Role of the small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni

Host cell invasion of the food-borne pathogen Campylobacter jejuni is one of the primary reasons of tissue damage in humans but molecular mechanisms are widely unclear. Here, we show that C. jejuni triggers membrane ruffling in the eukaryotic cell followed by invasion in a very specific manner first with its tip followed by the flagellar end. To pinpoint important signalling events involved in the C. jejuni invasion process, we examined the role of small Rho family GTPases. Using specific GTPase-modifying toxins, inhibitors and GTPase expression constructs we show that Rac1 and Cdc42, but not RhoA, are involved in C. jejuni invasion. In agreement with these observations, we found that internalization of C. jejuni is accompanied by a time-dependent activation of both Rac1 and Cdc42. Finally, we show that the activation of these GTPases involves different host cell kinases and the bacterial fibronectin-binding protein CadF. Thus, CadF is a bifunctional protein which triggers bacterial binding to host cells as well as signalling leading to GTPase activation. Collectively, our results suggest that C. jejuni invade host target cells by a unique mechanism and the activation of the Rho GTPase members Rac1 and Cdc42 plays a crucial role in this entry process.     

ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting

Activation of Rac1, a member of the Rho family of GTPases, is associated with multiple cellular responses, including membrane ruffling and focal complex formation. The mechanisms by which Rac1 is coupled to these functional responses are not well understood. It was recently shown that ARF6, a GTPase implicated in cytoskeletal alterations and a membrane recycling pathway, is required for Rac1-dependent phagocytosis in macrophages (Q. Zhang et al., J. Biol. Chem. 273:19977-19981, 1998). To determine whether ARF6 is required for Rac1-dependent cytoskeletal responses in macrophages, we expressed wild-type (WT) or guanine nucleotide binding-deficient alleles (T27N) of ARF6 in macrophages coexpressing activated alleles of Rac1 (Q61L) or Cdc42 (Q61L) or stimulated with colony-stimulating factor 1 (CSF-1). Expression of ARF6 T27N but not ARF6 WT inhibited ruffles mediated by Rac1 Q61L or CSF-1. In contrast, expression of ARF6 T27N did not inhibit Rac1 Q61L-mediated focal complex formation and did not impair Cdc42 Q61L-mediated filopodial formation. Cryoimmunogold electron microscopy demonstrated the presence of ARF6 in membrane ruffles induced by either CSF-1 or Rac1 Q61L. Addition of CSF-1 to macrophages led to the redistribution of ARF6 from the interior of the cell to the plasma membrane, suggesting that this growth factor triggers ARF6 activation. Direct targeting of Rac1 to the plasma membrane did not bypass the blockade in ruffling induced by ARF6 T27N, indicating that ARF6 regulates a pathway leading to membrane ruffling that occurs after the activation and membrane association of Rac. These data demonstrate that intact ARF6 function is required for coupling activated Rac to one of several effector pathways and suggest that a principal function of ARF6 is to coordinate Rac activation with plasma membrane-based protrusive events.     

Serine-71 phosphorylation of rac1 modulates downstream signaling

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.     

Implication of a small GTPase Rac1 in the activation of c-Jun N-terminal kinase and heat shock factor in response to heat shock

Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat shock protein (HSP) expression through activation of the heat shock factor (HSF), but its signal pathway is not clearly understood. Since a small GTPase Rac1 has been suggested to participate in the cellular response to stresses, we examined whether Rac1 is involved in the heat shock response. Here we show that moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant negative mutant of Rac1, significantly inhibited JNK activation by heat shock. Since Rac1V12 was able to activate JNK, it is suggested that heat shock may activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat shock-induced JNK and HSF activation were reduced as the heat shock temperature increased. Rac1N17 also inhibited HSF activation by l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)), suggesting that Rac1 may be linked to HSF activation by denaturation of polypeptides in response to various proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation of HSF1 in response to these proteotoxic stresses. Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for heat shock-inducible HSF activation and HSP expression, or otherwise a signal pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In sum, we suggest that Rac1 may play a critical role(s) in several aspects of the heat shock response.     

Potentiation of cell migration by adhesion-dependent cooperative signals from the GTPase Rac and Raf kinase

The small GTPase Rac is thought to regulate cell movement by influencing actin cytoskeletal organization and membrane ruffling. However, cell migration also depends on the activation of mitogen-activated protein kinase (MAPK), which can regulate myosin motor function, an event critical for cell contraction. Evidence is provided that, during active cell adhesion to the extracellular matrix, Rac potentiates the MAPK pathway and influences cell migration by selectively synergizing with Raf kinase but not with Ras or MAPK kinase. In fact, the synergy between Rac and Raf kinase increases the chemotactic sensitivity of cells to epidermal growth factor by 1000-fold. Therefore, the role of Rac in cell migration not only depends on its ability to regulate actin cytoskeletal organization but also on its capacity to potentiate chemokine activation of MAPK in a manner that depends on active cell adhesion to the extracellular matrix.     

Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress-induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear-induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow-induced stimulation of nuclear factor kappaB (NF-kappaB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM-1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.     

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGKζ initiates RhoGDI release and Rac1 activation. In DGKζ-deficient fibroblasts PAK1 phosphorylation and Rac1–RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGKζ, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGKζ stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGKζ, and PA to the activation of Rac1: the PA generated by DGKζ activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility.     

Endogenous ARF6 interacts with Rac1 upon angiotensin II stimulation to regulate membrane ruffling and cell migration

ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT(1)R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT(1)R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.     

Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K(+) channels

Arachidonic acid (AA) is generated via Rac-mediated phospholipase A2 (PLA2) activation in response to growth factors and cytokines and is implicated in cell growth and gene expression. In this study, we show that AA activates the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in a time- and dose-dependent manner. Indomethacin and nordihydroguaiaretic acid, potent inhibitors of cyclooxygenase and lipoxygenase, respectively, did not exert inhibitory effects on AA-induced SAPK/JNK activation, thereby indicating that AA itself could activate SAPK/JNK. As Rac mediates SAPK/JNK activation in response to a variety of stressful stimuli, we examined whether the activation of SAPK/JNK by AA is mediated by Rac1. We observed that AA-induced SAPK/JNK activation was significantly inhibited in Rat2-Rac1N17 dominant-negative mutant cells. Furthermore, treatment of AA induced membrane ruffling and production of hydrogen peroxide, which could be prevented by Rac1N17. These results suggest that AA acts as an upstream signal molecule of Rac, whose activation leads to SAPK/JNK activation, membrane ruffling and hydrogen peroxide production.     

A kinase-deficient TrkC receptor isoform activates Arf6-Rac1 signaling through the scaffold protein tamalin

Neurotrophins play an essential role in mammalian development. Most of their functions have been attributed to activation of the kinase-active Trk receptors and the p75 neurotrophin receptor. Truncated Trk receptor isoforms lacking the kinase domain are abundantly expressed during development and in the adult; however, their function and signaling capacity is largely unknown. We show that the neurotrophin-3 (NT3) TrkCT1-truncated receptor binds to the scaffold protein tamalin in a ligand-dependent manner. Moreover, NT3 initiation of this complex leads to activation of the Rac1 GTPase through adenosine diphosphate-ribosylation factor 6 (Arf6). At the cellular level, NT3 binding to TrkCT1-tamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling and the formation of cellular protrusions. Thus, our data identify a new signaling pathway elicited by the kinase-deficient TrkCT1 receptor. Moreover, we establish NT3 as an upstream regulator of Arf6.     

Expression of Rac1b stimulates NF-kappaB-mediated cell survival and G1/S progression

The small GTPase Rac1 can stimulate various signaling pathways following a tightly controlled GDP-GTP exchange. A splicing variant designated Rac1b was found to exist predominantly in the active GTP-bound state but the functional consequences of its expression remain unknown. Here we used mouse fibroblasts as a model to assess the signaling properties of Rac1b. We show that, in contrast to Rac1, expression of wild-type Rac1b is sufficient to stimulate cyclin D1 accumulation and G1/S progression in these cells. Moreover, expression of wild-type Rac1b, but not of wild-type Rac1, dramatically increased cell survival in the presence of only minimal growth stimuli. Both cellular responses were blocked by the NF-kappaB super-repressor IkappaBalpha(A32A36). Active Rac1b induced the phosphorylation and membrane translocation of IkappaBalpha, a prerequisite for the activation of NF-kappaB. These data demonstrate that Rac1b is a highly active Rac1 variant that stimulates cell cycle progression and cell survival in pathways involving NF-kappaB.     

Coronin 1A promotes a cytoskeletal-based feedback loop that facilitates Rac1 translocation and activation

The activation of the Rac1 GTPase during cell signalling entails its translocation from the cytosol to membranes, release from sequestering Rho GDP dissociation inhibitors (RhoGDI), and GDP/GTP exchange. In addition to those steps, we show here that optimal Rac1 activation during cell signalling requires the engagement of a downstream, cytoskeletal-based feedback loop nucleated around the cytoskeletal protein coronin 1A and the Rac1 exchange factor ArhGEF7. These two proteins form a cytosolic complex that, upon Rac1-driven F-actin polymerization, translocates to juxtamembrane areas where it expands the pool of activated, membrane-bound Rac1. Such activity requires the formation of an F-actin/ArhGEF7-dependent physical complex of coronin 1A with Pak1 and RhoGDIα that, once assembled, promotes the Pak1-dependent dissociation of Rac1 from the Rac1/RhoGDIα complex and subsequent Rac1 activation. Genetic evidence demonstrates that this relay circuit is essential for generating sustained Rac1 activation levels during cell signalling.     

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration

CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.     

Abba promotes PDGF-mediated membrane ruffling through activation of the small GTPase Rac1

Abba is a member of the I-BAR-domain protein family that is characterized by a convex-shaped membrane-binding motif. Overexpression of GFP-tagged Abba in murine fibroblasts potentiated PDGF-mediated formation of membrane ruffles and lamellipodia. Immunofluorescent microscopy and pull-down analysis revealed that GFP-Abba colocalized with an active form of Rac1 in the membrane ruffles and enhanced the Rac GTPase activity in response to PDGF stimulation. Further immunoprecipitation assays demonstrated that GFP-Abba bound to both wild-type and constitutively active Rac1 and that the binding to either of the Rac1 forms was significantly enhanced upon PDGF stimulation. On the other hand, an Abba mutant deficient in Rac1 binding failed to promote membrane ruffling and Rac1 activation in response to PDGF. However, the cells overexpressing a truncated mutant carrying the I-BAR domain alone displayed numerous filopodia-like microspikes in a manner independent of growth factors. Also, the Rac-binding activity of the mutant was not affected by PDGF treatment. Our data indicates that the interaction between full-length Abba and Rac1 is implicated in membrane deformation and subjected to a growth factor-mediated regulation through the C-terminal sequence.     

Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.     

Adaptor protein Crk is required for ephrin-B1-induced membrane ruffling and focal complex assembly of human aortic endothelial cells

Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes membrane ruffling in human aortic endothelial cells, because membrane ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130(Cas), but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1-induced membrane ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1-induced membrane ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1-induced membrane ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1-induced human aortic endothelial cells migration.