Signaling through CD5 Activates a Pathway Involving Phosphatidylinositol 3-Kinase, Vav, and Rac1 in Human Mature T Lymphocytes

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell–B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca²⁺ levels, and subsequently in the activation of Ca²⁺/calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CD5-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated Rac1 mutant (Rac1 · N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. A role for Vav in the CD5-induced signaling pathway is further supported by the findings that the expression of a dominant negative Vav mutant (Vav-C) completely abolishes the response to CD5 costimulation while the expression of a constitutively active Vav mutant [Vav(Δ1–65)] makes the CD5 costimulation signal superfluous. Wortmannin is unable to block the Vav(Δ1–65)- or Rac1 · V12-induced signals, indicating that both Vav and Rac1 function downstream from PI 3-kinase. Vav and Rac1 both act upstream from the CD5-induced activation of CaM kinase IV, since KN-62, an inhibitor of CaM kinases, and a dominant negative CaM kinase IV mutant block the Vav(Δ1–65)-and Rac1 · V12-mediated signals. We propose a model for the CD5-induced signaling pathway in which the PI 3-kinase lipid products, together with tyrosine phosphorylation, activate Vav, resulting in the activation of Rac1 by the Vav-mediated exchange of GDP for GTP.     

Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.     

Protein kinase A-dependent phosphorylation modulates beta1Pix guanine nucleotide exchange factor activity through 14-3-3beta binding

β₁Pix is a guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42 which has been shown to mediate signaling pathways leading to cytoskeletal reorganization. In the present study, we show that the basal association between endogenous βPix and endogenous 14-3-3β was increased after forskolin stimulation and significantly inhibited by protein kinase A inhibitor. However, forskolin stimulation failed to increase the interaction between 14-3-3β and a β₁Pix mutant that is insensitive to protein kinase A phosphorylation, β₁Pix(S516A, T526A). We present evidence indicating that forskolin-induced binding of 14-3-3β to β₁Pix results in inhibition of Rac1 GTP loading in 293 cells and in vitro. Furthermore, we show that deletion of 10 amino acid residues within the leucine zipper domain is sufficient to block β₁Pix homodimerization and 14-3-3β binding and modulates β₁Pix-GEF activity. These residues also play a crucial role in β₁Pix intracellular localization. These results indicate that 14-3-3β negatively affects the GEF activity of dimeric β₁Pix only. Altogether, these results provide a mechanistic insight into the role of 14-3-3β in modulating β₁Pix-GEF activity.     

A Conserved Negative Regulatory Region in αPAK: Inhibition of PAK Kinases Reveals Their Morphological Roles Downstream of Cdc42 and Rac1

αPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129–1143, 1997) resembling those of Cdc42^G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in αPAK). αPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42^G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding αPAK^83-149 potently inhibited GTPγS-bound Cdc42-mediated kinase activation of both αPAK and βPAK. Coexpression of this PAK inhibitor with Cdc42^G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1^G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of αPAK^83-149 completely blocked the phenotypic effects of hyperactive αPAK^L107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.     

Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

Adenovirus (Ad) endocytosis via α_v integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

Involvement of Phosphatidylinositide 3′-Kinase and Rac in Platelet-Derived Growth Factor-Induced Actin Reorganization and Chemotaxis

Previous work has suggested a role for phosphatidylinositide 3′-kinase (PI3-kinase) in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. In support of this notion, we show in this report that the PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF β-receptor expressing porcine aortic endothelial (PAE/PDGFR-β) cells. Treatment with wortmannin resulted in a dose-dependent decrease in chemotaxis with an IC₅₀value of about 15–20 nM.Higher concentrations of wortmannin also reduced basal random migration of transfected cells in the absence of PDGF. We also investigated the role of Rac in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-β cells led to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-β cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurred in the absence of PDGF stimulation and was independent of PI3-kinase activity. On the other hand, PAE/PDGFR-β cells transfected with inducible N17Rac (a dominant negative Rac mutant) failed to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway.     

p21-activated Kinases (PAKs) Mediate the Phosphorylation of PREX2 Protein to Initiate Feedback Inhibition of Rac1 GTPase

Phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP₃ or Gβγ, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP₃ and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.     

Regulation of adaptor protein GIT1 in platelets, leading to the interaction between GIT1 and integrin alpha(IIb)beta3

GIT1 is an adaptor protein, which links signaling proteins to focal adhesion, thereby regulating cytoskeletal reorganization. Platelets undergo dynamic cytoskeletal reorganization during platelet activation, for which a large number of adaptor proteins are required. However, there has been no report of GIT1 in platelets. We found that GIT1 was abundantly expressed in platelets and underwent tyrosine phosphorylation downstream of integrin α_IIbβ₃, which was inhibited by the Src kinase inhibitor PP2. Furthermore, GIT1 constitutively associated with βPIX, a guanine nucleotide exchange factor (GEF) for Rac. The GIT1/βPIX complex associated with α_IIbβ₃, concomitantly with GIT1 tyrosine phosphorylation. Moreover, both GIT1 and α_IIbβ₃ rapidly translocated to the cytoskeletal fraction during platelet aggregation, which was not observed in the absence of aggregation. These results suggest that tyrosine phosphorylation of GIT1 by Src kinases may regulate cytoskeletal reorganization downstream of α_IIbβ₃ by bringing the Rac GEF βPIX to the vicinity of the integrin.     

The PI3K p110α isoform regulates endothelial adherens junctions via Pyk2 and Rac1

Endothelial cell–cell junctions control efflux of small molecules and leukocyte transendothelial migration (TEM) between blood and tissues. Inhibitors of phosphoinositide 3-kinases (PI3Ks) increase endothelial barrier function, but the roles of different PI3K isoforms have not been addressed. In this study, we determine the contribution of each of the four class I PI3K isoforms (p110α, -β, -γ, and -δ) to endothelial permeability and leukocyte TEM. We find that depletion of p110α but not other p110 isoforms decreases TNF-induced endothelial permeability, Tyr phosphorylation of the adherens junction protein vascular endothelial cadherin (VE-cadherin), and leukocyte TEM. p110α selectively mediates activation of the Tyr kinase Pyk2 and GTPase Rac1 to regulate barrier function. Additionally, p110α mediates the association of VE-cadherin with Pyk2, the Rac guanine nucleotide exchange factor Tiam-1 and the p85 regulatory subunit of PI3K. We propose that p110α regulates endothelial barrier function by inducing the formation of a VE-cadherin–associated protein complex that coordinates changes to adherens junctions with the actin cytoskeleton.     

Smad3 Regulates Rho Signaling via NET1 in the Transforming Growth Factor-β-induced Epithelial-Mesenchymal Transition of Human Retinal Pigment Epithelial Cells

We previously demonstrated that RhoA-dependent signaling regulates transforming growth factor-β1 (TGF-β1)-induced cytoskeletal reorganization in the human retinal pigment epithelial cell line ARPE-19. Smad pathways have also been shown to mediate TGF-β1 activity. Here, we examined what regulates Rho GTPase activity and tested whether Smad signaling cross-talks with Rho pathways during TGF-β1-induced actin rearrangement. Using small interfering RNAs, we found that NET1, the guanine nucleotide exchange factor of RhoA, is critical for TGF-β1-induced cytoskeletal reorganization, N-cadherin expression, and RhoA activation. In ARPE-19 cells lacking NET1, TGF-β1-induced stress fibers and N-cadherin expression were not observed. Interestingly, in dominant-negative Smad3-expressing or constitutively active Smad7 cells, TGF-β1 failed to induce NET1 mRNA and protein expression. Consistent with these results, both dominant-negative Smad3 and constitutively active Smad7 blocked the cytoplasmic localization of NET1 and inhibited interactions between NET1 and RhoA. Finally, we found that NET1 is a direct gene target of TGF-β1 via Smad3. Taken together, our results demonstrate that Smad3 regulates RhoA activation and cytoskeletal reorganization by controlling NET1 in TGF-β1-induced ARPE-19 cells. These data define a new role for Smad3 as a modulator of RhoA activation in the regulation of TGF-β1-induced epithelial-mesenchymal transitions.     

Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.     

Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→Rac1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”     

Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

Essential Role of Class II Phosphatidylinositol-3-kinase-C2α in Sphingosine 1-Phosphate Receptor-1-mediated Signaling and Migration in Endothelial Cells

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3′-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110β, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2β, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P_1–3. We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P₁ in ECs. Knockdown of either PI3K-C2α or class I p110β markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110β was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110β were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110β markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110β suppressed S1P-induced S1P₁ internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P₁ internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P₁ internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.     

Rac Inhibition Reverses the Phenotype of Fibrotic Fibroblasts

Background

Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce α−smooth muscle actin (α-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.

Methods and Findings

Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and α−SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and α−SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.

Conclusion

Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.     

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na⁺,K⁺-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na⁺,K⁺-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na⁺,K⁺-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D₁ receptor subtype and the sequential activation of phospholipase A₂, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na⁺,K⁺-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.     

Paxillin kinase linker (PKL) regulates Vav2 signaling during cell spreading and migration

The Rho family of GTPases plays an important role in coordinating dynamic changes in the cell migration machinery after integrin engagement with the extracellular matrix. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs). However, the mechanisms by which GEFs and GAPs are spatially and temporally regulated are poorly understood. Here the activity of the proto-oncogene Vav2, a GEF for Rac1, RhoA, and Cdc42, is shown to be regulated by a phosphorylation-dependent interaction with the ArfGAP PKL (GIT2). PKL is required for Vav2 activation downstream of integrin engagement and epidermal growth factor (EGF) stimulation. In turn, Vav2 regulates the subsequent redistribution of PKL and the Rac1 GEF β-PIX to focal adhesions after EGF stimulation, suggesting a feedforward signaling loop that coordinates PKL-dependent Vav2 activation and PKL localization. Of interest, Vav2 is required for the efficient localization of PKL and β-PIX to the leading edge of migrating cells, and knockdown of Vav2 results in a decrease in directional persistence and polarization in migrating cells, suggesting a coordination between PKL/Vav2 signaling and PKL/β-PIX signaling during cell migration.     

Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase

The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC(50) of 1.1 μM for Rac inhibition by EHop-016 is ～100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μM. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μM), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.     

Photo-enhancement of macrophage phagocytic activity via Rac1-mediated signaling pathway: Implications for bacterial infection

Phagocytosis and the subsequent destruction of invading pathogens by macrophages are indispensable steps in host immune responses to microbial infections. Low-power laser irradiation (LPLI) has been found to exert photobiological effects on immune responses, but the signaling mechanisms underlying this photobiomodulation of phagocytosis remains largely unknown. Here, we demonstrated for the first time that LPLI enhanced the phagocytic activity of macrophages by stimulating the activation of Rac1. The overexpression of constitutively activated Rac1 clearly enhanced LPLI-induced phagocytosis, whereas the overexpression of dominant negative Rac1 exerted the opposite effect. The phosphorylation of cofilin was involved in the effects of LPLI on phagocytosis, which was regulated by the membrane translocation and activation of Rac1. Furthermore, the photoactivation of Rac1 was dependent on the Src/PI3K/Vav1 pathway. The inhibition of the Src/PI3K pathway significantly suppressed LPLI-induced actin polymerization and phagocytosis enhancement. Additionally, LPLI-treated mice exhibited increased survival and a decreased organ bacterial load when challenged with Listeria monocytogenes, indicating that LPLI enhanced macrophage phagocytosis in vivo. These findings highlight the important roles of the Src/PI3K/Vav1/Rac1/cofilin pathway in regulating macrophage phagocytosis and provide a potential strategy for treating phagocytic deficiency via LPLI.     

The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 –/– fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27–42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin–rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 –/– fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice.