1. Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS
- Author
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Andrew Macdonald, Nick A. Morrice, J. Simon C. Arthur, David G. Campbell, Maria Deak, Joanne McIlrath, Claire E. McCoy, and Rachel Toth
- Subjects
Molecular Sequence Data ,Biology ,Mitogen-activated protein kinase kinase ,Ribosomal Protein S6 Kinases, 90-kDa ,Biochemistry ,Mass Spectrometry ,Cell Line ,MAP2K7 ,Phosphoserine ,Humans ,ASK1 ,Amino Acid Sequence ,c-Raf ,Phosphorylation ,Molecular Biology ,Conserved Sequence ,MAPK14 ,MAP kinase kinase kinase ,Cyclin-dependent kinase 2 ,Cell Biology ,Molecular biology ,Enzyme Activation ,Mutagenesis ,biology.protein ,Cyclin-dependent kinase 9 ,Mitogen-Activated Protein Kinases ,Sequence Alignment ,Research Article ,Protein Binding - Abstract
MSK1 (mitogen- and stress-activated kinase 1) is a dual kinase domain protein that acts downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in cells. MSK1, and its related isoform MSK2, phosphorylate the transcription factors CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor 1), and the chromatin proteins histone H3 and HMGN1 (high-mobility-group nucleosomal-binding protein 1) in response to either mitogenic stimulation or cellular stress. MSK1 activity is tightly regulated in cells, and activation requires the phosphorylation of MSK1 by either ERK1/2 or p38α. This results in activation of the C-terminal kinase domain, which then phosphorylates further sites in MSK1, leading to the activation of the N-terminal kinase domain and phosphorylation of substrates. Here, we use precursor ion scanning MS to identify five previously unknown sites in MSK1: Thr630, Ser647, Ser657, Ser695 and Thr700. One of these sites, Thr700, was found to be a third site in MSK1 phosphorylated by the upstream kinases ERK1/2 and p38α. Mutation of Thr700 resulted in an increased basal activity of MSK1, but this could be further increased by stimulation with PMA or UV-C radiation. Surprisingly, however, mutation of Thr700 resulted in a dramatic loss of Thr581 phosphorylation, a site essential for activity. Mutation of Thr700 and Thr581 to an alanine residue resulted in an inactive kinase, while mutation of both sites to an aspartic acid residue resulted in a kinase with a significant basal activity that could not be further stimulated. Together these results are consistent with a mechanism by which Thr700 phosphorylation relieves the inhibition of MSK1 by a C-terminal autoinhibitory helix and helps induce a conformational shift that protects Thr581 from dephosphorylation. more...
- Published
- 2007
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