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Table of contents

The Rac and Rho family of GTPases

The C. elegans genome contains three rac-like genes: ced-10, mig-2, and rac-2. ced-10, mig-2 and rac-2 act redundantly in axon pathfinding: inactivating one gene has little effect, but inactivating two or more genes perturbes both axon outgrowth and guidance. mig-2 and ced-10 also have redundant functions in some cell migrations. By contrast, ced-10 is uniquely required for cell-corpse phagocytosis, and mig-2 and rac-2 have only subtle roles in this process. Rac activators are also used differentially. The UNC-73 Trio Rac GTP exchange factor affects all Rac pathways in axon pathfinding and cell migration but does not affect cell-corpse phagocytosis. CED-5 DOCK180, which acts with CED-10 Rac in cell-corpse phagocytosis, acts with MIG-2 but not CED-10 in axon pathfinding. Thus, distinct regulatory proteins modulate Rac activation and function in different developmental processes (Lundquist, 2001).

The Rho subfamily of GTPases has been shown to regulate cellular morphology. A new Drosophila member of the Rho family, named RhoL, is equally similar to Rac, Rho (see Drosophila Rho1), and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary causes nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimic this effect. Expression of constitutively active RhoL leads to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity is specifically required for migration of border cells, a subset of follicle cells. All three activities are necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis (Murphy, 1996).

Previous studies on small GTP-binding proteins of the Rho family have revealed their involvement in the organization of cell actin cytoskeleton. The function of these GTPases during vertebrate development is not known. With the aim of understanding the possible role of these proteins during neuronal development, five members expressed in developing chick neural retinal cells were cloned and sequenced. Four chicken genes, cRhoA, cRhoB, cRhoC, and cRac1A, and a novel Rac gene, cRac1B, have been identified that are homologous to known human genes. Analysis of the distribution of four of the identified transcripts in chicken embryos shows, for the first time, high levels of expression of Rho family genes in the vertebrate developing nervous system, with distinct patterns of distribution for the different transcripts. In particular, cRhoA and cRac1A gene expression appear ubiquitous in the whole embryo, and the cRhoB transcript is more prominent in populations of neurons actively extending neurites, whereas the newly identified cRac1B gene is homogeneously expressed only in the developing nervous system. Temporal analysis of the expression of the five genes suggests a correlation with the morphogenetic events occurring within the developing retina and the retinotectal pathway. Expression of an epitope-tagged cRac1B in retinal neurons shows a diffuse distribution of the protein in the cell body and along neurites. Taken as a whole, these results suggest important roles for ubiquitous and neural-specific members of the Rho family in the acquisition of the mature neuronal phenotype (Malosio, 1997).

Signaling proteins from the same family can have markedly different roles in a given cellular context. Expression of one hundred constitutively active human small GTPases is found to induce cell morphologies that fall into nine distinct classes. An algorithm is developed for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues are mostly outside known interaction surfaces and are structurally far apart from one another. This study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants (Heo, 2003).

Fifty six of the expressed small GTPase constructs triggered no significant morphology changes, while 44 others induced marked morphology changes. The induced morphologies were clearly distinguishable from one another and fell into only nine distinct classes. The Rho family members Rho6, Rho7, RhoE, and ARHE induced a marked cell rounding. Cells transfected with CDC42, CDC42h, TC10, and TCL constructs showed extensions of thin processes that have been termed filopodia, while cells transfected with Rac1, Rac2, Rac3, and RhoG constructs extended lamellipodia that consisted of mostly circular membrane sheets. Transfection of RhoA, RhoB, and RhoC constructs induced polymerized actin bundles or stress fibers that reached across the cell. Only RhoD and RhoH did not show a significant morphology change (Heo, 2003).

Arf family small GTPases induced two types of morphologies. Several members of the Arl family induced a shrunken morphology, while Arf6 had one of the most distinct morphologies with multiple characteristics that include broader cell arms, local membrane spreading, filopodia extensions as well as actin polymerization throughout the cell body and along the cell periphery. Within the shrunken morphology class, Arl 1, Arl 2, and Arl 3 could be considered as a subclass with less pronounced shrinkage and occasional induction of short filopodia type processes that have been termed microspikes in other studies (Heo, 2003).

Cells transfected with Ras family small GTPases also show two distinguishable morphology classes. The oncogenic H-, K-, and N-Ras induce a marked polarized morphology with membrane ruffles and strong actin staining at a polar end of the cells, while cells transfected with most of the remaining members show cell spreading combined with hairlike filopodia formation with pronounced polymerized actin boutons at their ends. The spreading of these cells has a resemblance to eyelashes and looks markedly different from the morphology of lamellipodia induced by Rac or RhoG or the polarized morphology induced by Ras (Heo, 2003).

Finally, several of the Rab family members also have a strong effect on cell morphology. Rab4B, Rab13, Rab22A, Rab23, and Rab35 induce a local spread morphology characterized by local lamellipodia extensions and occasional filopodia induction. Rab8 and Rab8B have the most dramatic effect on cell morphology of all constructs tested and, like Arf6, fall into the multiple morphology class characterized by large branched structures with local lamellipodia and filopodia (Heo, 2003).

In conclusion, this study shows that the structural fold of Ras superfamily small GTPases can induce nine different morphology classes. Furthermore, the residues have been discovered that define the filopodia, lamellipodia, polar, and eyelash morphologies and it was unexpectedly found that the locations of the switch-of-function sites are mostly outside the known effector interaction surfaces and are far apart from each other. These engineered small GTPases with a changed functional selectivity will be useful as tools in pull-down assays to identify the function-specific binding partners as perturbation constructs to investigate crosstalk between signaling processes and for testing whether particular cell functions are physiologically relevant by creating mutant model organisms. Finally, this study introduced an algorithm and a genome-based experimental classification strategy that can be employed to classify the functional space of protein families and to understand the structural basis of functional specificity (Heo, 2003).

Cell-intrinsic adaptation arising from chronic ablation of a key rho GTPase regulator

Genome-editing technologies allow systematic inactivation of human genes. Whether knockout phenotypes always reflect gene functions as determined by acute RNAi is an important question. This study shows how the acute knockdown of the Adams-Oliver syndrome (AOS) gene DOCK6 (see Drosophila Zir), coding for a RAC1/CDC42 guanine nucleotide exchange factor, results in strikingly different phenotypes to those generated by genomic DOCK6 disruption. Cell-intrinsic adaptation compensates for loss of DOCK6 function. Prolonged DOCK6 loss impacts upon the MRTF-A/SRF (see Drosophila bs) transcription factor, reducing levels of the ubiquitin-like modifier ISG15 (see Drosophila Nedd8). Reduced ISGylation of the IQGAP1 protein increases levels of active CDC42 and RAC1 to compensate for DOCK6 disruption. Similar downregulation of ISG15 in cells from DOCK6 AOS patients indicates that such adaptation can compensate for genetic defects during development. Thus, phenotypes of gene inactivation are critically dependent on the timescale, as acute knockdown reflects a transient state of adjustment to a new equilibrium that is attained following compensation (Cerikan, 2016).

Rac2 in Drosophila

The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors, and both of them are implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, a gain-of-function (GOF) screen was performed for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Among the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of the screen. Observed GOF phenotypes have led to the identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Among them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Analysis of rac2 phenotypes clearly demonstrate that the altered mesodermal level of Rac2 can influence specification of a number of cardiac and muscular cell types, including those expressing ladybird. The finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicates a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinase (RTK) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types (Bidet, 2003).

The loss of both, the SBM and the LaPs, is also observed in embryos over-expressing Rac2. This was surprising as previous reports suggested the involvement of rac2 in myoblast fusion processes (Hakeda-Suzuki, 2002). Since loss of rac2 function confirms its role in cell fate specification decisions and leads to the overproduction of lb positive muscle cells, it is hypothesised that rac2 might exert this new function by interacting with RTK signalling components. One potential way by which rac2 might exert the cell fate specification functions is the control of growth factor receptor trafficking and degradation. This possibility is in agreement with the previously described implication of vertebrate Rho-GTPases, RhoA, RhoB and Rac in cellular trafficking of the EGFR. It has been shown that the ligand-bound EGFR undergoes trafficking events that relocalize the receptor to the clathrin coated pits on the cellular membrane and then promote its internalization. The most important step in intracellular processing of EGFR is the formation of Multivesicular Bodies (MVB), which direct the EGFR either to the recycling or to the degradation pathways. One of the small Rho-GTPases, RhoB, was found to be specifically associated with MVB, and when over-expressed, was able to promote the EGFR degradation. The potential RhoB-like role of Drosophila rac2 in directing the RTKs to degradation is in agreement with the overproduction of lb-expressing muscle cells in rac2 mutants. The phenotype is reminiscent of that observed in mutants for the negative RTK effector Yan (Bidet, 2003).

These data also demonstrate new roles for rho, yan and rac2 in the specification of cardiac lineages. Interestingly, mutations of rho and rac2 affect specification of pericardial cells with no major effects on cardioblast identity. yan loss and GOF leads to even more pronounced phenotypes suggesting that, in addition to EGFR, other RTKs are involved in diversification of cardiac fates. rho and Ras/MAPK pathway have been shown to influence specification of eve-expressing pericardial cells. In addition, this study shows that rho represses and yan promotes specification of lb-positive pericardial cells. Surprisingly, in rho mutants, the supernumerary lb-positive pericardial cells co-express eve, a situation never observed in wild type embryos because of mutual repressive activities of eve and lb. This suggests that cross-repression requires the co-ordinated action of identity gene products and effectors of RTK signalling pathway. The overproduction of tin/eve-positive pericardial cells observed in rho GOF and in rac2 loss of function mutants suggests that the diversification of this particular cell type involves a rac2-dependent trafficking of EGF receptor. A future challenge will be to unravel whether Drosophila rac2 indeed co-operates with cell fate specification machinery by controlling the intracellular processing of EGFR and others RTKs (Bidet, 2003).

Rhomboid belongs to a large family of intermembrane serine proteases regulating the EGF-like ligand maturation in different species from prokaryotes to Human. One of the mouse rho homologs, ventrhoid, exhibits a very dynamic expression in central nervous system and forming somites, suggesting it may regulate early cell fate specification genes in a manner similar to that in which rho regulates lb in Drosophila. Several yan-like genes have also been identified in vertebrates. Two human yan homologs, named tel1 and tel2 share similar mesodermal embryonic expression pattern restricted to hematopoietic lineages. In addition, in adult mouse, tel1 is expressed in the heart and in skeletal muscles. As in Drosophila, yan functions with its closely related partner pointed. It is important to note that the vertebrate pnt genes ets-1 and ets-2 are involved in early embryonic heart and muscle development. The numerous vertebrate homologs of the third candidate gene of this study, rac2, control a variety of cellular processes including actin polymerization, integrin complex formation, cell adhesion, membrane trafficking, cell cycle progression, and cell proliferation. The majority Rho-GTPases are ubiquitously expressed, including the developing muscular and cardiac tissues, but their myogenic functions have not yet been investigated. The vertebrate Rac2 gene is specifically required for hematopoiesis. Its mutation in mice leads to the defective neutrophil cellular functions reminiscent of human phagocyte immunodeficiency. The only described link between Rho-GTPases and muscle concerns the binding and activation of a Serine/Threonine protein kinase homologous to myotonic dystrophy kinase by a small GTP binding protein Rho. It is speculated, however, that given the involvement of RhoB in EGFR trafficking, the vertebrate Rho GTPase can contribute to RTK-controlled myogenic pathways (Bidet, 2003).

Altogether, these data suggest that the RTK signalling involving rho, yan and rac2 might play an important and at least partially conserved role in diversification of cardiac and muscular lineages (Bidet, 2003).

The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1, and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. This paper examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. It was found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions (Machacek, 2009).

Activation of Rac

The C. elegans gene ced-12 functions in the engulfment of apoptotic cells and in cell migration, acting in a signaling pathway with ced-2 CrkII, ced-5 DOCK180, and ced-10 Rac GTPase and acting upstream of ced-10 Rac. ced-12 encodes a protein with a pleckstrin homology (PH) domain and an SH3 binding motif, both of which are important for ced-12 function. CED-12 acts in engulfing cells for cell corpse engulfment and interacts physically with CED-5, which contains an SH3 domain. CED-12 has Drosophila (CG5336) and human counterparts. Expression of CED-12 and its counterparts in murine Swiss 3T3 fibroblasts induces Rho GTPase-dependent formation of actin filament bundles. It is proposed that through interactions with membranes and with a CED-2/CED-5 protein complex, CED-12 regulates Rho/Rac GTPase signaling and leads to cytoskeletal reorganization by an evolutionarily conserved mechanism (Zhou, 2001).

GTP-binding proteins of the Rho family are maintained as cytosolic complexes with RhoGDI in resting cells, but are released and translocate to the membrane during the course of cell activation. Membrane association of Rac/Rho/CDC42 is specifically induced by GTP analogs and requires a heat- and trypsin-labile membrane component. Translocation is associated with the release of Rho family proteins from RhoGDI, but such release does not occur in the absence of membranes, nor is release in the absence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) sufficient for membrane association. Membrane binding is correlated with exchange of GTP gamma S for GDP on Rac, and only GTP gamma S-bound Rac becomes membrane localized. It is proposed that translocation of Rac and other members of the Rho family is controlled by membrane-associated guanine nucleotide exchange factors, providing a mechanism to regulate the release and activation of individual members of the Rho family during cell stimulation (Bokoch, 1994).

The pertussis toxin (PTX) insensitive heterotrimeric G protein G12 has been implicated in mitogenesis and transformation, but its direct effectors remain unknown. To define potential signaling pathways utilized by G12, an activated mutant of its alpha subunit, Galpha12(Q229L), was expressed in HEK293 cells and its effects on Ras (See Drosophila Ras) and mitogen-activated protein kinases (MAPKs) were examined. Transient expression of activated Galpha12 increases the percentage of Ras in the active, GTP-bound state, stimulates c-Jun NH2-terminal kinase (JNK) activity, and enhances the transcriptional activity of c-Jun. Dominant negative Ras (N17Ras) inhibits Galpha12-mediated JNK activation in NIH3T3 cells but fails to do so in HEK293 cells. In contrast, dominant negative Rac (N17Rac1) inhibits JNK activation by Galpha12 in HEK293 cells as well as three other cell lines. In 1321N1 cells, where thrombin stimulates G12-dependent mitogenesis, coexpression of N17Rac1 or a dominant negative mutant of MEKK1 (MEKKDelta[K432M]) inhibits c-Jun/AP-1 sensitive reporter gene expression stimulated by thrombin or Galpha12. These data demonstrate that the alpha subunit of the heterotrimeric G protein G12, like tyrosine kinase growth factor receptors, activates Ras and recruits a signal transduction pathway involving the small GTP-binding protein Rac that leads to JNK activation (Collins, 1996).

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. Mutant RhoG proteins that fuse to the green fluorescent protein have been expressed and subsequent changes in cell surface morphology and modifications of cytoskeletal structures analyzed. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells may still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, these data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways (Gauthier-Rouviere, 1998).

Transforming growth factor beta (TGF-beta) is a multifunctional factor that induces a wide variety of cellular processes affecting growth and differentiation. TGF-beta exerts its effects through a heteromeric complex of two transmembrane serine/threonine kinase receptors, the type I and type II receptors. TGF-beta initiates a signaling cascade leading to stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. Expression of dominant-interfering forms of various components of the SAPK/JNK signaling pathways (including Rho-like GTPases, mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and c-Jun) abolishes TGF-beta-mediated signaling. Therefore, the SAPK/JNK activation most probably contributes to TGF-beta signaling (Atfi, 1997).

L-selectin mediated adhesion to endothelial cells is a crucial step in the immune response to pathogens and in lymphocyte homing. Selectin molecules mediate leukocyte rolling on endothelial cells, the initial step for adhesion to occur. Stimulation of Jurkat T-lymphocytes via L-selectin results in activation of the p21Ras pathway and synthesis of reactive oxygen intermediates. Cellular stimulation via L-selectin induces a change of cytoskeleton organization demonstrated by a tenfold increase in actin filament polymerization. This actin polymerization is mediated by a Ras and Rac2 regulated pathway, since inhibition of Ras by transient transfection of transdominant inhibitory N17Ras or suppression of Rac2 protein expression by antisense oligonucleotides prevents L-selectin triggered actin polymerization. These results point to a signaling cascade (from L-selectin via Ras and Rac2 to actin filaments) that might be crucial for leukocyte adhesion (Brenner, 1997a).

Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, it is clear that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling via the small GTPase Rac. In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant results in a dramatic stimulation of membrane ruffling without affecting pinocytosis. Expression of Ha-Ras(G12V), an activated Ras mutant, stimulates both membrane ruffling and pinocytosis. The Ha-Ras(G12V)-stimulated pinocytosis but not the membrane ruffling is abolished by either wortmannin or co-expression with a dominant negative mutant of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis but not on membrane ruffling. These results indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent membrane ruffling (Li, 1997).

Ras-mediated transformation of mammalian cells has been shown to activate multiple signaling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA are implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG were examined by expressing constitutively active or dominant-negative forms in NIH3T3 cells. Expression of Rac1 or RhoG modulates the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases are required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase is not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicit a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. It is concluded that Ras-mediated transformation induces extensive changes in cell morphology that require the activity of members of the Rho family of GTPases. These data show that the pattern of coordinated Rho family activation, which elicits a focus-forming activity in NIH3T3 cells, is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells (Roux, 1997).

The phenotype of hematopoietic cells transformed by the BCR/ABL oncoprotein of the Philadelphia chromosome is characterized by growth factor-independent proliferation, reduced susceptibility to apoptosis, and altered adhesion and motility. The mechanisms underlying this phenotype are not fully understood, but there is evidence that some of the properties of BCR/ABL-expressing cells are dependent on the activation of downstream effector molecules such as RAS, PI-3k, and bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue). It is shown that the small GTP-binding protein Rac is activated by BCR/ABL in a tyrosine kinase-dependent manner. Upon transfection with a vector carrying the dominant-negative N17Rac, BCR/ABL-expressing myeloid precursor 32Dcl3 cells retain the resistance to growth factor deprivation-induced apoptosis but show a decrease in proliferative potential in the absence of interleukin-3 (IL-3) and markedly reduced invasive properties. Moreover, compared with BCR/ABL-expressing cells, fewer BCR/ABL plus N17Rac double transfectants are capable of homing to bone marrow and spleen. Consistent with these findings, survival of SCID mice injected with the BCR/ABL plus N17Rac double transfectants is markedly prolonged, when compared with that of mice injected with BCR/ABL-expressing cells. Together, these data support the important role of a Rac-dependent pathway(s) controlling motility in BCR/ABL-mediated leukemogenesis (Skorski, 1998).

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell-cell junctions and subsequent cell scattering. In Madin-Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induce the loss of the adherens junction proteins E-cadherin and beta-catenin from intercellular junctions during cell spreading, and the HGF/SF response is blocked by dominant-negative N17Ras. Desmosomes and tight junctions are regulated separately from adherens junctions, because the adherens junctions are not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac are required downstream of Ras, because loss of adherens junctions is blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of either or both MAP kinase kinase 1 or Rac1. All of these inhibitors also prevent HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110alpha subunit of PI 3-kinase alone does not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras are required for adherens junction disassembly and that the disassembly process is essential for the motile response to HGF/SF (Potempa, 1998).

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun (TAM67), suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, it has been established that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. These data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion (Malliri, 1998).

c-Jun N-terminal kinases (JNKs) are potently activated by a number of cellular stimuli. Small GTPases, in particular Rac, are responsible for initiating the activation of the JNK pathways. So far, the signals leading from extracellular stimuli to the activation of Rac have remained elusive. The Src homology 2 (SH2)- and Src homology 3 (SH3)-containing adaptor protein Crk is capable of activating JNK when ectopically expressed. Transient expression of Crk induces JNK activation, and this activation is dependent on both the SH2- and SH3-domains of Crk. Expression of p130(Cas), a major binding protein for the Crk SH2-domain, also induces JNK activation, which is blocked by the SH2-mutant of Crk. JNK activation by Cas and Crk is effectively blocked by a dominant-negative form of Rac, suggesting a linear pathway from the Cas-Crk-complex to the Rac-JNK activation. Many of the stimuli that activate the Rac-JNK pathway enhance engagement of the Crk SH2-domain. JNK activation by these stimuli, such as epidermal growth factor, integrin ligand binding and v-Src, is efficiently blocked by dominant-negative mutants of Crk. In turn, a dominant-negative form of Cas blocks integrin-mediated JNK activation, but not that of epidermal growth factor nor v-Src. Together, these results demonstrate an important role for Crk in connecting multiple cellular stimuli to the Rac-JNK pathway, and a role for the Cas-Crk complex in integrin-mediated JNK activation (Dolfi, 1998).

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P3), a product of PI 3-kinase. Pten has also been implicated in controlling cell migration, but the exact mechanism is not very clear. Using isogenic Pten+/+ and Pten-/- mouse fibroblast lines, it is shown that Pten deficiency leads to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduces the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125FAK is not changed in Pten-/- cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton, are observed in Pten-/- cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reverses the cell migration phenotype of Pten-/- cells. Thus, these studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42. It is suggested that Pten exerts its tumor suppressor function not only at the stage of tumor initiation, but also in tumor progression and metastasis (Liliental, 2000).

Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration, spreading, proliferation, and differentiation. The details of the signaling events that induce these changes in cell behavior are not well understood but they appear to involve activation of Rho family members that activate signaling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. The result is the formation of focal complexes, focal adhesions, and bundles and networks of actin filaments that allow the cell to spread. The present study shows that mu-calpain is active in adherent cells, that it cleaves proteins known to be present in focal complexes and focal adhesions, and that overexpression of mu-calpain increases the cleavage of these proteins, induces an overspread morphology and induces an increased number of stress fibers and focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by expression of a dominant negative form of mu-calpain results in an inability of cells to spread or to form focal adhesions, actin filament networks, or stress fibers. Cells expressing constitutively active Rac1 can still form focal complexes and actin filament networks (but not focal adhesions or stress fibers) in the presence of calpain inhibitors; cells expressing constitutively active RhoA can form focal adhesions and stress fibers. Taken together, these data indicate that calpain plays an important role in regulating the formation of focal adhesions and Rac- and Rho-induced cytoskeletal reorganizations and that it does so by acting at sites upstream of both Rac1 and RhoA (Kulkarni, 1999).

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. Clusters of integrin that form upstream of Rac activation have been identified. These clusters contain a Rac-binding protein(s) and appear to be involved in Rac activation. The integrin clusters contain calpain and calpain-cleaved ß3 integrin, while the focal complexes and focal adhesions (that form once Rac and Rho are activated) do not. Moreover, the integrin clusters are dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases are dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurs even when calpain inhibitors are present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s) (Bialkowska, 2000).

The role of the protein kinase Akt in cell migration is incompletely understood. Sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T236 residue. Transactivation of EDG-1 by Akt is not required for Gi-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequesters Akt and acts as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration. How GPCRs regulate Rac is poorly understood. EDG-1 activates Rac activity in endothelial cells and transfected CHO cells. Akt activity is required for EDG-1 to activate Rac. Akt and Rac are involved in a complex regulatory network to modulate actin dynamics and cell migration. Since EDG-1 phosphorylation by Akt is needed for Rac activation, it is likely that phosphorylated EDG-1 interacts with upstream mediators of Rac -- for example, the exchange factors such as Tiam. Indeed, S1P treatment of endothelial cells results in translocation of Tiam I and Rac to cell membrane (Lee, 2001).

Regulation of Rho GTPases by crosstalk

Integrin-mediated adhesion is a critical regulator of cell migration. Integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha5beta1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibit a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. Cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases (Cox, 2001).

The Rac GTPase regulates Rho signaling in a broad range of physiological settings and in oncogenic transformation. This study reports a novel mechanism by which crosstalk between Rac and Rho GTPases is achieved. Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP), a major modulator of Rho signaling. p190B colocalizes with constitutively active Rac1 in membrane ruffles. Moreover, activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in GTP-bound RhoA from membranes. p190B is recruited to the plasma membrane in response to integrin engagement. Collagen type I, a potent inducer of Rac1-dependent cell motility in HeLa cells, counteracts cytoskeletal collapse resulting from overexpression of wild-type p190B, but not that resulting from overexpression of a p190B mutant specifically lacking the Rac1-binding sequence. Furthermore, this p190B mutant exhibits dramatically enhanced RhoGAP activity, consistent with a model whereby binding of Rac1 relieves autoinhibition of p190B RhoGAP function. Collectively, these observations establish that activated Rac1, through direct interaction with p190B, modulates subcellular RhoGAP localization and activity, thereby providing a novel mechanism for Rac control of Rho signaling in a broad range of physiological processes (Bustos, 2008).

The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). This study reports the development of a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins (Wu, 2009).

Table of contents

Rac1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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