2 research outputs found
Cellular ERK Phospho-Form Profiles with Conserved Preference for a Switch-Like Pattern
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
Cellular ERK Phospho-Form Profiles with Conserved Preference for a Switch-Like Pattern
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