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

Claire E. McCoy, Andrew Macdonald, Nick A. Morrice, David G. Campbell, Maria Deak, Rachel Toth, Joanne McIlrath, J. Simon C. Arthur

    Research output: Contribution to journalArticlepeer-review

    40 Citations (Scopus)

    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 p38a. 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 p38a. 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.
    Original languageEnglish
    Pages (from-to)491-501
    Number of pages11
    JournalBiochemical Journal
    Volume402
    Issue number3
    DOIs
    Publication statusPublished - 15 Mar 2007

    Keywords

    • Amino Acid Sequence
    • Cell Line
    • Conserved Sequence
    • Enzyme Activation
    • Humans
    • Mass Spectrometry
    • Mitogen-Activated Protein Kinases
    • Molecular Sequence Data
    • Mutagenesis
    • Phosphorylation
    • Phosphoserine
    • Protein Binding
    • Ribosomal Protein S6 Kinases, 90-kDa
    • Sequence Alignment

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