HIV-1 Nef Binds the DOCK2–ELMO1 Complex to Activate Rac and Inhibit Lymphocyte Chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.     

Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity

Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and βPix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or βPix, to membrane microdomains. Transient RNAi knockdown, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a βPix binding-deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or βPix in Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the Src homology 3 domain binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation of a catalytically active Nef-PAK2 complex.     

An alpha-helical extension of the ELMO1 pleckstrin homology domain mediates direct interaction to DOCK180 and is critical in Rac signaling

The mammalian DOCK180 protein belongs to an evolutionarily conserved protein family, which together with ELMO proteins, is essential for activation of Rac GTPase-dependent biological processes. Here, we have analyzed the DOCK180-ELMO1 interaction, and map direct interaction interfaces to the N-terminal 200 amino acids of DOCK180, and to the C-terminal 200 amino acids of ELMO1, comprising the ELMO1 PH domain. Structural and biochemical analysis of this PH domain reveals that it is incapable of phospholipid binding, but instead structurally resembles FERM domains. Moreover, the structure revealed an N-terminal amphiphatic α-helix, and point mutants of invariant hydrophobic residues in this helix disrupt ELMO1-DOCK180 complex formation. A secondary interaction between ELMO1 and DOCK180 is conferred by the DOCK180 SH3 domain and proline-rich motifs at the ELMO1 C-terminus. Mutation of both DOCK180-interaction sites on ELMO1 is required to disrupt the DOCK180-ELMO1 complex. Significantly, although this does not affect DOCK180 GEF activity toward Rac in vivo, Rac signaling is impaired, implying additional roles for ELMO in mediating intracellular Rac signaling.     

An evolutionarily conserved autoinhibitory molecular switch in ELMO proteins regulates Rac signaling

Dedicator of cytokinesis (DOCK) proteins are guanine nucleotide exchange factors (GEFs) controlling the activity of Rac1/Cdc42 during migration, phagocytosis, and myoblast fusion [1-4]. Engulfment and cell motility (ELMO) proteins bind a subset of DOCK members and are emerging as critical regulators of Rac signaling [5-10]. Although formation of a DOCK180/ELMO complex is not essential for Rac1 activation, ELMO mutants deficient in binding to DOCK180 are unable to promote cytoskeleton remodeling [11]. How ELMO regulates signaling through DOCK GEFs is poorly understood. Here, we identify an autoinhibitory switch in ELMO presenting homology to a regulatory unit described for Dia formins. One part of the switch, composed of a Ras-binding domain (RBD) and Armadillo repeats, is positioned N-terminally while the other is housed in the C?terminus. We demonstrate interaction between these fragments, suggesting autoinhibition of ELMO. Using a bioluminescence resonance energy transfer biosensor, we establish that ELMO undergoes conformational changes upon disruption of autoinhibition. We found that engagement of ELMO to RhoG, or with DOCK180, promotes the relief of autoinhibition in ELMO. Functionally, we found that ELMO mutants with impaired autoregulatory activity promote cell?elongation. These results demonstrate an unsuspected level of regulation for Rac1 signaling via autoinhibition of ELMO.     

Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions.

Nef of primate lentiviruses is critical for high levels of viremia and the progression to AIDS. Nef associates with and activates a serine/threonine kinase (Nef-associated kinase [NAK]) via the small GTPases Rac1 and Cdc42. We identified the protooncogene and guanine nucleotide exchange factor Vav as the specific binding partner of Nef proteins from HIV-1 and SIV. The interaction between Nef and Vav led to increased activity of Vav and its downstream effectors. Both cytoskeletal changes and the activation of c-Jun N-terminal kinase (JNK) were observed. Furthermore, a dominant-negative Vav protein inhibited NAK activation and viral replication. Thus, the interaction between Nef and Vav initiates a signaling cascade that changes structural and physiological parameters in the infected cell.     

The Arf family GTPase Arl4A complexes with ELMO proteins to promote actin cytoskeleton remodeling and reveals a versatile Ras-binding domain in the ELMO proteins family

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800-Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO-DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.     

C-terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac-GEF

Genetic studies in Caenorhabditis elegans identified an evolutionarily conserved CED-2 (CrkII), CED-5 (DOCK180), CED-12 (ELMO), CED-10 (Rac1) module important for cell migration and phagocytosis of apoptotic cells. Previous studies have shown that DOCK180 and ELMO comprise an unconventional bipartite Dbl homology domain-independent Rac guanine nucleotide exchange factor (Rac-GEF); but it is still unclear how CrkII functions in Rac-GEF activity. In this study, we have characterized a unique function of CrkII in phagocytosis and Rac activation mediated by the C-terminal SH3 domain, a region of CrkII that has no clear cellular or biochemical function. We found that mutations that disrupt the C-terminal SH3 domain of CrkII (CrkII-SH3-C) abrogate engulfment of apoptotic cells and impair cell spreading on extracellular matrix. Surprisingly, despite the effects on engulfment, W276K CrkII strongly potentiated Rac-GTP loading when ectopically expressed in HEK 293T cells. Contrary to the effects of the true dominant negative SH2 domain mutants (R38K CrkII) and SH3-N domain mutants (W170K CrkII) that prevent macromolecular assembly of signaling proteins, W276K CrkII increases association between DOCK180 and CrkII as well as constitutive tethering of the Crk/DOCK180/ELMO protein complex that interacted with RhoG. Our results indicate that while N-terminal SH3 of CrkII promotes assembly between CrkII and DOCK180, the C-terminal SH3 of CrkII regulates the stability and turnover of the DOCK180/ELMO complex. Studies with W276K CrkII may offer a unique opportunity to study the structure and function of the DOCK180/ELMO Rac-GEF.     

Upregulation of fibronectin expression by COX-2 is mediated by interaction with ELMO1

Engulfment and cell motility 1 (ELMO1), a bipartite guanine nucleotide exchange factor (GEF) for the small GTPase Rac 1, was identified as a susceptibility gene for glomerular disease. Here, we reported that ELMO1 interacted with COX-2 in human mesangial cells. Furthermore, we identified ELMO1 as a posttranslational regulator of COX-2 activity. We demonstrated that COX-2 cyclooxygenase activity increased fibronectin promoter activity. The protein-protein interaction between ELMO1 and COX-2 increased the cyclooxygenase activity of COX-2 and, correspondingly, fibronectin expression. We also found that ET625, the dominant negative form of ELMO1 lacking Rac1 activity, interacted with COX-2, increased cyclooxygenase activity of COX-2 and enhanced COX-2-mediated fibronectin upregulation. To further rule out Rac1 as an ELMO1-mediated regulator of COX-2 activity, we employed the constitutive active Rac1, Rac1^Q63E, and demonstrated that Rac1 signaling has no effect on COX-2-mediated fibronectin promoter activity. These results suggest that ELMO1 contributes to the development of glomerular injury through serving as a regulator of COX-2 activity. The interaction of ELMO1 with COX-2 could play an important role in the development and progression of renal glomerular injury.     

The HIV-1 Nef protein and phagocyte NADPH oxidase activation

Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli.     

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex

The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity.     

Nef proteins from diverse groups of primate lentiviruses downmodulate CXCR4 to inhibit migration to the chemokine stromal derived factor 1

Nef proteins of primate lentiviruses promote viral replication, virion infectivity, and evasion of antiviral immune responses by modulating signal transduction pathways and downregulating expression of receptors at the cell surface that are important for efficient antigen-specific responses, such as CD4, CD28, T-cell antigen receptor, and class I and class II major histocompatibility complex. Here we show that Nef proteins from diverse groups of primate lentiviruses which do not require the chemokine receptor CXCR4 for entry into target cells strongly downmodulate the cell surface expression of CXCR4. In contrast, all human immunodeficiency virus type 1 (HIV-1) and the majority of HIV-2 Nef proteins tested did not have such strong effects. SIVmac239 Nef strongly inhibited lymphocyte migration to CXCR4 ligand, the chemokine stromal derived factor 1 (SDF-1). SIVmac239 Nef downregulated CXCR4 by accelerating the rate of its endocytosis. Downmodulation of CXCR4 was abolished by mutations that disrupt the constitutively strong AP-2 clathrin adaptor binding element located in the N-terminal region of the Nef molecule, suggesting that Nef accelerates CXCR4 endocytosis via an AP-2-dependent pathway. Together, these results point to CXCR4 as playing an important role in simian immunodeficiency virus and possibly also HIV-2 persistence in vivo that is unrelated to viral entry into target cells. We speculate that Nef targets CXCR4 to disrupt ordered trafficking of infected leukocytes between local microenvironments in order to facilitate their dissemination and/or impair the antiviral immune response.     

Nuclear localization of the DOCK180/ELMO complex

DOCK180 protein plays a key role during development, cell motility, and phagocytosis. It forms a complex with another protein ELMO, and this complex acts as a guanine nucleotide exchange factor (GEF) for Rac. However, DOCK180-containing complexes have not been purified by unbiased biochemical approaches, and the nature and subcellular localization of these complexes remain unclear. Here, we show that a large fraction of endogenous DOCK180 is present as a 700kDa nuclear complex with ELMO proteins. In addition, this nuclear DOCK180/ELMO complex has functional Rac-GEF activity. Furthermore, endogenous DOCK180 could be found in complexes with different ELMO isoforms (ELMO1, 2 or 3) in different cell lines, depending on the ELMO isoforms expressed. These studies suggest that DOCK180 may associate with different ELMO proteins to form cell-type specific complexes and may have functions in both the nucleus and the cytoplasm.     

Activation of p21-activated kinase 2 by human immunodeficiency virus type 1 Nef induces merlin phosphorylation

The accessory human immunodeficiency virus type 1 (HIV-1) protein Nef activates the autophosphorylation activity of p21-activated kinase 2 (PAK2). Merlin, a cellular substrate of PAK2, is homologous to the ezrin-radixin-moesin family and plays a critical role in Rac signaling. To assess the possible impact on host cell metabolism of Nef-induced PAK2 activation, we investigated the phosphorylation of merlin in Nef expressing cells. Here we report that Nef induces merlin phosphorylation in multiple cell lines independently of protein kinase A. This intracellular phosphorylation of merlin directly correlates with in vitro assay of the autophosphorylation activity of Nef-activated PAK2. Importantly, merlin phosphorylation induced by Nef was also observed in human primary T cells. The finding that Nef induces phosphorylation of the key signaling molecule merlin suggests several possible roles for PAK2 activation in HIV pathogenesis.     

Ankyrin repeat domain 28 (ANKRD28), a novel binding partner of DOCK180, promotes cell migration by regulating focal adhesion formation

DOCK180 is a guanine exchange factor of Rac1 originally identified as a protein bound to an SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130^Cas, and recruits the Crk-p130^Cas complex to focal adhesions. To understand the role of DOCK180 in cell adhesion and migration, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and identified ANKRD28, a protein that contains twenty-six ankyrin domain repeats. Knockdown of ANKRD28 by RNA interference reduced the velocity of migration of HeLa cells, suggesting that this protein plays a physiologic role in the DOCK180-Rac1 signaling pathway. Furthermore, knockdown of ANKRD28 was found to alter the distribution of focal adhesion proteins such as Crk, paxillin, and p130^Cas. On the other hand, expression of ANKRD28, p130^Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130^Cas, and impaired detachment of the cell membrane during migration. Consequently, cells expressing ANKRD28 exhibited multiple long cellular processes. ANKRD28 associated with DOCK180 in an SH3-dependent manner and competed with ELMO, another protein bound to the SH3 domain of DOCK180. In striking contrast to ANKRD28, overexpression of ELMO induced extensive lamellipodial protrusion around the entire circumference. These data suggest that ANKRD28 specifies the localization and the activity of the DOCK180-Rac1 pathway.     

Structural insights into the small GTPase specificity of the DOCK guanine nucleotide exchange factors

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) regulates cytoskeletal dynamics by activating the GTPases Rac and/or Cdc42. Eleven human DOCK proteins play various important roles in developmental processes and the immune system. Of these, DOCK1–5 proteins bind to engulfment and cell motility (ELMO) proteins to perform their physiological functions. Recent structural studies have greatly enhanced our understanding of the complex and diverse mechanisms of DOCK GEF activity and GTPase recognition and its regulation by ELMO. This review is focused on gaining structural insights into the substrate specificity of the DOCK GEFs, and discuss how Rac and Cdc42 are specifically recognized by the catalytic DHR-2 and surrounding domains of DOCK or binding partners.     

Down-modulation of CD8αβ is a fundamental activity of primate lentiviral Nef proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.     

Activation of Rac1 by paxillin-Crk-DOCK180 signaling complex is antagonized by Rap1 in migrating NBT-II cells

Induction of epithelial cell motility is a fundamental morphogenetic event that is recapitulated during carcinoma metastasis. Random motility of NBT-II carcinoma cells on collagen critically depends on paxillin phosphorylation at Tyr-31 and Tyr-118, the binding sites for the adapter protein CrkII. Two constitutive partners of CrkII are the exchange factors DOCK180 and C3G. CrkII bound to DOCK180 formed a signaling complex with phosphorylated paxillin that was necessary for cell migration as inferred from the inhibition caused by a DOCK180-interfering mutant. DOCK180, which acts predominantly on the Rho family GTPase Rac1, restored cell locomotion in cells expressing Phe-31/118 paxillin mutants deficient in Rac1 GTP-loading, suggesting that formation of paxillin-Crk-DOCK180 signaling complex controls collagen-dependent migration mainly through Rac1 activation. In migrating cells, CrkII constitutive association with C3G was not sufficient to stimulate its GDP/GTP exchange activity toward the Ras family GTPase Rap1. However, when constitutively active RapV12 was overexpressed, it negatively regulated cell motility. Activation of the C3G/Rap1 signaling pathway resulted in down-regulation of the paxillin-Crk-DOCK180 complex and reduction of Rac1-GTP, suggesting that Rap1 activation could suppress the Rac1 signaling pathway in epithelial cells.     

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.