title: 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 us 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 toward 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 multisite phosphorylation.
authors.affiliation: Molecular Structure Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Hahn
authors.forename: Lorenza Alice
authors.initials: LA
authors.lastname: D'Alessandro
authors.forename: Sofia
authors.initials: S
authors.lastname: Depner
authors.forename: Katharina
authors.initials: K
authors.lastname: Waldow
authors.forename: Martin E
authors.initials: ME
authors.lastname: Boehm
authors.forename: Julie
authors.initials: J
authors.lastname: Bachmann
authors.forename: Marcel
authors.initials: M
authors.lastname: Schilling
authors.forename: Ursula
authors.initials: U
authors.lastname: Klingmüller
authors.forename: Wolf D
authors.initials: WD
authors.lastname: Lehmann
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002247:Carbon Isotopes; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D061251:Primary Cell Culture; D053719:Tandem Mass Spectrometry
chemical_list: D002247:Carbon Isotopes; C553307:HGF protein, mouse; D009587:Nitrogen Isotopes; D010750:Phosphoproteins; D017228:Hepatocyte Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; D010744:Phosphoric Monoester Hydrolases