title: Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS.
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.
authors.affiliation: MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Claire E
authors.initials: CE
authors.lastname: McCoy
authors.forename: Andrew
authors.initials: A
authors.lastname: macdonald
authors.forename: Nick A
authors.initials: NA
authors.lastname: Morrice
authors.forename: David G
authors.initials: DG
authors.lastname: Campbell
authors.forename: Maria
authors.initials: M
authors.lastname: Deak
authors.forename: Rachel
authors.initials: R
authors.lastname: Toth
authors.forename: Joanne
authors.initials: J
authors.lastname: McIlrath
authors.forename: J Simon C
authors.initials: JS
authors.lastname: Arthur
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D017124:Conserved Sequence; D004789:Enzyme Activation; D006801:Humans; D013058:Mass Spectrometry; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D010768:Phosphoserine; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C116919:mitogen and stress-activated protein kinase 1; D020928:Mitogen-Activated Protein Kinases