Nobuchika Suzuki

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

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.

Regulation and Physiological Functions of G12/13-Mediated Signaling Pathways

Accumulating data indicate that G12 subfamily (Gα12/13)-mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. It has been demonstrated that Gα12/13-mediated signals form networks with other signaling proteins at various levels, from cell surface receptors to transcription factors, to regulate cellular responses. Gα12/13 have slow rates of nucleotide exchange and GTP hydrolysis, and specifically target RhoGEFs containing an amino-terminal RGS homology domain (RH-RhoGEFs), which uniquely function both as a GAP and an effector for Gα12/13. In this review, we will focus on the mechanisms regulating the Gα12/13 signaling system, particularly the Gα12/13-RH-RhoGEF-Rho pathway, which can regulate a wide variety of cellular functions from migration to transformation.

Regulation of RGS-RhoGEFs by Gα12 and Gα13 Proteins

Abstract Three mammalian Rho guanine nucleotide exchange factors (RhoGEFs), leukemia-associated RhoGEF (LARG), p115RhoGEF, and PDZ-RhoGEF, contain regulator of G-protein signaling (RGS) domains within their amino-terminal regions. These RhoGEFs link signals from heterotrimeric G12/13 protein-coupled receptors to Rho GTPase activation, leading to various cellular responses, such as actin reorganization and gene expression. The activity of these RhoGEFs is regulated by Gα12/13 through their RGS domains. Because RhoGEFs stimulate guanine nucleotide exchange by Rho GTPases, RhoGEF activation can be measured by monitoring GTP binding to or GDP dissociation from Rho GTPases. This article describes methods used to perform reconstitution assays to measure the activity of RhoGEFs regulated by Gα12/13.

Activation of Leukemia-associated RhoGEF by Gα13 with Significant Conformational Rearrangements in the Interface

The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Gα13. However, the molecular mechanism of LARG activation upon Gα13 binding is not yet well understood. In this study, we analyzed the Gα13-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Gα13 interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Gα13 resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Gα13 proceeds with a large negative heat capacity change (ΔCp°), accompanied by a positive entropy change (ΔS°). These results likely indicate that the binding of Gα13 with the RH domain triggers conformational rearrangements between Gα13 and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Gα13.

Gα13/PDZ-RhoGEF/RhoA signaling is essential for gastrin-releasing peptide receptor-mediated colon cancer cell migration.

Gastrin-releasing peptide receptor (GRPR) is ectopically expressed in over 60% of colon cancers. GRPR expression has been correlated with increased colon cancer cell migration. However, the signaling pathway by which GRPR activation leads to increased cancer cell migration is not well understood. We set out to molecularly dissect the GRPR signaling pathways that control colon cancer cell migration through regulation of small GTPase RhoA. Our results show that GRP stimulation activates RhoA predominantly through G13 heterotrimeric G-protein signaling. We also demonstrate that postsynaptic density 95/disk-large/ZO-1 (PDZ)-RhoGEF (PRG), a member of regulator of G-protein signaling (RGS)-homology domain (RH) containing guanine nucleotide exchange factors (RH-RhoGEFs), is the predominant activator of RhoA downstream of GRPR. We found that PRG is required for GRP-stimulated colon cancer cell migration, through activation of RhoA–Rho-associated kinase (ROCK) signaling axis. In addition, PRG-RhoA-ROCK pathway also contributes to cyclo-oxygenase isoform 2 (Cox-2) expression. Increased Cox-2 expression is correlated with increased production of prostaglandin-E2 (PGE2), and Cox-2-PGE2 signaling contributes to total GRPR-mediated cancer cell migration. Our analysis reveals that PRG is overexpressed in colon cancer cell lines. Overall, our results have uncovered a key mechanism for GRPR-regulated colon cancer cell migration through the Gα13-PRG-RhoA-ROCK pathway.

G protein-dependent basal and evoked endothelial cell vWF secretion

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1

Rho GTPases are integral to the regulation of actin cytoskeleton-dependent processes, including mitosis. Rho and leukemia-associated Rho guanine-nucleotide exchange factor (LARG), also known as ARHGEF12, are involved in mitosis as well as diseases such as cancer and heart disease. Since LARG has a role in mitosis and diverse signaling functions beyond mitosis, it is important to understand the regulation of the protein through modifications such as phosphorylation. Here we report that LARG undergoes a mitotic-dependent and cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Additionally, LARG is phosphorylated at the onset of mitosis and dephosphorylated as cells exit mitosis, concomitant with Cdk1 activity. Furthermore, using an in vitro kinase assay, we show that LARG can be directly phosphorylated by Cdk1. Through expression of phosphonull mutants that contain non-phosphorylatable alanine mutations at potential Cdk1 S/TP sites, we demonstrate that LARG phosphorylation occurs in both termini. Using phosphospecific antibodies, we confirm that two sites, serine 190 and serine 1176, are phosphorylated during mitosis in a Cdk1-dependent manner. In addition, these phosphospecific antibodies show phosphorylated LARG at specific mitotic locations, namely the mitotic organizing centers and flanking the midbody. Lastly, RhoA activity assays reveal that phosphonull LARG is more active in cells than phosphomimetic LARG. Our data thus identifies LARG as a phosphoregulated RhoGEF during mitosis.

Modification of p115RhoGEF Ser330 regulates its RhoGEF activity

p115RhoGEF is a member of a family of Rho-specific guanine nucleotide exchange factors that also contains a regulator of G protein signaling homology domain (RH-RhoGEFs) that serves as a link between Gα13 signaling and RhoA activation. While the mechanism of regulation of p115RhoGEF by Gα13 is becoming well-known, the role of other regulatory mechanisms, such as post-translational modification or autoinhibition, in mediating p115RhoGEF activity is less well-characterized. Here, putative phosphorylation sites on p115RhoGEF are identified and characterized. Mutation of Ser(330) leads to a decrease in serum response element-mediated transcription as well as decreased activation by Gα13 in vitro. Additionally, this study provides the first report of the binding kinetics between full-length p115RhoGEF and RhoA in its various nucleotide states and examines the binding kinetics of phospho-mutant p115RhoGEF to RhoA. These data, together with other recent reports on regulatory mechanisms of p115RhoGEF, suggest that this putative phosphorylation site serves as a means for initiation or relief of autoinhibition of p115RhoGEF, providing further insight into the regulation of its activity.

A method of generating antibodies against exogenously administered self-antigen by manipulating CD4+CD25+ regulatory T cells

The generation of antibodies against self-antigens or antigens having a high degree of structural homology with self-antigens is a difficult task because of immunological tolerance. CD4(+)CD25(+) regulatory T cells play an important role in maintaining peripheral tolerance. Sakaguchi et al. previously reported that the transfusion of CD25(+) cell-depleted mouse splenocytes into syngeneic nude mice results in a breaking of peripheral tolerance that leads to the development of autoimmunity. In this study, we attempted to apply this mouse model to the generation of antibodies against self-antigens. We depleted CD25(+) cells from BALB/c mouse splenocytes and transferred the rest of the cells into syngeneic nude mice. The animals were immunized with mouse thyroglobulin. We observed a significant increase of the anti-mouse thyroglobulin antibody titer in the group of mice immunized twice within 10 days after the cell transfer (P<0.05). From these mice, we established hybridoma cell lines producing anti-mouse thyroglobulin monoclonal antibodies, including a clone with a dissociation constant of 10(-8)M. Control nude mice which received CD25(+) cell-containing BALB/c splenocytes did not produce anti-mouse thyroglobulin antibodies. When the CD25(-)cell-transferred nude mice were immunized with mouse Gα12, another self-antigen, anti-Gα12 antibodies were produced in the sera. This method should prove highly useful in the generation of antibodies against self-antigens or antigens for which the structure is highly conserved across species.