Figure 4.

Tyrosine phosphorylation of LARG by Tec. (A) Tyrosine phosphorylation of LARG by Tec in vitro. Myc-tagged Tec or Tec-KD was overexpressed in COS1 cells, immunoprecipitated by anti-myc antibody, and used for kinase assays. Recombinant ΔPDZ-LARG or p115RhoGEF (600 nM each) with or without 1 μM AlF-activated Gα12 or Gα13 were incubated with immunoprecipitated Tec in the presence of ATP for 20 min at 30°C. Proteins were separated by SDS/PAGE, followed by immunoblotting with antiphosphotyrosine antibody. (B) Tyrosine phosphorylation of LARG by Tec in vivo. HEK293 cells were cotransfected with myc-tagged ΔPDZ-LARG, ΔN-LARG, or p115 with or without mHTec. RhoGEFs were immunoprecipitated by anti-myc antibody from the cell lysates, and the immunoprecipitates were separated by SDS/PAGE, followed by immunoblotting using antiphosphotyrosine antibody or anti-myc antibody. (C) Stimulation of GDP dissociation from RhoA by Gα12, LARG, and Tec. GDP dissociation from RhoA by ΔPDZ-LARG was assayed for 20 min at 20°C in the presence of the indicated proteins (as described in Methods): 10 nM ΔPDZ-LARG, 200 nM AlF-activated Gα12, and 200 nM AlF-activated Gα13. *, P < 0.01, significant difference from the data with Gα12 + LARG + Tec. The results shown are from a representative experiment of three such experiments with similar results. (D) Time course of GDP dissociation from RhoA. GDP dissociation from RhoA was measured with the indicated proteins: ○, control; □, 10 nM ΔPDZ-LARG; ◊, 10 nM ΔPDZ-LARG + Tec; ▵, 10 nM ΔPDZ-LARG + 200 nM Gα12; ▿, 10 nM ΔPDZ-LARG + 200 nM Gα13; ▴, 10 nM ΔPDZ-LARG + 200 nM Gα12 + Tec; or ▾, 10 nM ΔPDZ-LARG + 200 nM Gα13 + Tec.