T1.7 Combinatorial Use of Chemical Genetics and Proteomics to identify kinase substrates of Mps1
| Project leaders |
Dr. Geert JPL Kops Prof. Dr. Albert J. R. Heck |
| Address |
UMC Utrecht Dept. of Physiological Chemistry Universiteitsweg 100 3584 CG Utrecht The Netherlands |
| Phone | 0887555163 |
| Fax | 0887568101 |
| This e-mail address is being protected from spambots. You need JavaScript enabled to view it |
Summary
Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by a cell cycle checkpoint that allows chromosome segregation only when all chromosomes are aligned for equal division to daughter cells. The process of chromosome attachment and the mitotic checkpoint are thus important to prevent aneuploidy and subsequent genetic imbalances that contribute to carcinogenesis and developmental defects. The dynamic regulation of chromosome segregation and mitotic checkpoint signaling is coordinated by a set of multifunctional kinases, one of which is Mps1. It is currently unknown which proteins are functional substrates of Mps1 in checkpoint signaling. Similarly, Mps1 may have mitotic roles other than checkpoint signaling and chromosome alignment, and identifying substrates for these functions will be crucial to our understanding of the dynamic coordination of processes in mitosis. We will use chemical genetics combined with quantitative phospho-proteomics to identify direct substrates for Mps1 in mitosis. We have generated cell lines in which endogenous Mps1 was replaced by an engineered kinase that can accept bulky ATP analogs or ATP-like molecules. We will search for direct substrates in four ways:
Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by a cell cycle checkpoint that allows chromosome segregation only when all chromosomes are aligned for equal division to daughter cells. The process of chromosome attachment and the mitotic checkpoint are thus important to prevent aneuploidy and subsequent genetic imbalances that contribute to carcinogenesis and developmental defects. The dynamic regulation of chromosome segregation and mitotic checkpoint signaling is coordinated by a set of multifunctional kinases, one of which is Mps1. It is currently unknown which proteins are functional substrates of Mps1 in checkpoint signaling. Similarly, Mps1 may have mitotic roles other than checkpoint signaling and chromosome alignment, and identifying substrates for these functions will be crucial to our understanding of the dynamic coordination of processes in mitosis. We will use chemical genetics combined with quantitative phospho-proteomics to identify direct substrates for Mps1 in mitosis. We have generated cell lines in which endogenous Mps1 was replaced by an engineered kinase that can accept bulky ATP analogs or ATP-like molecules. We will search for direct substrates in four ways:
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Direct labelling and purification of substrates by loading the engineered kinase with N6-benzyl-ATPγS.
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Quantitative phospho-proteomics on mitotic lysates in which the kinase was or was not inhibited by bulky ATP-competitive compounds.
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Quantitative phospho-proteomics on kinase-inhibited mitotic lysates in which the kinase was or was not re-activated
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Quantitative phospho-proteomics to identify preferred phosphorylation sites of recombinant Mps1 from a cellular peptide mixture.