Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

Abstract : MSK1 is a dual kinase domain protein that acts downstream of the ERK1/2 and p38 MAPK signalling pathways in cells. MSK1, and its related isoform MSK2, phosphorylate the transcription factors CREB and ATF1, and the chromatin proteins histone H3 and HMGN1 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 p38{alpha}. 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 mass spectrometry to identify 5 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 p38{alpha}. Mutation of Thr700 resulted in an increased basal activity of MSK1, but this could be further increased by stimulation with PMA or UV-C. Surprisingly however mutation of Thr700 resulted in a dramatic loss of Thr581 phosphorylation, a site essential for activity. Mutation of Thr700 and Thr581 to alanine resulted in an inactive kinase, while mutation of both sites to aspartic acid resulted in a kinase with a significant basal activity but which could not be further stimulated. Together these results are consistent with a mechanism by which Thr700 phosphorylation relieves the inhibition of MSK1 and a C-terminal autoinhibitory helix and helps induce a conformational shift that protects Thr581 from dephosphorylation.
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Claire E Mccoy, Andrew Macdonald, Nick A Morrice, David G Campbell, Maria Deak, et al.. Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS. Biochemical Journal, Portland Press, 2006, 402 (3), pp.491-501. ⟨10.1042/BJ20061183⟩. ⟨hal-00478646⟩

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