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.
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Research Article|
February 26 2007
Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS
Claire E. Mccoy;
Claire E. Mccoy
1
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
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Andrew Macdonald;
Andrew Macdonald
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
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Nick A. Morrice;
Nick A. Morrice
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
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David G. Campbell;
David G. Campbell
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
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Maria Deak;
Maria Deak
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
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Rachel Toth;
Rachel Toth
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
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Joanne Mcilrath;
Joanne Mcilrath
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
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J. Simon C. Arthur
J. Simon C. Arthur
2
1MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
2To whom correspondence should be addressed (email j.s.c.arthur@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
August 02 2006
Revision Received:
November 10 2006
Accepted:
November 22 2006
Accepted Manuscript online:
November 22 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2007
Biochem J (2007) 402 (3): 491–501.
Article history
Received:
August 02 2006
Revision Received:
November 10 2006
Accepted:
November 22 2006
Accepted Manuscript online:
November 22 2006
Citation
Claire E. Mccoy, Andrew Macdonald, Nick A. Morrice, David G. Campbell, Maria Deak, Rachel Toth, Joanne Mcilrath, J. Simon C. Arthur; Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS. Biochem J 15 March 2007; 402 (3): 491–501. doi: https://doi.org/10.1042/BJ20061183
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