Figure 5: Gα_12/13 signalling reduces the interaction of 14-3-3 with GEF-H1 and can stimulate exchange activity independent of Gβ.

(a) Gα_13QL disrupts 14-3-3 protein interaction with GEF-H1. HEK293T cells were transfected with Myc-GEF-H1 with or without Flag-14-3-3 and Gα_13QL, or with the dominant-negative RGS peptide (rgRGS). Myc-protein complexes were immunoblotted with anti-GEF-H1, anti-Flag or anti-Gα₁₃ antibodies. Western blots of GEF-H1, Gα₁₃ and 14-3-3 in the whole-cell lysate are shown. (b) Gα₁₃-bound GEF-H1 is dephosphorylated at Ser885. HEK293T cells were transfected with GEF-H1, Flag-Tctex-1, Myc-14-3-3 and Gα_13QL. Myc- Flag- and Gα₁₃ protein complexes were precipitated from lysates and immunoblotted with anti-Tctex-1, anti-14-3-3, anti-Gα₁₃, anti-GEF-H1 or anti-phosphoSer885 antibodies (right panel). Western blots of total cell lysates are shown (left). (c) Inhibition of both Gα and Gβ are required to inhibit thrombin-induced disruption of the GEF-H1–dynein complex. Cells were transfected with Myc-GEF-H1 with or without rgRGS and/or βARKct. Cells were treated with thrombin and Myc-complexes were immunoprecipitated from lysates and blotted for endogenous GEF-H1 and DIC. Western blots of total GEF-H1 and DIC are shown below. (d) Inhibition of both Gα and Gβ are required to block thrombin-induced relocalization of GEF-H1. GEF-H1^−/− cells were transfected with GEF-H1–eGFP alone or with rgRGS and/or βARKct. Cells were treated with thrombin, fixed and stained with anti-α-tubulin antibodies and imaged by confocal microscopy. Scale bar, 20 μm. (e) Inhibition of both Gα and Gβ are required to block LPA-induced activation of GEF-H1 exchange activity. RhoA nucleotide exchange rates were measured in lysates from HEK293T cells expressing combinations of GEF-H1–eGFP, Tctex-1, rgRGS domain and βARKct peptide, with or without LPA stimulation, as indicated. The s.d. of at least three independent replicates are shown along with P values from pairwise t-tests.