Rac1

EVOLUTIONARY HOMOLOGS

Table of contents

Microtubules interact with Rac

Members of the Ras-related Rho family are involved in controlling actin-based changes in cell morphology. Microinjection of Rac1, RhoA, and Cdc42Hs into Swiss 3T3 cells induces pinocytosis and membrane ruffling, stress fiber formation, and filopodia formation, respectively. To identify target proteins involved in these signaling pathways, cell extracts immobilized on nitrocellulose have been probed with [gamma-32P]GTP-labeled Rac1, RhoA, and Cdc42Hs. Two 55-kDa brain proteins that bind Rac1 but not RhoA or Cdc42Hs have been identified. These 55-kDa proteins are abundant, have pI values of around 5.5, and can be purified by Q-Sepharose chromatography. The characteristics evident upon two-dimensional gel analysis suggest that the proteins comprise alpha- and beta-tubulin. Indeed, beta-tubulin specific antibodies detect one of the purified 55-kDa proteins. Rac1 binds pure tubulin (purified by cycles of polymerization and depolymerization) only in the GTP-bound state. The GTPase negative Rac1 point mutants, G12V and Q61L, do not significantly affect the ability of Rac1 to interact with tubulin while the "effector-site" mutant D38A prevents interaction. These results suggest that the Rac1-tubulin interaction may play a role in Rac1 function (Best, 1996).

Migrating fibroblasts in culture exhibit an elongated, polarized shape, with a wide flat lamella that terminates in a ruffling lamellipodium at the leading edge, facing the direction of migration, and a trailing cell body tapering back to an extended tail. The tapering cell sides are relatively inactive. Membrane protrusions and ruffles are continuously initiated at the leading edge and undergo a smooth centripetal movement, known as retrograde flow, towards the cell body. Localized actin polymerization, nucleated at the leading edge, is required for ruffling activity, retrograde flow and protrusion of the leading edge to drive directed cell migration. Microtubules emanate out from the cell center toward the leading edge, aligned with the direction of migration, where their plus ends exhibit random changes between periods of growth and shortening; such fluctuations are termed dynamic instability. When treated with agents that cause microtubule disassembly, fibroblasts lose their extended shape, and the protrusive lamellipodial activity that is normally confined to the leading edge is reduced and distributed to random sites around the cell periphery. Thus, microtubules are thought to promote lamellipodial protrusion and direct sites of actin polymerization. Indeed, correlative video and immunofluorescence microscopy has shown that when microtubules enter a protrusion of the leading edge, the edge becomes stabilized against retraction, thereby promoting the cell's forward advance (Waterman-Storer, 1999 and references therein).

The prevailing hypothesis is that microtubules, extending from the cell center to the leading edge, serve as tracks for the directed delivery of membrane vesicles to the lamellipodium, where the membrane of the vesicles is inserted into the plasma membrane at the leading edge to drive lamellipodial protrusion and retrograde flow during cell migration. However, cell migration is stopped by pharmacological stabilization of microtubule dynamic instability without disassembly of microtubule tracks, indicating that migration requires some aspect of microtubule dynamic instability. In contrast to the prevailing view, the growth of microtubules induced in fibroblasts by removal of the microtubule destabilizer nocodazole has been shown to activate Rac1 GTPase, leading to the polymerization of actin in lamellipodial protrusions. Lamellipodial protrusions are also activated by the rapid growth of a disorganized array of very short microtubules induced by the microtubule-stabilizing drug taxol. Thus, neither microtubule shortening nor long-range microtubule-based intracellular transport is required for activating protrusion. It is suggested that the growth phase of microtubule dynamic instability at leading-edge lamellipodia locally activates Rac1 to drive actin polymerization and lamellipodial protrusion required for cell migration. Thus, protrusion of the leading edge can occur independently of microtubule-based organelle transport. However, the long-term maintenance of protrusive activity and the establishment of fibroblast polarity may also require that microtubules are organized into a radial array (Waterman-Storer, 1999).

How does the growth phase of microtubule dynamic instability activate Rac1? One possibility is that microtubule growth directly generates cytoplasmic Rac1-GTP. Rac1-GTP, but not Rac1-GDP, is known to bind to tubulin dimers. Thus, if Rac1-GTP and tubulin were to compete for the same binding site on a microtubule, it is possible that Rac1-GTP could be displaced from tubulin and released into the cytoplasm when the tubulin is added to the end of a microtubule during growth. This would explain how growing microtubule ends could provide active Rac1 to the leading cell edge. Alternatively, there is biochemical evidence to indicate that microtubules may enhance Rac1 activity by mediating the assembly of microtubule-bound Rac1 signaling complexes. Indeed, both upstream guanine-nucleotide-exchange-factor activators of Rac1, including GEF-H1, Vav and Lfc, and the downstream Rac1 effectors MLK2 and JNK either bind to tubulin or localize to microtubules in cells. It is also possible that these signaling complexes could associate preferentially with growing microtubule plus ends in vivo, making their assembly dependent on microtubule growth, similar to the behaviour observed for the microtubule-endosome linking protein, CLIP-170. Finally, it may be that the activity of some other Rho-family member is responsible for microtubule-growth-dependent Rac1 activity. RhoG activates both Rac1 and the Rho-family member Cdc42Hs in a microtubule-dependent way. Thus, microtubule-growth-induced lamellipodial protrusions could be due to RhoG activity. However, it is not clear how RhoG activity could depend on fresh microtubule growth. Another member of the Rho family of GTPases, RhoA, is also important in the relationship between microtubules and F-actin in cell contractility and adhesion. RhoA inhibitors block the assembly of F-actin stress fibers and the formation of focal adhesions that are induced by microtubule depolymerization. Not surprisingly, direct measurement of RhoA-GTP levels in cells has also shown that depolymerization of microtubules with colchicine activates RhoA. This observation, together with the results presented here, indicates that Rho-family members may underlie the elusive crosstalk between microtubules and actin that is required for the regulation of cell motility and cytokinesis (Waterman-Storer, 1999).

Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 (Drosophila homolog: CLIP-190) binds to the growing ends of microtubules and plays pivotal roles in orientation. IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization (Fukata, 2002).

Miscellaneous proteins interacting with Rac

The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in the progression of cells through G1 phase of the cell cycle. However, little is known of the signaling molecules that mediate its activation. Rho family G proteins regulate pp70S6k activity in vivo. Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6k activity in multiple cell types. Activation requires an intact effector domain and isoprenylation of Cdc42 and Rac1. Coexpression of Dbl, an exchange factor for Cdc42, also activates pp70S6k. Growth factor-induced activation of pp70S6k is abrogated by dominant negative alleles of Cdc42 and Rac1. In addition, Cdc42 and Rac1 form GTP-dependent complex with the catalytically inactive form of pp70S6k in vitro and in vivo, suggesting a mechanism by which these G proteins activate pp70S6k (Chou, 1996).

Neutrophils contain a soluble guanine-nucleotide binding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). The two components of the stimulatory heterodimer has been identified by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, it has been demonstrated that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which is also observed for GTP[S]-activated recombinant Rac1, is independent of LyGDI, but requires C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulates PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C fails to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins (Illenberger, 1998).

The Ras-related Rho family GTPases mediate signal transduction pathways that regulate a variety of cellular processes. Like Ras, the Rho proteins (which include Rho, Rac, and CDC42) interact directly with protein kinases likely to serve as downstream effector targets of the activated GTPase. Activated RhoA has recently been reported to interact directly with several protein kinases: p120 PKN, p150 ROK alpha and -beta, p160 ROCK, and p164 Rho kinase. A novel Rho-associated kinase, p140, appears to be the major Rho-associated kinase activity in most tissues. Peptide microsequencing reveals that p140 is probably identical to the previously reported PRK2 kinase, a close relative of PKN. However, unlike the previously described Rho-binding kinases that are Rho specific, p140 associates with Rac as well as Rho. Moreover, the interaction of p140 with Rho in vitro is nucleotide independent, whereas the interaction with Rac is completely GTP dependent. The association of p140 with either GTPase substantially promotes kinase activity and expression of a kinase-deficient form of p140 in microinjected fibroblasts disrupts actin stress fibers. These results indicate that p140 may be a shared kinase target of both Rho and Rac GTPases, mediating their effects on the rearrangements of the actin cytoskeleton (Vincent, 1997).

It has been suggested that Pak1 is the most upstream kinase connecting Rac and CDC42 to JNK; however, coexpression of Pak1 with activated forms of Cdc42 or Rac1 diminishes rather than enhances JNK activation. This prompted an exploration of the possibility that kinases other than Pak might participate in signaling from GTP-binding proteins to JNK. A computer-assisted search was carried out for proteins containing areas of homology to those in Pak1 involved in binding to Rac1 and Cdc42. This led to the identification of mixed lineage kinase 3 (MLK3), also known as protein-tyrosine kinase 1, as a potential candidate for this function. MLK3 overexpression is sufficient to activate JNK potently without affecting the phosphorylating activity of MAPK or p38. MLK3 binds the GTP-binding proteins Cdc42 and Rac1 in vivo and MLK3 mediates activation of the MEKK-SEK-JNK kinase cascade by Rac1 and Cdc42. Taken together, these findings strongly suggest that members of the novel MLK family of highly related kinases link small GTP-binding proteins to the JNK signaling pathway (Teramoto, 1996).

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, it has been shown that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, a yeast two-hybrid cDNA library has been screened. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function (Nagata, 1998).

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, is localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacts with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induces the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, results in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway (Kuroda, 1998).

Rho family GTPases appear to play an important role in the regulation of the actin cytoskeleton, but the mechanism of regulation is unknown. Since phosphoinositide 3-kinase and phosphatidylinositol 4,5-bisphosphate have also been implicated in actin reorganization, the possibility that Rho family members interact with phosphoinositide kinases was investigated. Both GTP- and GDP-bound Rac1 associate with phosphatidylinositol-4-phosphate 5-kinase (Drosophila homolog: skittles) in vitro and in vivo. Phosphoinositide 3-kinase also bind to Rac1 and Cdc42Hs, and these interactions are GTP-dependent. Stimulation of Swiss 3T3 cells with platelet-derived growth factor induces the association of PI 3-kinase with Rac in immunoprecipitates. PI 3-kinase activity was also detected in Cdc42 immunoprecipitates from COS7 cells. These results suggest that phosphoinositide kinases are involved in Rho family signal transduction pathways and raise the possibility that the effects of Rho family members on the actin cytoskeleton are mediated in part by phosphoinositide kinases (Tolias, 1995).

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the presence of the effector domains of G proteins. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. A diacylglycerol kinase (DGK) also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrate that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases is enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and that they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways. It is suggested that PtdInsP 5-kinase, DGK and Rac may exist as a preformed complex bound to RhoGDI. Upon stimulation, the complex may be shuttled to the membrane, where the DGK could synthesize phosphatidic acid, which would stimulate phosphotidylinositol-4,5-diphosphate production. These phospholiplid products may mediate dissociation of the Rac-RhoGDI complex and/or stimulate nucleotide exchange. Newly synthesized lipids could also bind to actin regulatory proteins and induce actin uncapping and new actin polymerization as well as target other Rac signaling pathwys such as the NADPH oxidase (Tolias, 1998).

Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA does not exhibit this activity, and none of the proteins activate the ERK subgroup of MAPKs. JNK, but not ERK, activation is also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 as an intermediate between Ha-Ras and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation (Minden, 1995).

Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. The interaction between Rac and p67(phox) has been explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex has been demonstrated measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) has been shown. Rac1 and Rac2 bind to p67(phox) with a 1:1 stoichiometry. Mutational studies have identified two regions in Rac1 that are important for activity: the "effector region" (residues 26-45) and the "insert region" (residues 124-135). Proteins mutated in the effector region show a marked increase in both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations, while showing markedly elevated EC50 values, bind with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b558 (Nisimoto, 1997).

Rho regulates the myosin light chain phosphatase and Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton (Tapon, 1997).

A novel Rac1-interacting protein, POR1, binds directly to Rac1; the interaction of POR1 with Rac1 is GTP dependent. A mutation in the Rac1 effector binding loop shown to abolish membrane ruffling also abolishes interaction with POR1. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1 (V12Rac1) in quiescent rat embryonic fibroblast REF52 cells. POR1 synergizes with an activated mutant of Ras, V12Ras, in the induction of membrane ruffling. These results suggest a potential role for POR1 in Rac1-mediated signaling pathways (Van Aelst, 1996).

The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Expression of the GTPase-defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement is inhibited by co-expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1-interacting protein involved in membrane ruffling, but not with the dominant-negative mutant of Rac1, Rac1(S17N). A synergistic effect has been detected between POR1 and ARF6 for the induction of actin polymerization. ARF6 interacts directly with POR1; this interaction is GTP dependent. These findings indicate that ARF6 and Rac1 function in distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1 (D'Souza-Schorey, 1997).

The small GTPase Rac has been implicated in a wide range of cellular processes, including the organization of the actin cytoskeleton, transcriptional control and endocytic vesicle trafficking. The signaling components that mediate these functions downstream of Rac largely remain to be identified. In this study, synaptojanin 2, a polyphosphoinositide phosphatase, has been identified as a novel Rac1 effector. Synaptojanin 2 directly and specifically interacts with Rac1 in a GTP-dependent manner. Expression of constitutively active Rac1 causes the translocation of synaptojanin 2 from the cytoplasm to the plasma membrane. Both activated Rac1 and a membrane-targeted version of synaptojanin 2 inhibits endocytosis of the epidermal growth factor (EGF) and transferrin receptors, a process that is known to be dependent on polyphosphoinositide lipids. Endocytosis of growth factor receptors is thought to play an important role in the regulation of cell proliferation. Thus, these results suggest that synaptojanin 2 may mediate the inhibitory effect of Rac1 on endocytosis and could contribute to Rac1-mediated control of cell growth (Malecz, 2000).

Adhesion to fibronectin through the alpha5beta1 integrin enables endothelial cells to proliferate in response to growth factors, whereas adhesion to laminin through alpha2beta1 results in growth arrest under the same conditions. On laminin, endothelial cells fail to translate Cyclin D1 mRNA and activate CDK4 and CDK6. Activated Rac, but not MEK1, PI-3K, or Akt, rescues biosynthesis of cyclin D1 and progression through the G1 phase. Conversely, dominant negative Rac prevents these events with culture on fibronectin. Mitogens promote activation of Rac when cells are grown on fibronectin but not laminin. This process is mediated by SOS and PI-3K and requires coordinate upstream signals through Shc and FAK. These results indicate that Rac is a crucial mediator of the integrin-specific control of cell cycle in endothelial cells (Mettouchi, 2001).

What is the mechanism by which growth factor receptors and specific integrins jointly activate Rac? This process requires the DH domain of SOS as well as PI-3K. Structural and functional studies indicate that the PH domain of SOS exerts an allosteric inhibition on the adjacent DH domain. Upon interaction of the PH domain with PIP-3 in the plasma membrane, SOS is thought to undergo a conformational transition that exposes the DH domain and allows it to activate Rac. It is proposed that alpha5beta1 and other Shc-linked integrins cooperate with growth factor receptors to recruit the Grb2/SOS complex at sites of integrin-mediated adhesion, where FAK-PI3K signaling increases the local concentration of PIP-3 and activates the exchange activity of SOS toward Rac. This model accounts for the ability of both dominant negative Shc and FAK to suppress activation of Rac. The mechanism by which PI-3K is activated upon recruitment by FAK remains to be examined, but the ability of dominant negative Ras to inhibit activation of Rac suggests an involvement of Ras (Mettouchi, 2001).

The small GTPase Rac has been implicated in growth cone guidance mediated by semaphorins and their receptors. Plexin-B1, a receptor for Semaphorin4D (Sema4D), and p21-activated kinase (PAK) can compete for the interaction with active Rac and plexin-B1 can inhibit Rac-induced PAK activation. Expression of active Rac enhances the ability of plexin-B1 to interact with Sema4D. Active Rac stimulates the localization of plexin-B1 to the cell surface. The enhancement in Sema4D binding depends on the ability of Rac to bind plexin-B1. These observations support a model where signaling between Rac and plexin-B1 is bidirectional; Rac modulates plexin-B1 activity and plexin-B1 modulates Rac function (Vikis, 2002).

Sema4D enhances the interaction between plexin-B1 and active Rac. A model is proposed by which Sema4D binds the plexin-B1 receptor and stimulates the recruitment of Rac-GTP. Sequestration of Rac results in the inactivation of PAK and growth cone collapse/turning. This model conflicts with studies on the role of Rac downstream of the plexin-A1 receptor where dominant negative Rac inhibits collapse in response to Sema3A; this suggests that Rac activation is required for Sema3A-mediated growth cone collapse. Perhaps plexin-A and -B signal via different mechanisms since plexin-A does not interact with active Rac. However, in Drosophila, Rac functions downstream of plexA even though it does not interact with plexA. It is possible that a yet unidentified protein couples plexin-A with Rac (Vikis, 2002).

These results indicate that another consequence of the plexin-B1/Rac interaction is to modulate Sema4D ligand binding. This effectively classifies plexin-B1 as a downstream effector of Rac and is the first example of a small GTPase that directly regulates receptor function. An enhancement in the quantity of receptor at the cell membrane and minor changes in affinity for ligand contribute to this enhancement. Whether this is a result of enhanced recruitment to the cell surface and/or inhibition of receptor endocytosis is presently unclear. RhoA does not interact with plexin-B1 and does not stimulate Sema4D ligand binding, yet it has been reported to be activated by clustering of plexin-B1 receptor. In Drosophila, plexB interacts with Rho and stimulates its activity. It appears that humans and flies use different mechanisms for plexin-B stimulation of Rho activity (Vikis, 2002).

These data also suggest that endogenous Rac-GTP is necessary for the maintenance of plexin-B1 at the cell surface. This is based on the observation that dominant negative Rac (RacN17), which inhibits endogenous Rac activation, effectively inhibits the plexin-B1/Sema4D interaction. Furthermore, the Rac binding defective mutant plexin-B1-GGA is compromised in the interaction with Sema4D. This led the authors to postulate that factors that modulate Rac activation can enhance the sensitivity of the receptor/ligand interaction. It is possible that activation of a Rac-specific GEF and/or inactivation of a GAP may modulate the levels of plexin-B1 at the cell surface and its affinity for ligand. Hence, this suggests that engagement of plexin-B1 by Sema4D may be regulated by intracellular levels of Rac-GTP. What the biological consequence of this is remains unknown, however this may be a mechanism by which Rac activation by one axon guidance cue can modulate the responsiveness of the axon growth cone to another guidance cue, such as Sema4D. It is worth noting that whether this model operates in axon growth cone guidance requires further analysis in neurons. Under physiological conditions the axon growth cone is exposed to multiple guidance cues. Therefore, Rac may act as a mediator for cross-talk between different axon guidance cues. Furthermore, the data suggest that signaling between plexin-B1 and Rac is bidirectional. Ligand-gated plexin-B1 can sequester Rac from activating other downstream effectors whereas active Rac can enhance the activity of plexin-B1 (Vikis, 2002).

Rac pathway targets JNK

MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrates differential MEKK subcellular localization: MEKK1 is nuclear and found in post-Golgi vesicular-like structures; MEKK2 and 4 are localized to distinct Golgi-associated vesicles that are dispersed by brefeldin A. MEKK1 and 2 are activated by EGF; kinase-inactive mutants of each MEKK partially inhibit EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibits EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associates with Rac and Cdc42; kinase-inactive mutants block Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 do not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42, independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli (Fanger, 1997).

In COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation both by oncogenic exchange factors and after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway (Coso, 1995).

The stress-activated p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that appear to play a key role in regulating inflammatory responses. Co-expression of constitutively active forms of Rac and Cdc42 leads to activation of p38 while dominant negative Rac and Cdc42 inhibit the ability of interleukin-1 to increase p38 activity. p21-activated kinase 1 (Pak1) is a potential mediator of Rac/Cdc42 signaling, and Pak1 stimulates p38 activity. A dominant negative Pak1 suppresses both interleukin-1- and Rac/Cdc42-induced p38 activity. Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of Pak and leading to activation of p38 and JNK (Zhang, 1995).

A genetic mosaic system has been used to conduct an in vivo analysis of the effects of Rac1 activation on the developing intestinal epithelium. Expression of a constitutively active human Rac1 (Rac1Leu61) in the 129/Sv-derived small intestinal epithelium of C57Bl/6-ROSA26 -> 129/Sv chimeric mice leads to precocious differentiation of some lineages with accompanying alterations in their apical actin. The underlying mechanisms have been explored in this study. Rac1Leu61 leads to accumulation of the 46 kDa form of phosphorylated Jun N-terminal kinase (p-Jnk) in the apical cytoplasm, but not in the nucleus of E18.5 proliferating and differentiating intestinal epithelial cells. The effect is cell-autonomous, selective for this mitogen-activated protein kinase family member, and accompanied by apical cytoplasmic accumulation of p21-activated kinase. c-Jun, a downstream nuclear target of p-Jnk, does not show evidence of enhanced phosphorylation, providing functional evidence for cytoplasmic sequestration of p-Jnk in Rac1Leu61-expressing epithelium. In adult chimeras, Rac1 activation augments cell proliferation in crypts of Lieberkühn, without a compensatory change in basal apoptosis and produces a dramatic, very unusual widening of villi. These results reveal a novel in vivo paradigm for Rac1 activation involving p-Jnk-mediated signaling at a distinctive extra-nuclear site, with associated alterations in the actin cytoskeleton. They also provide a new perspective about the determinants of small intestinal villus morphogenesis (Stappenbeck, 2001).

CrkII, a cellular homolog of v-crk, belongs to a family of adaptor proteins that play a central role in signal transduction cascades. CrkII interacts directly with c-Jun N-terminal kinase 1 (JNK1). A proline-rich sequence of JNK1 is critical for the interaction of the kinase with the N-terminal Src homology 3 (SH3) domain of CrkII. JNK1 is localized with CrkII in membrane ruffles of Crk-overexpressing cells in a Rac1-dependent manner. A JNK1 mutant (K340A) that fails to interact with CrkII is defective in Rac/epidermal growth factor-induced activation, but remains responsive to UVC irradiation. Furthermore, CrkII recruits JNK1 to a p130Cas multiprotein complex (see CAS/CSE1 segregation protein) where it may be activated through a hematopoietic progenitor kinase 1- and mitogen-activated protein kinase kinase 4-dependent pathway. Together, the results presented here argue for a new mechanism of regulation of the JNK pathway through the CrkII-p130Cas adaptor complex (Girardin, 2001).

The data presented here strongly support the hypothesis that the CrkII-JNK1 interaction is functionally linked to JNK1 activation by CrkII. (1) By using CrkDelta(N)SH3, a CrkII mutant protein harboring single mutations which alter the structure of the N-terminal SH3 domain, it has been shown that JNK1 interacts with CrkII through CrkII's N-terminal SH3 domain. While wild-type CrkII activates JNK1, CrkDelta(N)SH3 overexpression fails to do so. (2) Since mutation in the CrkII N-terminal SH3 domain could also affect other signaling pathways, the role of CrkII-JNK1 interaction in JNK1 activation was directly assessed by mutating a lysine residue in a proline-rich cluster of JNK1 that shares high homology with the CrkII N-terminal SH3 binding domain motif. Indeed, this lysine to alanine substitution strongly decreases both the CrkII-JNK1 interaction and CrkII-induced JNK activation. Since the activation of JNK1 by CrkII is concomitant with the appearance of N-terminally phosphorylated c-Jun in the nucleus, it is very likely that CrkII regulates the transcription of genes dependent upon the activity of c-Jun. However, the possibility that JNK1 also phosphorylates cytoplasmic proteins cannot be excluded (Girardin, 2001).

In conclusion, these results argue for a role of the p130Cas-CrkII interface as a scaffolding complex regulated by Rac1, which is involved in a specific signaling pathway leading to JNK1 activation. These findings suggest that different routes leading to JNK activation may be physically separated by different scaffolding proteins, such as JIP-1 or the p130Cas-CrkII complex. The sequestration of kinase cascades could, in turn, allow cells to modulate their responses to various stimuli (Girardin, 2001).

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