Ezrin ubiquitylation by WWP1.
(A) Lysates from 293T cells transfected with the indicated combinations of wild type flag-WWP1, flag-WWP1 C890A (C/A) and Myc-ubiquitin were immunoprecipitated with an anti-ezrin antibody followed by a blot with a Myc antibody. (B) 293T cells expressing Flag-WWP1, ezrin-VSVG and Myc-His-ubiquitin were lysed either in RIPA buffer or RIPA buffer containing 1% SDS and the ubiquitylated proteins were purified on TALON beads. The immunoblot was performed with an anti-VSVG antibody. (C) In vitro ubiquitylation was performed in presence of the indicated proteins. Left panel shows a Ponceau staining. Right panel: the blot was performed with an anti-Myc antibody to detect ubiquitylated protein. The brackets indicate the extent ubiquitylation of ezrin Δ29 and WWP1 (D) Ezrin is multi-ubiquitylated. 293T cells were transfected with the indicated plasmids. Upper panels: Immunoprecipitation of ezrin-Myc was followed by immunoblotswith the anti-VSVG (ubiquitin) and anti-ezrin antibodies. The asterisk indicates the position of non ubiquitylated ezrin. Lower panels: the immunoblots were performed on cell lysates with anti-Flag (WWP1) and anti-ezrin antibodies. (E) Ezrin ubiquitylation requires the PPXY motif. Lysates from 293T cells transfected with the indicated combinations of wild type flag-WWP1, flag-WWP1 C890A, wild type ezrin-VSVG, P474A, P475A, Y477F ezrin-VSVG and Myc-ubiquitin were immunoprecipitated with an anti-ezrin antibody followed by immunoblot with anti-Myc and -VSVG antibodies. (F) Ezrin steady state levels are not affected by WWP1 expression. 293T cells were transfected with the indicated combinations of plasmids and treated or not with the proteasomal inhibitor MG132. Immunoblots were performed with the indicated antibodies. Tubulin serves as a loading control. (G) Ezrin ubiquitylation with MG132 treatment. 293T cells were transfected with the indicated plasmids and treated or not with MG132. Immunoprecipitation with anti-VSVG antibody was followed by immunoblotting with anti-Myc and -VSVG antibodies. (H) Expression of WWP1 does not affect the half-life of ezrin. Stable Flp-In™ T-REx™ 293 cell lines expressing wild type PC- tagged WWP1 or vector alone were pulse-labeled for 15 min with 35Smethionine/cysteine and chased for 0, 2, 24, 48, and 72 hours. Autoradiography of ezrin immunoprecipitates is shown.
