title: Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase.
abstract: A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs.
authors.affiliation: Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4330, USA.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Chikumi
authors.forename: Shigetomo
authors.initials: S
authors.lastname: Fukuhara
authors.forename: J Silvio
authors.initials: JS
authors.lastname: Gutkind
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C577222:ARHGEF12 protein, human; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; C403525:RAPGEF2 protein, human; D064067:Rho Guanine Nucleotide Exchange Factors; D014443:Tyrosine; D019833:Genistein; D013917:Thrombin; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins