Crystal Structure of the Catalytic Domain of the Mitotic Checkpoint Kinase Mps1 in Complex with SP600125

Chu, Matthew L. H.; Chavas, Leonard M. G.; Douglas, Kenneth T.; Eyers, Patrick A.; Tabernero, Lydia

doi:10.1074/jbc.m803026200

Cited by 53 publications

(83 citation statements)

References 27 publications

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“…Peptide sequence m/z the Mps1 protein kinase [21]. Our analyses failed to detect any nonphosphorylated T676, while the stoichiometry of phosphorylation of T686 was 95% Ϯ 1%, indicating that both sites are highly modified.…”

Section: Workflowcontrasting

confidence: 48%

mentioning

confidence: 99%

“…Mps1 is a mitotic protein kinase required for the spindle assembly checkpoint (SAC) in many organisms including yeast, flies, zebrafish, frogs, and humans [4,21]. Autophosphorylation of Mps1 is required for full activity in vitro with complex changes in the extent and location of phosphorylation known to be responsible for regulating protein kinase function [4,21]. We previously identified 16 sites of autophosphorylation using liquid chromatography (LC)-MS/MS, incorporating the complementary fragmentation strategies of collision-induced dissociation and electron-transfer dissociation [3].…”

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confidence: 99%

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Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry

Johnson

Eyers

et al. 2009

J. Am. Soc. Mass Spectrom.

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Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment.

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Section: Workflowcontrasting

confidence: 48%

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confidence: 99%

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confidence: 99%

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Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry

Johnson

Eyers

et al. 2009

J. Am. Soc. Mass Spectrom.

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“…The carboxyl terminus of Mps1 is disordered in both of the reported Mps1 crystal structures (23,29). The carboxyl terminus of Mps1 does not appear to be required for its autophosphorylation but is critical for transphosphorylation of the substrates (Fig.…”

Section: Table 2 Enzyme Kinetic Constants From the Two Substrate Kinementioning

confidence: 99%

“…Crystallographic analysis of Mps1 indicated that whereas the kinase domain in the carboxyl-terminal region of Mps1 adopts a canonical kinase fold, the 65-residue carboxyl-terminal tail of Mps1 is unstructured (23,29) (Fig. 2A).…”

Section: Cellular Abundance Of Mps1 Cdk1mentioning

confidence: 99%

Cellular Abundance of Mps1 and the Role of Its Carboxyl Terminal Tail in Substrate Recruitment

Sun

Yang

Wang

et al. 2010

Journal of Biological Chemistry

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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.

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Selective killing of p53‐deficient cancer cells by SP600125

et al. 2012

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The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53+/+ and TP53−/− human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53−/− (but not TP53+/+) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53−/− cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53−/− cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.

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Crystal Structure of the Catalytic Domain of the Mitotic Checkpoint Kinase Mps1 in Complex with SP600125

Cited by 53 publications

References 27 publications

Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry

Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry

Cellular Abundance of Mps1 and the Role of Its Carboxyl Terminal Tail in Substrate Recruitment

Selective killing of p53‐deficient cancer cells by SP600125

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