Fig. 3

AnxA6 SUMOylation inhibits EGFR/ERK pathway by promoting EGFR-PKCα interaction. A-B The UBC9 expression enhanced AnxA6-mediated inhibition of pY-EGFR and p-ERK1/2 levels, whereas the inhibitor ML792 treatment abolished suppression of EGFR/ERK phosphorylation due to AnxA6 upon EGF stimulation. HeLa or A431 cells were transfected with pFlag-AnxA6 plasmids for 24 h, followed to enhance or inhibit total SUMOylation by pHA-UBC9 plasmid transfection (A) or ML792 inhibitor incubation (B). Cells were serum-starved for overnight and then stimulated with 200 ng/ml (A) or 100 ng/ml (B) EGF for 3 min, then cell lysates were harvested to detect protein expression of pY-EGFR, EGFR, pERK1/2, ERK1/2 and AnxA6. C-D The K299R mutation of AnxA6 impaired its ability to inactivate pY-EGFR and pERK1/2. HeLa (C) or A431 (D) cells were transfected with pFlag-AnxA6 or pFlag-AnxA6^K299R plasmids for 36 h, starved overnight and followed with/without 100 ng/ml EGF treatment for 3 min. Cell lysates were immunoblotted with the indicated antibodies. E The K299R mutation impaired its recruitment ability of AnxA6 to promote EGFR-PKCα interaction. A431 cells were transfected with pFlag-AnxA6 or pFlag-AnxA6^K299R plasmids for 36 h, starved overnight and followed with 100 ng/ml EGF treatment for 3 min. One portion of cell lysates was performed IP to capture EGFR-binding complex, detected by western blot analysis with Flag, EGFR and PKCα antibodies. Another part of cell lysates was detected cellular protein level of Flag-AnxA6, EGFR and PKCα. F HeLa cells were co-transfected with the indicated plasmids for 24 h, followed ML792 treatment for 12 h, and cells were subjected to serum deprivation for overnight to stimulate with 100 ng/ml EGF for 3 min. One portion of cell lysates was performed IP to capture Flag-tagging AnxA6 binding protein complex, and detected protein expression by western blot analysis with Flag, SUMO1, EGFR and PKCα antibodies. Another part of cell lysates was detected cellular level of Flag-AnxA6, UbC9, SUMO1, EGFR and PKCα. G The K299R mutation of AnxA6 impaired its ability to interact EGFR but not affect its binding to PKCα. A431 cells were transfected with pFlag-AnxA6 or pFlag-AnxA6^K299R plasmids for 36 h, starved overnight and followed with 100 ng/ml EGF treatment for 3 min. Cell lysates were immunoprecipitated to capture Flag-tagging AnxA6 and subsequently immunoblotted with indicated antibodies. S-Flag-AnxA6: SUMOylated Flag-tagging AnxA6, IP: immunoprecipitation, IB: immunoblot, Input: same account of cell lysate to load