Shigetomo Fukuhara

Cbl-b regulates the CD28 dependence of T-cell activation

Whereas co-stimulation of the T-cell antigen receptor (TCR) and CD28 triggers T-cell activation, stimulation of the TCR alone may result in an anergic state or T-cell deletion, both possible mechanisms of tolerance induction. Here we show that T cells that are deficient in the adaptor molecule Cbl-b (ref. 3) do not require CD28 engagement for interleukin-2 production, and that the Cbl-b-null mutation (Cbl-b-/-) fully restores T-cell-dependent antibody responses in CD28-/-mice. The main TCR signalling pathways, such as tyrosine kinases Zap-70 and Lck, Ras/mitogen-activated kinases, phospholipase Cγ-1 and Ca2+ mobilization, were not affected in Cbl-b-/- T cells. In contrast, the activation of Vav, a guanine nucleotide exchange factor for Rac1/Rho/CDC42, was significantly enhanced. Our findings indicate that Cbl-b may influence the CD28 dependence of T-cell activation by selectively suppressing TCR-mediated Vav activation. Mice deficient in Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis, suggesting that the dysregulation of signalling pathways modulated by Cbl-b may also contribute to human autoimmune diseases such as multiple sclerosis.

A Novel PDZ Domain Containing Guanine Nucleotide Exchange Factor Links Heterotrimeric G Proteins to Rho

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Gα12 family, Gα12 and Gα13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Gα12 and Gα13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Gα12 family which, in turn, activate an exchange factor acting on Rho.

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G12 family to Rho

A putative guanine nucleotide exchange factor (GEF), termed leukemia‐associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein‐coupled receptors (GPCRs) and heterotrimeric G proteins of the Gα12 family stimulate Rho‐dependent signaling pathways.

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.

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 α subunits of the Gα12family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Gα12 and Gα13 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, Gα12, Gα13, and Gαq 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.

Divergent Signaling Pathways Link Focal Adhesion Kinase to Mitogen-activated Protein Kinase Cascades EVIDENCE FOR A ROLE OF PAXILLIN IN c-Jun NH2-TERMINAL KINASE ACTIVATION

Stimulation of a number of cell surface receptors, including integrins and G protein-coupled receptors, results in the activation of a non-receptor tyrosine kinase known as focal adhesion kinase (FAK). In turn, this kinase is believed to play a critical role in signaling to intracellular kinase cascades controlling gene expression such as extracellular signal-regulated kinases (ERKs), by a yet poorly defined mechanism. Furthermore, whether this tyrosine kinase also mediates the activation of other mitogen-activated protein kinase family members, such as c-Jun NH2-terminal kinases (JNKs), is still unclear. We show here that the activation of FAK by anchoring to the cell membrane is itself sufficient to stimulate potently both ERK and JNK. These effects were found to be phosphatidylinositol 3-kinase-independent, as FAK effectively stimulated Akt, and wortmannin suppressed Akt but not ERK or JNK activation. As previously reported by others, activation of ERK correlated with the ability of FAK to induce tyrosine phosphorylation of Shc. Surprisingly, however, stimulation of JNK was not dependent on the kinase activity of FAK or on the ability to induce tyrosine phosphorylation of FAK substrates. Instead, we provide evidence that FAK may stimulate JNK through a novel pathway involving the recruitment of paxillin to the plasma membrane and the subsequent activation of a biochemical route dependent on small GTP-binding proteins of the Rho family.

Novel Molecular Mediators in the Pathway Connecting G-protein-coupled Receptors to MAP Kinase Cascades

The family of receptors coupled to heterotrimeric GTP-binding proteins (G proteins) constitutes the largest group of integral membrane proteins involved in signal transduction. These receptors participate in many important biological functions, ranging from photoreception to neurotransmission and exocytosis, as well as in processes such as embryogenesis, angiogenesis, tissue regeneration and normal and aberrant cell growth. Initial studies addressing the functioning of these receptors had focused primarily on second messenger-generating systems. Here, the authors survey the current knowledge on how this family of receptors transduces signals to the nucleus through an intricate network of nucleotide exchange factors, small GTPases, and cytoplasmic kinases which, in turn, control gene expression by phosphorylating nuclear regulatory molecules.

PDZ-RhoGEF and LARG Are Essential for Embryonic Development and Provide a Link between Thrombin and LPA Receptors and Rho Activation

Background: Many Rho GEFs have been proposed to link GPCRs to Rho activation. Results: PDZ-RhoGEF and LARG double deficiency leads to embryonic lethality and, along with p115 RhoGEF, are required for thrombin-induced cell migration, proliferation, and RhoA activation. Conclusion: PDZ-RhoGEF, LARG, and p115 RhoGEF mediate Gα12/13 signaling to RhoA. Significance: These findings identify novel targets for pharmacological intervention in diseases involving aberrant GPCR signaling. G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.

A new twist for the tumour suppressor hamartin

Ezrin/radixin/moesin (ERM) proteins form crosslinks between cortical actin filaments and the plasma membrane. New findings indicate that they may also bind to the tumour-suppressor protein hamartin, thereby regulating Rho GTPases, the actin-based cytoskeleton and cell adhesion. Loss of hamartin function disrupts cell adhesion, which may contribute to the formation of tumours in individuals carrying hamartin mutations.

Signaling from G-Protein-Coupled Receptors to the Nucleus

With more than 1000 members, the family of G protein-coupled receptors (GPCRs) represents the largest group of cell surface receptors. The vast majority of these receptors exhibit a common structural motif consisting of the presence of seven membrane-spanning regions (1) (Fig. 1) that, when activated, undergo a dramatic conformational change, resulting in the exposure of previously masked G-protein binding sites in the intracellular loops (2–4). This causes the exchange of GDP for GTP bound to the G-protein α subunit and the dissociation of Gα from the βγ heterodimers. Then GTP-bound G-protein α subunits and βγ complexes initiate intracellular signaling responses by activating a variety of effector molecules, including adenylyl cyclases, phosphodiesterases, phospholipases, ion channels, ion transporters, and several intracellular kinases (5–8).