Cell Science at a Glance 1149 Rho activation at a family of small GTPases. Rho GTPases (GDP-bound) conformations. (Four Rho control multiple cellular processes, family members are GTPase deficient and glance including actin and microtubule bind GTP constitutively; little is known Rachel J. Buchsbaum dynamics, gene expression, the cell about their regulation.) There are three cycle, cell polarity and membrane classes of regulatory proteins that affect Division of Hematology/Oncology and Molecular Oncology Research Institute, Tufts-New England transport, through their ability to bind to the activation state of these cycling Rho Medical Center, 750 Washington Street, Boston, numerous downstream effectors, which molecules: guanine nucleotide exchange MA 02111, USA lead to diverse parallel downstream factors (GEFs), which promote exchange e-mail: [email protected] signaling pathways (Schwartz, 2004). of GTP for GDP; GTPase-activating Journal of Cell Science 120, 1149-1152 proteins (GAPs), which enhance the Published by The Company of Biologists 2007 Over 20 members of the Rho family have intrinsic GTP-hydrolysis activity, leading doi:10.1242/jcs.03428 been identified in mammalian cells to GTPase inactivation; and guanine- (Wennerberg and Der, 2005). These are nucleotide-dissociation inhibitors (GDIs), Cells receive a multitude of stimuli – represented here by the canonical proteins which bind to prenylated GDP-bound chemical (such as cytokines and growth Rho, Rac and Cdc42 and have been the Rho proteins and allow translocation factors) and physical (such as subject of numerous excellent reviews between membranes and the cytosol. mechanical stresses or adhesion to (Bishop and Hall, 2000; Ridley, 2001; Currently, the best understood regulators extracellular matrix or other cells) – that Boettner and Van Aelst, 2002; Ridley et of Rho activation in response to upstream influence cell function by affecting al., 2003; Burridge and Wennerberg, stimuli are the GEFs. intracellular signaling pathways. Very 2004; Jaffe and Hall, 2005). Like the often these stimuli involve cell surface classic monomeric Ras GTPase, most GEFs for Rho family proteins have been receptors or other molecules that Rho proteins act as switches by cycling identified in bacteria, plants, yeast, function through activation of the Rho between active (GTP-bound) and inactive worms, fruit flies and humans. Most RSKRho and MSKActivation at a Glance at a Glance Rachel J. Buchsbaum ECM GF LPA Thrombin Eph Semaphorin S. typhimurium Plexins LPA, Thrombin, etc. Integrins RTK GPCRs Eph receptors Plasma membrane Gα Gβ Gγ PIP2 PIP3 TKs Journal of Cell Science R-Ras CrkII DOCK180 SopE, SopE2 Key p130CAS Src Fak RhoG GTPases DOCK180 Dbs Ack HIV-1gp41 P ELMO Dbl Ephexin Vav Ack Ephexin Cbl-b hSiah1 Ephexin Classical GEFs p115RhoGEF LARG PDZ-RhoGEF APC PKA Ubi-Ubi PKA Lbc Asef Non-classical GEFs Kalirin 14-3-3 Trio GEF-H1 RasGTP nm23HI Eps8 GEF activators ITSN WASP Abi1 SOS P-Rex1 Microtubules Tiam1 GEF inhibitors IRSp53 RasGRF in il γ h β p o in 3 p PAK Fgd1 S GTPase effectors p115RhoGEF Net1 Par COOL-2 COOL-2 JIP2 CNK1 WAVE2 COOL-2 Scaffolds and Rho aPKC S6KF MLK3 Rac Cdc42 protein complexes Par6 Actin MLK2 MKK7 LMW p190RhoGAP ROS PTPASE COOL-1 Nucleus Golgi Cbl Fgd1 Net1 jcs.biologists.org Ect2 Effectors Ubi-Ubi © Journal of Cell Science 2007 (120, pp. 1149-1152) (See poster insert) 1150 Journal of Cell Science 120 () RhoGEF activity is mediated by catalytic well as a DH domain to promote GTP- the A-kinase-anchoring protein (AKAP) DH (Dbl homology) domains, which GDP exchange on Rac. PIP3 binding Lbc through C-terminal leucine zipper stabilize GTP-free Rho intermediates, relieves a similar DH-PH interaction that sequences is required for inhibition of its effectively leading to GTP loading blocks GTP-GDP exchange on Rac, exchange activity by PKA and 14-3-3. In owing to high intracellular GTP levels. while the DH-PH region itself blocks an addition, Rho family GEFs may also be DH domains contain three conserved allosteric Ras-binding site on SOS that downregulated by being targeted for regions and form related structures of regulates Ras activity. Recently degradation. Examples include SOCS1- elongated bundles of ␣-helices, in which published work indicates that EGF- stimulated Vav polyubiquitylation, Ras- amino acid variations confer specificity receptor-dependent phosphorylation of a stimulated polyubiquitylation of the towards individual GTPases. Almost all tyrosine residue near a downstream dual Ras-RacGEF RasGRF2, the DH-containing Rho family GEFs regulatory region is required to unmask ubiquitylation of murine SOS2, and the contain a PH (pleckstrin homology) the exchange activity of the Cdc42 GEF Cbl-directed ubiquitylation of COOL- domain immediately C-terminal to the COOL-1/␤PIX (Feng et al., 2006). 1/␤PIX. DH domain. DH-associated PH domains have several regulatory roles with regard GEFs have a number of other functional Multiple levels of regulation have been to DH domain and GEF function, domains, many of which couple to identified in the case of some Rho family including modulation of exchange upstream receptors or other signaling GEFs. Interaction of the PH domain of activity, interactions with phospholipids molecules. It is thus not surprising that RacGEF Vav with PIP3, for example, and proteins, and membrane targeting. protein-protein interactions also regulate allows tyrosine phosphorylation of GEF activity. Activated G␣13, released residues interacting with the GTPase- The identification of over 60 mammalian from its ␤␥ subunits by LPA- or binding pocket by Src/Syk tyrosine Rho family GEFs to date has revealed a thrombin-mediated stimulation of G- kinases (TKs) in response to T cell complex array of regulatory mechanisms protein-coupled receptors (GPCRs), can receptor signaling, further opening up for these proteins. The poster depicts the bind to and stimulate several RhoGEFs, access to the Rac-binding site. The best-studied GEFs and major themes in including Dbl, p115RhoGEF, PDZ- activation of P-Rex1 by PIP3 and G␤␥ their regulation. A comprehensive RhoGEF and LARG. The G␤␥ units bind subunits is blunted by PKA-mediated description is beyond the scope of this and activate Dbl, as mentioned above. The phosphorylation, and the exchange overview and I apologize to those hematopoietic RacGEF P-Rex1 is activity of the RacGEF Tiam1 is authors whose work is not included. activated by binding to both PIP3 and modulated by both phosphoinositide However, several excellent reviews have specific G␤␥ subunits. In the RacGEF binding and by phosphorylation on been published (Schmidt and Hall, 2002; Asef, interaction between the N-terminal threonine and tyrosine. Erickson and Cerione, 2004; Rossman et ABR region and the armadillo repeat al., 2005). domain of the adenomatous polyposis coli Change in intracellular location and protein (APC) relieves autoinhibition and localized activation of Rho GTPases is Journal of Cell Science A common theme in the regulation of promotes Rac exchange activity. Some another mechanism for regulation of Rho Rho family GEFs is relief of GEFs, such as Dbl, Dbs, and RasGRF1 family GEFs. Sometimes the PH domain intramolecular inhibitory interactions. and RasGRF2, form homo- or hetero- mediates translocation – for example, the This is illustrated by the constitutive oligomers through DH domain localization of Dbl and Lbc to actin stress activation of N-terminally truncated interactions that are required for full fibers. Some GEFs, such as Tiam1 and Dbl, Vav, Asef, Tiam1, Ect2 and Net1 function of the protein, whereas Fgd1 (a Cdc42GEF), contain a second mutants and C-terminally truncated dimerization of others (PDZ-RhoGEF, PH domain. In the case of Tiam1, the p115RhoGEF and Lbc mutants. Often LARG, p115RhoGEF) is inhibitory. In second PH-domain is required for the associated PH domain is involved in the case of the multidomain GEFs Kalirin membrane translocation; in Fgd1, a this intramolecular inhibition. In the and Trio that contain separate DH proline-rich N-terminal region, rather RhoGEF Dbl, for example, the N- domains for Rac and Rho, alternative than the PH domains, localizes the terminus binds to the PH domain, splicing leads to multiple isoforms that protein to subcortical actin and Golgi blocking access of the GTPase to the DH have different functional activities during structures. Other Rho family GEFs are domain. Phosphorylation by Ack1 or development. recruited to membranes by adaptor interaction with heterotrimeric G protein proteins, direct binding, or other protein ␤␥ subunits may relieve the inhibition. In GEF activity can also be downregulated interactions. Adaptor proteins such as the RacGEF Vav, an interaction between by interaction with inhibitory proteins Grb2 and SLP-76 are required for DH and PH domains that masks the Rac- – for example, the inhibition of Vav by localization of Vav to activated B- and T- binding site is induced by binding of binding of the C-terminus to Cbl-b or cell receptors. The DH-PH domains of phosphatidylinositol 4,5-bisphosphate hSiah1, the inhibition of p115RhoGEF ephexin directly interact with the (PIP2) to the PH domain and relieved by by binding of the C-terminus to the HIV- transmembrane receptor tyrosine kinase binding of phosphatidylinositol 3,4,5- 1gp41 protein, and the inhibition of EphA4. Binding of G␣13 after LPA or trisphosphate (PIP3). Complex inhibitory Tiam1 by binding of the N-terminus to thrombin stimulation induces intramolecular interactions have been the tumor suppressor