Fig 1.
SGEF is tyrosine-phosphorylated by Src.
(A) The domain structure of SGEF and Ephexin4. Tyrosine residues in the LYQ motif correspond to the phosphorylation sites of Ephexin1 and Eohexin5 by Src. (B, C) Cell lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Flag antibody, and tyrosine phosphorylation (p-Tyr) and total cell lysates were analyzed by immunoblotting with antibodies against phospho-tyrosine, Flag, and HA.
Fig 2.
Expression of constitutively active Src suppresses SGEF-mediated promotion of cell migration and RhoG activation.
(A) Motility of HEK293T cells was evaluated by Transwell cell migration assays. HEK293T cells transfected with YFP alone or together with the indicated plasmids were plated in the upper chamber of the filters, and cells that had migrated to the underside of the filters were analyzed at 6 h after plating. Results are expressed as means ± SEM (n = 5) of relative cell migration, with SGEF-WT-transfected cell migration set at 1 (*P < 0.05, ***P < 0.001, t-test). (B) HEK293T cells transfected with YFP alone or together with the indicated plasmids were plated in the upper chamber of the filters, and cells that had migrated to the underside of the filters were analyzed at 6 h after plating. Results are expressed as means ± SEM (n = 4) of relative cell migration, with Ephexin4-WT-transfected cell migration set at 1 (ns, not significant, ***P < 0.001, t-test). (C) Cell lysates from HEK293T cells transfected with indicated plasmids were incubated with GST-ELMO-NT, and bound Myc-RhoG (GST-ELMO-NT pull-down) and total cell lysates were analyzed with antibodies against Myc, Flag, and HA. The amount of bound Myc-RhoG was normalized to the total amount of Myc RhoG in cell lysates, and relative RhoG activity was expressed as fold change relative to the basal activity (None). Data are presented as means ± SEM from four independent experiments (*P < 0.05, ***P < 0.001, t-test).
Fig 3.
Phosphorylation of SGEF by Src blocks the interaction of SGEF with RhoG.
(A) Cell lysates from HEK293T cells transfected with the indicated plasmids were incubated with GST-RhoG-G15A, and bound Flag-SGEF (GST-RhoG-G15A pull-down) and total cell lysates were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. (B) HEK293T cells were transfected with the indicated plasmids and treated for 30 min with 20 μM PP2 or PP3. Then cell lysates were incubated with GST-RhoG-G15A, and bound Flag-SGEF and total cell lysates were analyzed by immunoblotting with anti-Flag and anti-HA antibodies.
Fig 4.
Y530 is an important phosphorylation site in SGEF.
(A) Cell lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Flag antibody, and tyrosine phosphorylation (p-Tyr) and total cell lysates were analyzed by immunoblotting with antibodies against phospho-tyrosine, Flag, and HA. (B) Cell lysates from HEK293T cells transfected with the indicated plasmids were incubated with GST-RhoG-G15A, and bound Flag-SGEF and total cell lysates were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. (C) Cell lysates from HEK293T cells transfected with indicated plasmids were incubated with GST-ELMO-NT, and bound Myc-RhoG and total cell lysates were analyzed with antibodies against Myc, Flag, and HA.
Fig 5.
Src does not block the SGEF-Y530F-mediated promotion of cell migration.
(A) HEK293T cells transfected with YFP alone or together with the indicated plasmids were plated in the upper chamber of the filters, and cells that had migrated to the underside of the filters were analyzed at 6 h after plating. Results are expressed as means ± SEM (n = 3) of relative cell migration, with SGEF-Y530F-transfected cell migration set at 1 (*P < 0.05, **P < 0.01, t-test). (B) Cell lysates from HeLa cells transfected with the indicated plasmids were immunoprecipitated with anti-Flag antibody, and tyrosine phosphorylation (p-Tyr) and total cell lysates were analyzed by immunoblotting with antibodies against phospho-tyrosine, Flag, and HA. (C) HeLa cells transfected with YFP alone or together with the indicated plasmids were plated in the upper chamber of the filters, and cells that had migrated to the underside of the filters were analyzed at 6 h after plating. Results are expressed as means ± SEM (n = 3) of relative cell migration, with SGEF-WT or Y530F-transfected cell migration set at 1 (ns, not significant, *P < 0.05, **P < 0.01, t-test).