent tissue. Mps1 targets and phosphorylates T115 in the molecular chaperone Hsp90 and this regulates the chaperone activity. Ex vivo data showed that FL-ganetespib selectively accumulates in ccRCC and not in the normal tissue. Pharmacologic inhibition of Mps1 reverts this drug uptake by ccRCC. CONCLUSIONS Mps1 protein kinase is an Hsp90 client and it is involved in post-translational regulation of the chaperone function. Over-expression of Mps1 in the ccRCC hyperphosphorylates Hsp90 and this is the molecular basis of the tumor selectivity of Hsp90 inhibitors. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e455 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mourad Abouelleil More articles by this author Diana Dunn More articles by this author Mark Woodford More articles by this author Sandra Jensen More articles by this author Mahmoud Chehab More articles by this author Alosh Madala More articles by this author Tiffany Caza More articles by this author Steve Landas More articles by this author William G. Stetler-Stevenson More articles by this author Len Neckers More articles by this author Dimitra Bourboulia More articles by this author Gennady Bratslavsky More articles by this author Mehdi Mollapour More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint

The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint.

Structure-Based Design of Orally Bioavailable 1H-Pyrrolo[3,2-c]pyridine Inhibitors of Mitotic Kinase Monopolar Spindle 1 (MPS1)

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Hydrophobin gene expression in the maize pathogen Cochliobolus heterostrophus

Filamentous fungi produce hydrophobins, small proteins localized on the outer surface of their cell walls and involved in growth and development. Hydrophobin gene expression depends on nutrient availability, light, and the activity of conserved signal-transduction pathways. We found four hydrophobins, one class I and three class II family members, in the Cochliobolus heterostrophus genome with high homology to other ascomycete hydrophobins, which present a typical conserved array of cysteines. The expression profile of a selected gene from each class was determined in a series of signaling-deficient mutants. Loss of either of two mitogen-activated protein kinase (MAPK) genes, CHK1 and MPS1, led to decreased hydrophobin class I (CHYD1) gene expression. Mutants in both MAPK genes had easily wettable colonies, but decreased CHYD1 expression was not the sole explanation for this phenotype. A significant elevation of hydrophobin class II (CHYD3) gene expression was measured in the chk1 mutant, suggesting a complex role for MAPK in controlling the expression of these hydrophobins. Similar but less marked tendencies were observed in G-protein α and β subunit loss-of-function mutants; however these showed no alteration in colony hydrophobicity. Overexpression of CHYD1 in the wild-type background caused a change in colony morphology and a small but significant increase in aerial growth. Thus G-protein and MAPK signal transduction influence hydrophobin gene expression and colony hydrophobicity. The connection between colony hydrophobicity and expression of the hydrophobin genes CHYD1 and CHYD3, however, is not one-to-one, indicating that additional factors determine colony-surface properties. The approach of using hydrophobin-overexpression mutants to investigate their role may be generalized to other hydrophobins and small secreted proteins.

Identification of Anaphase Promoting Complex Substrates in S. cerevisiae

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an essential ubiquitin ligase that targets numerous proteins for proteasome-mediated degradation in mitosis and G1. To gain further insight into cellular pathways controlled by APC/CCdh1, we developed two complementary approaches to identify additional APC/CCdh1 substrates in budding yeast. First, we analyzed the stabilities of proteins that were expressed at the same time in the cell cycle as known APC/C substrates. Second, we screened for proteins capable of interacting with the Cdh1 substrate-binding protein in a yeast two-hybrid system. Here we characterize five potential APC/C substrates identified using these approaches: the transcription factors Tos4 and Pdr3; the mRNA processing factor Fir1; the spindle checkpoint protein kinase Mps1; and a protein of unknown function, Ybr138C. Analysis of the degradation motifs within these proteins revealed that the carboxyl-terminal KEN box and D-boxes of Tos4 are important for its interaction with Cdh1, whereas the N-terminal domain of Ybr138C is required for its instability. Functionally, we found that a stabilized form of Mps1 delayed cell division upon mild spindle disruption, and that elevated levels of Ybr138C reduced cell fitness. Interestingly, both Tos4 and Pdr3 have been implicated in the DNA damage response, whereas Mps1 regulates the spindle assembly checkpoint. Thus, the APC/CCdh1-mediated degradation of these proteins may help to coordinate re-entry into the cell cycle following environmental stresses.

Structural and Functional Characterization of the Protein Kinase Mps1 in Arabidopsis thaliana

In eukaryotes, protein kinases catalyze the transfer of a gamma-phosphate from ATP (or GTP) to specific amino acids in protein targets. In plants, protein kinases have been shown to participate in signaling cascades driving responses to environmental stimuli and developmental processes. Plant meristems are undifferentiated tissues that provide the major source of cells that will form organs throughout development. However, non-dividing specialized cells can also dedifferentiate and re-initiate cell division if exposed to appropriate conditions. Mps1 (Monopolar spindle) is a dual-specificity protein kinase that plays a critical role in monitoring the accuracy of chromosome segregation in the mitotic checkpoint mechanism. Although Mps1 functions have been clearly demonstrated in animals and fungi, its role in plants is so far unclear. Here, using structural and biochemical analyses here we show that Mps1 has highly similar homologs in many plant genomes across distinct lineages (e.g. AtMps1 in Arabidopsis thaliana). Several structural features (i.e. catalytic site, DFG motif and threonine triad) are clearly conserved in plant Mps1 kinases. Structural and sequence analysis also suggest that AtMps1 interact with other cell cycle proteins, such as Mad2 and MAPK1. By using a very specific Mps1 inhibitor (SP600125) we show that compromised AtMps1 activity hampers the development of A. thaliana seedlings in a dose-dependent manner, especially in secondary roots. Moreover, concomitant administration of the auxin IAA neutralizes the AtMps1 inhibition phenotype, allowing secondary root development. These observations let us to hypothesize that AtMps1 might be a downstream regulator of IAA signaling in the formation of secondary roots. Our results indicate that Mps1 might be a universal component of the Spindle Assembly Checkpoint machinery across very distant lineages of eukaryotes.

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.

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.