Mass spectrometry based characterization of differential phosphorylation of signaling proteins by epidermal growth factor and nucleotides
Kehasse, Amanuel Yohannes
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Corneal wound repair involves several signaling events that are critical for successful wound closure. Physical injury, mechanical stimuli and cellular stress induce release of endogenous nucleotides and growth factors to the extracellular milieu. Upon activation of their respective receptors, the nucleotides and growth factors activate a wide range of signaling events. Previous studies demonstrated that injury and exposure to exogenous nucleotides result in phosphorylation of epidermal growth factor receptor (EGFR). Phosphorylation features, e.g., site occupancy and the dynamics of modifications at specific residues of proteins, are known to affect protein stability and function. The hypothesis tested is that injury/nucleotides and EGF stimulation result in distinct phosphorylation patterns on EGFR, leading to differential recruitment and subsequent phosphorylation of downstream signaling proteins. Mass spectrometry-based quantitative analysis of the phosphorylation profiles showed distinct profiles for tyrosine residues on EGFR, protein kinase C, mitogen-activated protein kinase, and other proteins of the EGFR and purinergic-signaling network. In addition to the difference in recruitment of the adaptor protein growth factor receptor-bound protein 2 (Grb2) and SHC-adaptor protein (Shc) isoforms, UTP and EGF stimulation resulted in distinct phosphorylation of phospholipase Cγl, Shc and Src. It was demonstrated that, in comparison to EGF, a scratch wound injury or stimulation with UTP caused brief internalization of EGFR, minimal association with Grb2 and lesser phosphorylation of EGFR. Previously the P2Y2 receptor was shown to play a critical role in injury induced Ca2+ mobilization and migration. Therefore, cells were transfected with siRNA to P2Y2 receptor and analysis of pEGFR and downstream signaling proteins was analyzed using stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometric analysis. P2Y2 knockdown resulted in decreased phosphorylation of Y992, Y1086 and Y1148 residues on EGFR. Consistent with this result, Western blots on cultures transfected with siRNA using site-specific antibodies to P2Y2 receptor showed a decrease in phosphorylation of EGFR-Y845, EGFR-Y1068, and EGFR-Y1086 residues. Taken together, our fmdings show that injury and activation of purinergic receptors and direct activation of the EGFR receptor via EGF induce distinct downstream pathways in which phosphorylation of signaling proteins may dictate respective downstream signaling events.
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