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. 2005 Mar;16(3):1481–1490. doi: 10.1091/mbc.E04-08-0721

Figure 3.

Figure 3.

Ezrin Y145 phosphorylation by Src. (A) Tyrosine phosphorylation ((P)Y) and phosphorylated Y145 ezrin ((P)Y145) were detected on immunoprecipitated VSVG-ezrin from LLC-PK1 cells untreated or treated with 0.1 mM pervanadate for 5 min (-/+ pvd). Pretreatment with the Src kinase inhibitor, PP2 (20 min; 10 μM) inhibits ezrin Y145 phosphorylation. (B) Endogenous ezrin immunoprecipitated from A431 cells. EGF treatment (3 min; 100 nM) induces ezrin Y145 phosphorylation, which is sensitive to the PP2 inhibitor. (C) Immunoprecipitated ezrin from MDCK ts v-Src cells at different times after v-Src activation (40.5°C switched to 35°C). Immunoblots detect time-dependent induced ezrin tyrosine phosphorylation and ezrin (P)Y145 upon v-Src activation. (D) Immunoprecipitated ezrin from SYF-/- cells. Only in cells transfected with Src Y527F is ezrin phosphorylated at Y145. (E) Src directly phosphorylates ezrin at Y145 in vitro. Top: kinase assays show phosphorylation of GST-truncated ezrin (Ez wt Δ52), but not of GST-wild-type ezrin (Ez wt). Reduced phosphorylation was detected on GST-truncated ezrin with the Y145F mutation. Bottom: Coomassie gel stains GST-purified ezrin variants used in the assay. Results are representative of three independent experiments. (F) VSVG-tag immunoprecipitations from cells expressing wild-type ezrin or the PIP2- mutant of ezrin. Pervanadate treatment does not induce Y145 phosphorylation of the ezrin PIP2- mutant.