Typical Phospho-form Abundance Profile of Cellular ERK Discovered by Quantitative UPLC-MS
Conference Recording Apr 07, 2013
AbstractExtracellular signal-regulated kinase (ERK) has essential functions in cell differentiation, proliferation, and survival. ERK activation consists of phosphorylation at threonine (T) and tyrosine (Y) at the activation motif TEY by the kinase MEK. The TEY motif can occur in four forms, TY, pTY, TpY, and pTpY. The relative abundances of these four forms were profiled by an extended version of the one-source peptide/phosphopeptide standard methodology in combination with nanoUPLC-MS which gives phospho-form abundance data with low variability of = 5 %. Following cellular ERK stimulation, we observed that the distribution of ERK phospho-forms in general showed a preference toward the active, doubly phosphorylated and the inactive, unphosphorylated species. This result suggests a switch-like activation/deactivation mechanism. ERK phospho-form distribution profiles were found to be independent of the stimulus and highly similar for the isoforms ERK1 and ERK2. Screening of several murine and human cell types revealed that the relative abundances of TY- and pTpY-ERK are a general property, while the relative abundances of pT- and pY-ERK are more variable. Additionally, we show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases rather than the MEK kinase shape the ERK phospho-form distribution.