摘要：

BACKGROUND: Cell migration is required for the physiological processes of wound repair, resolution of inflammation, and organogenesis, but is also involved in the pathology of tumor invasion. Thus, investigation of the regulatory mechanisms of cell migration is important for both understanding basic intestinal biology and identifying potential therapeutic targets for disorders such as inflammatory bowel diseases and colon cancer. HYPOTHESIS: Upon ligand binding, epidermal growth factor (EGF) receptor (R) autophosphorylates on c-terminal tyrosines, generating docking sites for signaling partners that stimulate proliferation, restitution, and chemotaxis. Specificity for individual EGFR tyrosines in cellular responses has been hypothesized but not well documented. Here we tested the requirement for particular tyrosines, and associated downstream pathways, in mouse colon epithelial cell chemotactic migration. We compared these requirements to those for the phenotypically distinct restitution (wound healing) migration. METHODS: Wild-type, Y992/1173F, Y1045F, Y1068F, and Y1086F EGFR constructs were expressed in EGFR-/- cells; EGF-induced chemotaxis or restitution were determined by Boyden chamber or modified scratch wound assay, respectively. Pharmacological inhibitors of p38, phospholipase C (PLC), Src, MEK, JNK/SAPK, phosphatidylinositol 3-kinase (PI 3-kinase), and protein kinase C (PKC) were used to block EGF-stimulated signaling. Pathway activation was determined by immunoblot analysis. RESULTS: Unlike wild-type EGFR, Y992/1173F and Y1086F EGFR did not stimulate colon epithelial cell chemotaxis towards EGF; Y1045F and Y1068F EGFR partially stimulated chemotaxis. Only wt EGFR promoted colonocyte restitution. Inhibition of p38, PLC, and Src blocked chemotaxis; JNK, PI 3-kinase, and PKC inhibitors blocked restitution but not chemotaxis. All four EGFR mutants stimulated downstream signaling in response to EGF, but Y992/1173F EGFR was partially defective in PLCγ activation while both Y1068F and Y1086F EGFR failed to activate Src. CONCLUSIONS: Specific EGFR tyrosines play key roles in determining cellular responses to ligand. Chemotaxis and restitution, which have different migration phenotypes and physiological consequences, have overlapping but not identical EGFR signaling requirements.

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