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

Abstract

Activated G␣13 stimulated the RhoGEF activity of p115 through interaction with the N-terminal RGS domain. However, G␣12 could not activate Rho through p115, although it interacted with the RGS domain of p115. The biochemical mechanism from G␣12 to Rho activation remained unknown. In this study, we analyzed the interaction of leukemia-associated RhoGEF (LARG), which also contains RGS domain, with G␣12 and G␣13. RGS domain of LARG demonstrated G␣12-and G␣13-specific GAP activity. LARG synergistically stimulated SRF activation by G␣12 and G␣13 in HeLa cells, and the SRF activation by G␣12-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 G␣13 but not G␣12. However, when LARG was phosphorylated by Tec, G␣12 effectively stimulated the RhoGEF activity of LARG. These results demonstrate the biochemical mechanism of Rho activation through G␣12 and that the regulation of RhoGEFs by heterotrimeric G proteins G12͞13 is further modulated by tyrosine phosphorylation.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase

“…Activated Ga 12/13 in turn binds to and activate rhoGEFs Suzuki et al, 2003), which then activate RhoA (Dhanasekaran and Dermott, 1996;Kurose, 2003). Ga 13 has been directly implicated in GPCRstimulated cell migration (Offermanns et al, 1997;Radhika et al, 2004).…”

“…Transient transfection PC-3 cells were transiently transfected with mammalian expression vectors encoding FRNK, the dominant-negative mutant form of FAK (kindly provided by Dr Filippo Giancotti) or Ga 13 -CT, the dominant-negative mutant form of Ga 13 (Suzuki et al, 2003) using Lipofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. Cytomegalovirus promoter driven pGFP-C1 plasmid (CLONTECH, CA, USA) was used as a transfection control to evaluate the transfection efficiency.…”

CD98hc (SLC3A2) regulation of skin homeostasis wanes with age

“…It was recently shown that Fyn may stimulate LARG activity downstream of integrins (Guilluy et al, 2011), but we observe, rather, an inhibition of Src family kinases upon loss of CD98hc in our experimental model, which suggests that LARG has to be activated by other means. These may possibly include phosphorylation by kinases such as PKC or FAK, for instance (Chikumi et al, 2002;Disatnik et al, 2002;Suzuki et al, 2003;Dovas et al, 2006), as well as regulation of LARG homodimerization by CD98hc, which has long been known to regulate its activity (Chikumi et al, 2004). Further molecular investigations, in vitro, in cell culture should be performed to dissect this regulation.…”

Identification and molecular characterization of the Gα12–Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans

“…After 4 h, FBS was supplemented into the culture to a final concentration of 10%. Cells were harvested after 20 h. Immunoprecipitation by myc antibody in the presence or absence of AlF 4 Ϫ was performed as described (14). The immunoprecipitates were loaded onto 10% SDS͞polyacrylamide gel for electrophoresis and immunoblotted with anti-myc antibody (Sigma) or anti-flag M2 antibody (Sigma).…”

“…Recently, two other mammalian RhoGEFs with the RGS domain (RGS-RhoGEFs), PSD-95͞Dlg͞ZO-1 (PDZ)-RhoGEF, and leukemia-associated RhoGEF (LARG), have been shown to mediate Rho activation through G␣12͞13 (12)(13)(14).…”