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Extracellular signal-regulated kinase-1 phosphorylates early growth response-1 at serine 26
- Santiago, Fernando S., Sanchez-Guerrero, Estella, Zhang, Guishui, Zhong, Ling, Raftery, Mark J., Khachigian, Levon M.
- Biochemical and biophysical research communications 2019 v.510 no.3 pp. 345-351
- antigens, genes, mass spectrometry, mice, mitogen-activated protein kinase, monoclonal antibodies, mutants, peptides, phosphoproteins, point mutation, polyacrylamide gel electrophoresis, precipitin tests, protein phosphorylation, serine
- Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser12 and Ser26 with the latter ∼12 times more abundant than Ser12. Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser26>Ala26 mutant Egr-1) revealed that Ser26 accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser26. pS26 MAb recognised inducible ∼75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. Immunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the ∼75 kDa species but not ∼100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1.