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Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry.
- Source :
-
Proteomics [Proteomics] 2019 Sep; Vol. 19 (17), pp. e1900086. Date of Electronic Publication: 2019 Aug 07. - Publication Year :
- 2019
-
Abstract
- Mitogen-activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three-tiered kinase module-MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase-that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine-glutamic acid-tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal-regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)-selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID-SRM-MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono-phosphorylatable mutants ERK <superscript>T202A</superscript> and ERK <superscript>Y204F</superscript> indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.<br /> (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Subjects :
- Animals
Extracellular Signal-Regulated MAP Kinases chemistry
Extracellular Signal-Regulated MAP Kinases genetics
Glutamic Acid chemistry
Glutamic Acid genetics
HeLa Cells
Humans
Mitogen-Activated Protein Kinase Kinases chemistry
Mitogen-Activated Protein Kinase Kinases genetics
Mutation
PC12 Cells
Phosphorylation
Rats
Signal Transduction
Threonine chemistry
Threonine genetics
Tyrosine chemistry
Tyrosine genetics
Extracellular Signal-Regulated MAP Kinases metabolism
Glutamic Acid metabolism
Mitogen-Activated Protein Kinase Kinases metabolism
Threonine metabolism
Tyrosine metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1615-9861
- Volume :
- 19
- Issue :
- 17
- Database :
- MEDLINE
- Journal :
- Proteomics
- Publication Type :
- Academic Journal
- Accession number :
- 31318149
- Full Text :
- https://doi.org/10.1002/pmic.201900086