Gα12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF

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Abstract
Heterotrimeric G proteins, G12 and G13, have been shown to transduce signals from G protein-coupled receptors to activate Rho GTPase in cells. Recently, we identified p115RhoGEF, one of the guanine nucleotide exchange factors (GEFs) for Rho, as a direct link between Gα13 and Rho [Kozasa, T., et al. (1998) Science 280, 2109–2111; Hart, M. J., et al. (1998) Science 280, 2112–2114]. Activated Gα13 stimulated the RhoGEF activity of p115 through interaction with the N-terminal RGS domain. However, Gα12 could not activate Rho through p115, although it interacted with the RGS domain of p115. The biochemical mechanism from Gα12 to Rho activation remained unknown. In this study, we analyzed the interaction of leukemia-associated RhoGEF (LARG), which also contains RGS domain, with Gα12 and Gα13. RGS domain of LARG demonstrated Gα12- and Gα13-specific GAP activity. LARG synergistically stimulated SRF activation by Gα12 and Gα13 in HeLa cells, and the SRF activation by Gα12-LARG was further stimulated by coexpression of Tec tyrosine kinase. It was also found that LARG is phosphorylated on tyrosine by Tec. In reconstitution assays, the RhoGEF activity of nonphosphorylated LARG was stimulated by Gα13 but not Gα12. However, when LARG was phosphorylated by Tec, Gα12 effectively stimulated the RhoGEF activity of LARG. These results demonstrate the biochemical mechanism of Rho activation through Gα12 and that the regulation of RhoGEFs by heterotrimeric G proteins G12/13 is further modulated by tyrosine phosphorylation.