Rho Activation at a Glance Rho Activation at a Glance

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

(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 nm23H1. redistribution of p115RhoGEF from the described for the dual Ras-Rac exchange Association of microtubules with the cytosol to the plasma membrane. And factor SOS, which possesses both a RhoGEF GEF-H1 inhibits its exchange upon nuclear envelope breakdown prior CDC25-like exchange domain for Ras as activity toward Rho. Oligomerization of to mitosis, the RhoGEFs Ect2 and Net1 Journal of Cell Science 120 (7) 1151

are released from the nucleus, where they beginning. For example, the human and function upstream of Rho GTPases. are otherwise sequestered, to activate ortholog of Connector Enhancer of Ksr1 In humans the CZH/DOCK180 the Rho-mediated contraction of the (hCNK1), a multi-domain adaptor protein superfamily members lack DH-PH actomyosin ring that drives cytokinesis. first implicated in Ras signaling domains altogether but possess DOCK pathways, may also function as a scaffold homology domains required for GTPases themselves often regulate Rho protein for Rho-dependent Jnk activation nucleotide exchange through an as-yet- family GEF activity. For example, through binding RhoGEFs Net1 and unidentified mechanism. Canonical several RhoGEFs activated by G␣-GTP, p115RhoGEF, activated Rho, MLK2 and members act as RacGEFs, others are including p115RhoGEF, LARG and MKK7 (Jaffe et al., 2005). Cdc42GEFs, and some remain to be PDZ-RhoGEF, also contain an RGS characterized. Bacteria, which lack their domain. This domain functions as a GAP In some cases where GEFs activate more own Rho GTPases, have multiple for G␣ subunits, thus allowing the GEFs than one GTPase, formation of a complex mechanisms for modulating the activity to fine-tune signals coming from can also direct GEFs towards specific of mammalian Rho family proteins. The GPCRs. The RacGEF Tiam1 is activated GTPases. The dual Ras-Rac-GEF SOS, bacteria Salmonella typhimurium injects by Ras-GTP through its Ras-binding for example, contains a proline-rich C- the non-DH-containing SopE (Rac and domain. Similarly, binding of Rac-GTP terminus that can interact with the SH3 Cdc42) and SopE2 (Rac) proteins into to the PH domain of the RhoGEF Dbs domains of either Grb2 or Abi1/E3b1. mammalian cells, where they function as promotes Rho activation. During the Interaction of SOS with Grb2 enables GEFs in order to facilitate bacterial process of actin remodeling and cell recruitment of the complex to tyrosine internalization. And in plants, which spreading, Rac activation generates kinase receptors and activation of Ras. By contain numerous Rop (Rho of plants) reactive oxygen species that inhibit the contrast, interaction of SOS with Abi1 proteins, a new family of Rho GEFs has low-molecular-weight protein tyrosine leads to formation of a complex with Eps8 recently been identified. These contain a phosphatase (LMW-PTPase), leading to and activation of Rac. Similarly, the novel PRONE (plant-specific Rop increased tyrosine phosphorylation and GTPase activation profile of ephexin is nucleotide exchanger) domain (Berken activation of p190RhoGAP and modulated depending on the activation et al., 2005). subsequent downregulation of Rho state of the EphA4 receptor that it binds. activity. In a complex process probably When unstimulated, ephexin can activate GEF activation of Rho GTPases, like all involving both allosteric and direct Cdc42 and possibly Rac in addition to signaling pathways, does not take place effects, Cdc42-GTP activates and Rac- Rho, but EphA4 clustering leads to in an isolated linear fashion but occurs GTP inhibits the RacGEF activity of enhanced Rho activation and suppresses through the serial formation of dimeric COOL-2/␣PIX (Baird et al., Cdc42/Rac activation. The GEF macromolecular complexes at precise 2005). specificity of COOL-2/␣PIX depends on points in time and space that are its monomer-dimer equilibrium. As noted currently incompletely understood. Another emerging theme in Rho family above, the dimer promotes Rac activation. Further insight into how Rho proteins are ␤␥ Journal of Cell Science GEF function is a role specifying However, interaction with G -PAK activated by upstream signals will signaling downstream of Rho GTPases, complexes promotes dissociation to require more knowledge of how the often through participation in monomers, which can activate either Rac effects of GEFs are coordinated with multimolecular complexes. This can be or Cdc42. Finally, although Ras-GRF1 those of GAPs and GDIs, along with through direct binding of the GEF to the and Ras-GRF2 can each potentially other signaling molecules. An example immediate GTPase effector protein activate both Ras and Rac GTPases, of this is integrin signaling, itself a downstream – for example, binding of RasGRF1 preferentially activates Rac to complex system involving multiple COOL-2/␣PIX to Pak and binding of promote LTD (long-term depression) and ligands and multiple receptors. There are intersectin (ITSN) to Wiskott-Aldrich Ras-GRF2 preferentially activates Ras to many examples of integrin activation syndrome protein (WASP). Alternatively promote LTP (long-term potentiation) in leading to signaling through Rho this can be through binding of the GEF to the hippocampus, at least in part because proteins, but the details of the regulatory scaffold proteins that also form a complex they are associated with different subsets proteins involved are often unclear. with GTPase effectors – for example, of NMDA glutamate receptors in the Integrin ligation is known to trigger binding of Tiam1 and RasGRF1 to the brain (Li et al., 2006). The fact that GEFs signaling through activation of Vav and p38 scaffold JIP2, and binding of Tiam1 both turn on GTPase signaling and formation of DOCK180 complexes to the F-actin–protein-phosphatase-1– specifically direct the downstream effects containing either p130CAS and CrkII or S6kinase scaffold spinophilin and the of that signaling may explain why higher the Rac-like RhoG and the DOCK180- Par3 component of the Par polarity organisms have evolved so many more binding protein Elmo (Hsia et al., 2003; complex (Mertens et al., 2006). A GEFs than GTPases. Katoh and Negishi, 2003; Gakidis et al., combination of both mechanisms is 2004). However, integrin signaling also displayed by Tiam1, which can also There are proteins that lack the classical leads to Rho inactivation through Src- interact with IRSp53, a small adaptor tandem DH-PH motif but exhibit mediated activation of RhoGAP and Rac molecule that binds activated Rac as well nucleotide exchange activity toward Rho activation through release of RhoGDI as the Arp2/3–G-actin scaffold WAVE2. family members. SWAP-70, part of the (Dib et al., 2001; Del Pozo et al., 2002). Our understanding of the complex B cell DNA recombination complex, and contributions of scaffold proteins to GEF- related family members, possess PH Furthermore, pathways involving directed GTPase signaling is just domains N-terminal to DH-like domains multiple GTPases are often coordinately 1152 Journal of Cell Science 120 (7)

Bishop, A. L. and Hall, A. (2000). Rho GTPases and their Semaphorins command cells to move. Nat. Rev. Mol. Cell triggered during complex biological effector proteins. Biochem. J. 348, 241-255. Biol. 6, 789-800. processes. An example of this is seen Boettner, B. and Van Aelst, L. (2002). The role of Rho Li, S., Tian, X. and Feig, L. A. (2006). Distinct roles for during axon guidance in the developing GTPases in disease development. Gene 286, 155-174. Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) Burridge, K. and Wennerberg, K. (2004). Rho and Rac and Ras-GRF2 in the induction of long-term potentiation nervous system. Binding of the take center stage. Cell 116, 167-179. and long-term depression. J. Neurosci. 26, 1721-1729. transmembrane glycoprotein Del Pozo, M. A., Kiosses, W. B., Alderson, N. B., Meller, Mertens, A. E. E., Pegtel, D. M. and Collard, J. G. N., Hahn, K. M. and Schwartz, M. A. (2002). Integrins (2006). Tiam1 takes PARt in cell polarity. Trends Cell Biol. semaphorin 4D to its transmembrane regulate GTP-Rac localized effector interactions through 16, 308-316. receptor plexin-B1 leads to Rho dissociation of Rho-GDI. Nat. Cell Biol. 4, 232-239. Ridley, A. (2001). Rho family proteins: coordinating cell activation through stimulation of the Dib, K., Melander, F. and Andersson, T. (2001). Role of responses. Trends Cell Biol. 11, 471-477. p190RhoGAP in beta 2 integrin regulation of RhoA in Ridley, A., Schwartz, M. A., Burridge, K., Firtel, R. A., RhoGEFs LARG and PRG, human neutrophils. J. Immunol. 166, 6311-6322. Ginsberg, M. H., Borisy, G., Parsons, J. T. and Horwitz, sequestration of active Rac by a GTPase- Erickson, J. W. and Cerione, R. A. (2004). Structural A. R. (2003). Cell migration: integrating signals from front binding domain on plexin B1, and elements, mechanism, and evolutionary convergence of to back. Science 302, 1704-1709. Rho protein-guanine nucleotide exchange factor Rossman, K. L., Der, C. J. and Sondek, J. (2005). GEF inactivation of the Ras family member complexes. Biochemistry 43, 837-842. means go: turning on Rho GTPases with guanine R-Ras through a plexin GAP domain, Feng, Q., Baird, D., Peng, X., Wang, J., Ly, T., Guan, nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6, J.-L. and Cerione, R. A. (2006). Cool-1 functions as an 167-180. which leads to integrin detachment, essential regulatory node for EGF receptor- and Src- Schmidt, A. and Hall, A. (2002). Guanine nucleotide repulsion of the axonal growth cone, and mediated cell growth. Nat. Cell Biol. 8, 945-956. exchange factors for Rho GTPases: turning on the switch. turning of the developing axon (Kruger Gakidis, M. A., Cullere, X., Olson, T., Wilsbacher, J. Genes Dev. 16, 1587-1609. L., Zhang, B., Moores, S. L., Ley, K., Swat, W., Schwartz, M. (2004). Rho signalling at a glance. J. Cell et al., 2005). Understanding the Mayadas, T. and Brugge, J. S. (2004). Vav GEFs are Sci. 117, 5457-5458. coordinated regulation of RhoGEF required for beta2 integrin-dependent functions of Wennerberg, K. and Der, C. J. (2005). Rho-family activity with that of GAPs, GDIs and neutrophils. J. Cell Biol. 166, 273-282. GTPases: it’s not only Rac and Rho (and I like it). J. Cell Hsia, D. A., Mitra, S. K., Hauck, C. R., Streblow, D. N., Sci. 117, 1301-1312. other Ras family proteins will constitute Nelson, J. A., Ilic, D., Huang, S., Li, E., Nemerow, G. the next frontier in the study of Rho R. and Leng, J. (2003). Differential regulation of cell motility and invasion by FAK. J. Cell Biol. 160, 753-767. GTPases. Jaffe, A. B. and Hall, A. (2005). Rho GTPases: biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21, 247-269. Jaffe, A. B., Hall, A. and Schmidt, A. (2005). Cell Science at a Glance on the Web References Association of CNK1 with Rho guanine nucleotide Baird, D., Feng, Q. and Cerione, R. A. (2005). The Cool- exchange factors controls signaling specificity Electronic copies of the poster insert are 2/␣-Pix protein mediates a Cdc42-Rac signaling cascade. downstream of Rho. Curr. Biol. 15, 405-412. available in the online version of this article Curr. Biol. 15, 1-10. Katoh, H. and Negishi, M. (2003). RhoG activates Rac1 at jcs.biologists.org. The JPEG images can Berken, A., Thomas, C. and Wittinghofer, A. (2005). A by direct interaction with the Dock180-binding protein be downloaded for printing or used as new family of RhoGEFs activates the Rop molecular Elmo. Nature 424, 461-464. slides. switch in plants. Nature 436, 1176-1180. Kruger, R. P., Aurandt, J. and Guan, K. L. (2005). Journal of Cell Science