Vascular Biology and Translational Research Laboratory, School of Medical Sciences, University of New South Wales, Sydney, Australia.
UNSW Medicine, University of New South Wales, Sydney, Australia.
Biochem Biophys Res Commun. 2019 Mar 12;510(3):345-351. doi: 10.1016/j.bbrc.2019.01.019. Epub 2019 Jan 31.
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 Ser and Ser with the latter ∼12 times more abundant than Ser. Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser>Ala mutant Egr-1) revealed that Ser 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 Ser. 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.
Egr-1 是一种即刻早期基因产物和主调控因子，最初在 20 世纪 80 年代被发现时被描述为一种磷酸化蛋白。然而，Egr-1 中磷酸化的特定残基仍然难以捉摸。在这里，我们使用 ERK1 在体外对重组 Egr-1 进行磷酸化，然后进行质谱分析，结果鉴定出 Egr-1 中 Ser 和 Ser 的磷酸化，后者的丰度约为 Ser 的 12 倍。与 Ser>Ala 突变型 Egr-1 相比，野生型重组 Egr-1 的磷酸化表明，Ser 占 ERK1 对 Egr-1 磷酸化的大部分。使用 N-FGSFPH(pS)PTMDNYC-C 作为抗原生成小鼠单克隆抗体（pS26 MAb）。pS26 MAb 识别 ERK1 磷酸化的 Egr-1，但不识别 Ser 点突变的 Egr-1。pS26 MAb 识别在暴露于 FGF-2 的细胞的核提取物中诱导的约 75 kDa 和 100 kDa 物种。肽阻断揭示了两种诱导的物种都是磷酸化特异性的。用 pS26 MAb 免疫沉淀暴露于 FGF-2 的细胞的核提取物，然后进行 SDS-PAGE 和质谱分析，鉴定出与约 75 kDa 物种相对应的 Egr-1 序列，但不与约 100 kDa 物种相对应。本研究鉴定了内源性 Egr-1 中磷酸化的特定氨基酸。
