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Cellular ERK Phospho-form Profiles with Conserved Preference for a Switch-like Pattern

Abstract:

ERK is a member of the MAPK pathway with essential functions in cell proliferation, differentiation and survival. Complete ERK activation by the kinase MEK requires dual phosphorylation at T and Y within the activation motif TEY. We show that exposure of primary mouse hepatocytes to hepatocyte growth factor (HGF) results in phosphorylation at the activation motif, but not of other residues nearby. To determine the relative abundances of unphosphorylated ERK and the three ERK phospho-forms pT, pY and pTpY, we employed an extended one-source peptide/phosphopeptide standard method in combination with nanoUPLC-MS. This method enabled to determine the abundances of phospho-forms with a relative variability of ≤ 5 % (SD). We observed a switch-like preference of ERK phospho-form abundances towards the active, doubly phosphorylated and the inactive, unphosphorylated form. Interestingly, ERK phospho-form profiles were similar upon growth factor and cytokine stimulation. A screening of several murine and human cell systems revealed that the balance between TY- and pTpY-ERK is conserved while the abundances of pT- and pY-ERK are more variable within cell types. We show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases determine the ERK phospho-form distribution. This study provides novel quantitative insights into multi-site phosphorylation.

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J. Proteome Res.
J. Proteome Res. 12(2): 637-46
6th Dec 2012

Bettina Hahn, Lorenza Alice D Alessandro, Sofia Depner, Katharina Waldow, Martin Erich Boehm, Julie Bachmann, Marcel Schilling, Ursula Klingmüller, Wolf Dieter Lehmann

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[Lorenza D'Alessandro] [Katharina Waldow] [Ursula Klingmüller]

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Views: 1691
  • Created: 10th Jan 2013 at 14:19
  • Last updated: 24th Oct 2013 at 16:15

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