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

Biochem J. 2007 Mar 15;402(3):491-501. doi: 10.1042/BJ20061183.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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


  • Phosphoserine
  • Ribosomal Protein S6 Kinases, 90-kDa
  • mitogen and stress-activated protein kinase 1
  • Mitogen-Activated Protein Kinases