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Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphenylfuropyrimidine was identified as a strong inhibitor of Nef-dependent Hck activation. This compound also exhibited remarkable antiretroviral effects, blocking Nef-dependent HIV replication in cell culture. Structurally related analogs were synthesized and shown to exhibit similar Nef-dependent anti-viral activity, identifying the diphenylfuropyrimidine substructure as a new lead for antiretroviral drug development. This study demonstrates that coupling non-catalytic HIV accessory factors with host cell target proteins addressable by high-throughput assays may afford new avenues for the discovery of anti-HIV agents.

Activation of Src family kinases by HIV-1 Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site allosterically influence Nef interactions with a key effector protein linked to AIDS progression.

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.

Nef is an HIV accessory protein that plays an important role in the progression of disease after viral infection. It interferes with numerous signaling pathways, one of which involves serine/threonine kinases. Here, we report the results of an NMR structural investigation on full-length Nef and its interaction with the entire regulatory domain of Hck (residues 72–256; Hck32L). A helical conformation was found at the N-terminus for residues 14–22, preceding the folded core domain. In contrast to the previously studied truncated Nef (Nef Δ1–39), the full-length Nef did not show any interactions of Trp57/Leu58 with the hydrophobic patch formed by helices α1 and α2. Upon Hck32L binding, the N-terminal anchor domain as well as the well-known SH3-binding site of Nef exhibited significant chemical shift changes. Upon Nef binding, resonance changes in the Hck spectrum were confined mostly to the SH3 domain, with additional effects seen for the connector between SH3 and SH2, the N-terminal region of SH2 and the linker region that contains the regulatory polyproline motif. The binding data suggest that in full-length Nef more than the core domain partakes in the interaction. The solution conformation of Hck32L was modeled using RDC data and compared with the crystal structure of the equivalent region in the inactivated full-length Hck, revealing a notable difference in the relative orientations of the SH3 and SH2 domains. The RDC-based model combined with 15N backbone dynamics data suggest that Hck32L adopts an open conformation without binding of the polyproline motif in the linker to the SH3 domain.

Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intra-molecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.

The HIV-1 accessory factor Nef is essential for high-titer viral replication and AIDS progression. Nef function requires interaction with many host cell proteins, including specific members of the Src kinase family. Here we explored whether Src-family kinase activation is a conserved property of Nef alleles from a wide range of primary HIV-1 isolates and their sensitivity to selective pharmacological inhibitors. Representative Nef proteins from the major HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J and K strongly activated Hck and Lyn as well as c-Src to a lesser extent, demonstrating for the first time that Src-family kinase activation is a highly conserved property of primary M-group HIV-1 Nef isolates. Recently, we identified 4-amino substituted diphenylfuropyrimidines (DFPs) that selectively inhibit Nef-dependent activation of Src-family kinases as well as HIV replication. To determine whether DFP compounds exhibit broad-spectrum Nef-dependent antiretroviral activity against HIV-1, we first constructed chimeric forms of the HIV-1 strain NL4-3 expressing each of the primary Nef alleles. The infectivity and replication of these Nef chimeras was indistinguishable from that of wild-type virus in two distinct cell lines (U87MG astroglial cells and CEM-T4 lymphoblasts). Importantly, the 4-aminopropanol and 4-aminobutanol derivatives of DFP potently inhibited the replication of all chimeric forms of HIV-1 in both U87MG and CEM-T4 cells in a Nef-dependent manner. The antiretroviral effects of these compounds correlated with inhibition of Nef-dependent activation of endogenous Src-family kinases in the HIV-infected cells. Our results demonstrate that the activation of Hck, Lyn and c-Src by Nef is highly conserved among all major clades of HIV-1 and that selective targeting of this pathway uniformly inhibits HIV-1 replication.

Chronic myelogenous leukemia (CML) is driven by Bcr-Abl, a constitutively active protein-tyrosine kinase that stimulates proliferation and survival of myeloid progenitors. Global inhibition of myeloid Src family kinase (SFK) activity with the broad-spectrum pyrrolo-pyrimidine inhibitor, A-419259, blocks proliferation and induces apoptosis in CML cell lines, suggesting that transformation by Bcr-Abl requires SFK activity. However, the contribution of Hck and other individual SFKs to Bcr-Abl signaling is less clear. Here, we developed an A-419259-resistant mutant of Hck by replacing the gatekeeper residue (Thr-338; c-Src numbering) in the inhibitor-binding site with a bulkier methionine residue (Hck-T338M). This substitution reduced Hck sensitivity to A-419259 by more than 30-fold without significantly affecting kinase activity in vitro. Expression of Hck-T338M protected K-562 CML cells and Bcr-Abl-transformed TF-1 myeloid cells from the apoptotic and antiproliferative effects of A-419259. These effects correlated with persistence of Hck-T338M kinase activity in the presence of the compound, and were accompanied by sustained Erk and Stat5 activation. In contrast, control cells expressing equivalent levels of wild-type Hck retained sensitivity to the inhibitor. We also show for the first time that A-419259 induces cell-cycle arrest and apoptosis in primary CD34+ CML cells with equal potency to imatinib. These data suggest that Hck has a nonredundant function as a key downstream signaling partner for Bcr-Abl and may represent a potential drug target in CML.

Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication in both primary T lymphocytes and monocyte-derived macrophages. This enhancement phenotype has been linked to the ability of Nef to modulate the activity of cellular kinases. We find that despite the reported high-affinity interaction between Nef and the Src kinase Hck in vitro, a Nef-Hck interaction in the context of HIV-1-infected primary macrophages is not detectable. However, Nef binding and activation of the PAK-related kinase and phosphorylation of its substrate could be readily detected in both infected primary T lymphocytes and macrophages. Furthermore, we show that this substrate is a complex composed of the recently characterized PAK interacting partner PIX (PAK-interacting guanine nucleotide exchange factor) and its tightly associated p95 protein. PAK and PIX-p95 appear to be differentially activated and phosphorylated depending on the intracellular environment in which nef is expressed. These results identify the PIX-p95 complex as a novel effector of Nef in primary cells and suggest that the regulation of the PAK signaling pathway may differ in T cells and macrophages.

HIV-1 Nef is essential for AIDS pathogenesis, but this viral protein is not targeted by antiviral strategies. The functions of Nef are largely related to perturbations of intracellular trafficking and signaling pathways through leucine-based and polyproline motifs that are required for interactions with clathrin-associated adaptor protein complexes and SH3 domain-containing proteins, such as the phagocyte-specific kinase Hck. We previously described a single-domain antibody (sdAb) targeting Nef and inhibiting many, but not all, of its biological activities. We now report a further development of this anti-Nef strategy through the demonstration of the remarkable inhibitory activity of artificial Nef ligands, called Neffins, comprised of the anti-Nef sdAb fused to modified SH3 domains. The Neffins inhibited all key activities of Nef, including Nef-mediated CD4 and major histocompatibility complex class I (MHC-I) cell surface downregulation and enhancement of virus infectivity. When expressed in T lymphocytes, Neffins specifically inhibited the Nef-induced mislocalization of the Lck kinase, which contributes to the alteration of the formation of the immunological synapse. In macrophages, Neffins inhibited the Nef-induced formation of multinucleated giant cells and podosome rosettes, and it counteracted the inhibitory activity of Nef on phagocytosis. Since we show here that these effects of Nef on macrophage and T cell functions were both dependent on the leucine-based and polyproline motifs, we confirmed that Neffins disrupted interactions of Nef with both AP complexes and Hck. These results demonstrate that it is possible to inhibit all functions of Nef, both in T lymphocytes and macrophages, with a single ligand that represents an efficient tool to develop new antiviral strategies targeting Nef.

Src family kinases (SFKs) are modular signaling proteins possessing SH3, SH2, and tyrosine kinase domains. The SH3 and SH2 domains of SFKs have dual roles: they regulate the activity of the kinases, and they also target SFKs to their cellular substrates. We generated a series of novel SFKs by replacing the SH2 and SH3 domains of Hck with the syntrophin PDZ domain. In some constructs, the negative regulatory tyrosine in the C-terminal tail was also replaced with a PDZ ligand sequence. When expressed in mammalian cells, the substrate specificity of the PDZ-kinases was directed to a different group of proteins than wild-type Hck. The PDZ-kinases phosphorylate neuronal nitric oxide synthase (nNOS), a known binding partner of the syntrophin PDZ domain. We also introduced a PDZ ligand at the C-terminus of the adaptor protein Cas. PDZ-Hck kinases phosphorylate the engineered Cas protein in Cas–/– cells and restore the migration defect of these cells. A PDZ-kinase was also functional in rewiring MAPK signaling via an engineered ErbB2 construct containing a PDZ ligand sequence. Several of the PDZ-kinases show autoregulatory properties similar to natural SFKs. Thus, the PDZ–ligand interaction is able to functionally replace the normal SH2–pY527 interaction that regulates SFKs. Our data highlight the modularity and evolvability of signaling proteins.

Background

p73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73.

Results

Exogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP), a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation.

Conclusion

We have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73-dependent transcriptional activation of a target gene, Ipaf, as well as apoptosis.

Abstract

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated neurocognitive disorders (HAND). The role of HIV Nef and other accessory proteins in HAND pathogenesis is unclear. To determine whether HIV nef undergoes adaptive selection in brain, we cloned 100 nef sequences (n = 30 brain and n = 70 lymphoid) from four patients with AIDS and HIV-associated dementia (HAD). Normalized nonsynonymous substitutions were more frequent at the divergence of lymphoid and brain sequences, indicating stronger adaptive selection in brain compared to lymphoid tissue. Brain-specific nonsynonymous substitutions were found within an NH3-terminal CTL epitope, the PACS1 binding motif, or positions predicted to be important for activation of the myeloid-restricted Src family tyrosine kinase Hck. These results suggest that adaptive selection of HIV nef in brain may reflect altered requirements for efficient replication in macrophages and brain-specific immune selection pressures.

Background

The HIV-1 accessory protein Nef is an important determinant of lentiviral pathogenicity that contributes to disease progression by enhancing viral replication and other poorly understood mechanisms. Nef mediates diverse functions including downmodulation of cell surface CD4 and MHC Class I, enhancement of viral infectivity, and enhancement of T cell activation. Nef interacts with a multiprotein signaling complex that includes Src family kinases, Vav1, CDC42, and activated PAK2 (p21-activated kinase 2). Although previous studies have attempted to identify a biological role for the Nef-PAK2 signaling complex, the importance of this complex and its constituent proteins in Nef function remains unclear.

Results

Here, we show that Nef mutants defective for PAK2-association, but functional for CD4 and MHC Class I downmodulation and infectivity enhancement, are also defective for the ability to enhance viral replication in primary T cells that are infected and subsequently activated by sub-maximal stimuli (1 μg/ml PHA-P). In contrast, these Nef mutants had little or no effect on HIV-1 replication in T cells activated by stronger stimuli (2 μg/ml PHA-P or anti-CD3/CD28-coated beads). Viruses bearing wild-type Nefs, but not Nef mutants defective for PAK2 association, enhanced NFAT and IL2 receptor promoter activity in Jurkat cells. Moreover, expression of wild-type Nefs, but not mutant Nefs defective for PAK2 association, was sufficient to enhance responsiveness of primary CD4 and CD8 T cells to activating stimuli in Nef-expressing and bystander cells. siRNA knockdown of PAK2 in Jurkat cells reduced NFAT activation induced by anti-CD3/CD28 stimulation both in the presence and absence of Nef, and expression of a PAK2 dominant mutant inhibited Nef-mediated enhancement of CD25 expression.

Conclusion

Nef-mediated enhancement of cellular activation and viral replication in primary T cells is dependent on PAK2 and on the strength of the activating stimuli, and correlates with the ability of Nef to associate with PAK2. PAK2 is likely to play a role in Nef-mediated enhancement of viral replication and immune activation in vivo.

To study the role of Src family tyrosine kinases in infection with human immunodeficiency virus type 1 (HIV-1), we constructed an Hck mutant, HckN, that hinders signaling from wild-type Hck. HIV-1 produced in HckN-expressing cells was significantly less infectious to HeLa–CD4–LTR–β-gal (MAGI) cells than HIV-1 produced in mock-transfected cells. The inhibitory effect of HckN was compensated for by the expression of vesicular stomatitis virus G protein. Finally, we found that the HIV-1 produced in the HckN-expressing cells entered into the cells less efficiently than did the control HIV-1. These results suggest that the Src family tyrosine kinases regulate entry of HIV-1 into target cells.

It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.

Background

HIV-1 Nef is a multifunctional protein required for full pathogenicity of the virus. As Nef has no known enzymatic activity, it necessarily functions through protein-protein interaction interfaces. A critical Nef protein interaction interface is centered on its polyproline segment (P69VRPQVPLRP78) which contains the helical SH3 domain binding protein motif, PXXPXR. We hypothesized that any Nef-SH3 domain interactions would be lost upon mutation of the prolines or arginine of PXXPXR. Further, mutation of the non-motif “X” residues, (Q73, V74, and L75) would give altered patterns of inhibition for different Nef/SH3 domain protein interactions.

Results

We found that mutations of either of the prolines or the arginine of PXXPXR are defective for Nef-Hck binding, Nef/activated PAK2 complex formation and enhancement of virion infectivity (EVI). Mutation of the non-motif “X” residues (Q, V and L) gave similar patterns of inhibition for Nef/activated PAK2 complex formation and EVI which were distinct from the pattern for Hck binding. These results implicate an SH3 domain containing protein other than Hck for Nef/activated PAK2 complex formation and EVI. We have also mutated Nef residues at the N-and C-terminal ends of the polyproline segment to explore interactions outside of PXXPXR. We discovered a new locus GFP/F (G67, F68, P69 and F90) that is required for Nef/activated PAK2 complex formation and EVI.

MHC Class I (MHCI) downregulation was only partially inhibited by mutating the PXXPXR motif residues, but was fully inhibited by mutating the C-terminal P78. Further, we observed that MHCI downregulation strictly requires G67 and F68. Our mutational analysis confirms the recently reported structure of the complex between Nef, AP-1 μ1 and the cytoplasmic tail of MHCI, but does not support involvement of an SH3 domain protein in MHCI downregulation.

Conclusion

Nef has evolved to be dependent on interactions with multiple SH3 domain proteins. To the N- and C- terminal sides of the polyproline helix are multifunctional protein interaction sites. The polyproline segment is also adapted to downregulate MHCI with a non-canonical binding surface. Our results demonstrate that Nef polyproline helix is highly adapted to directly interact with multiple host cell proteins.

Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program.

HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1–mediated MHC-I down-regulation in primary CD4+ T-cells. 2c did not interfere with the PACS-2–dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1–dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4+ T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P104A/P107A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.

Signaling cascades are managed in time and space by interactions between and among proteins. These interactions are often aided by adaptor proteins, which guide enzyme-substrate pairs into proximity. Miniature proteins are a class of small, well-folded protein domains possessing engineered binding properties. Here we make use of two miniature proteins with complementary binding properties to create a synthetic adaptor protein that effectively redirects a ubiquitous signaling event: tyrosine phosphorylation. We report that miniature-protein-based adaptor 3 uses templated catalysis to redirect the Src family kinase Hck to phosphorylate hDM2, a negative regulator of the p53 tumor suppressor and a poor Hck substrate. Phosphorylation occurs with multiple turnover and at a single site targeted by c-Abl kinase in the cell.

Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef+ wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.

Background

HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown.

Results

To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery.

Conclusions

Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.

Interleukin-6 (IL-6) induces the activation of the Src family kinase Hck, which is associated with the IL-6 receptor β-chain, gp130. Here we describe the identification of an “acidic” domain comprising amino acids 771 to 811 of gp130 as a binding region for Hck, which mediates proliferative signaling. The deletion of this region of gp130 (i.e., in deletion mutant d771-811) resulted in a significant reduction of Hck kinase activity and cell proliferation upon stimulation of gp130 compared to wild-type gp130. In addition, d771-811 disrupted the growth factor-stimulated activation of Erk and the dephosphorylation of Pyk2. Based on these findings, we propose a novel, acidic domain of gp130, which is responsible for the activation of Hck, Erk, and Pyk2 and signals cell proliferation upon growth factor stimulation.

The nef gene product of human immunodeficiency virus type 1 (HIV-1) is important for the induction of AIDS, and key to its function is its ability to manipulate T-cell function by targeting cellular signal transduction proteins. We reported that Nef coprecipitates a multiprotein complex from cells which contains tumor suppressor protein p53. We now show that Nef interacts directly with p53. Binding assays showed that an N-terminal, 57-residue fragment of Nef (Nef 1-57) contains the p53-binding domain. Nef also interacted with p53 during HIV-1 infection in vitro. As p53 plays a critical role in the regulation of apoptosis, we hypothesized that Nef may alter this process. Nef inhibited UV light-induced, p53-dependent apoptosis in MOLT-4 cells, with Nef 1-57 being as effective as its full-length counterpart. The inhibition by Nef of p53 apoptotic function is most likely due its observed ability to decrease p53 protein half-life and, consequently, p53 DNA binding activity and transcriptional activation. These data show that HIV-1 Nef may augment HIV replication by prolonging the viability of infected cells by blocking p53-mediated apoptosis.

Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1β (IL-1β), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn−/− mice 3 days postchallenge, although the level was less than that observed in Hck−/− Fgr−/− Lyn−/− mice. A correlate to augmented clearance of P. murina in Hck−/− Fgr−/− Lyn−/− mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.

Protein tyrosine kinases are thought to participate in signal transduction pathways in a variety of cell types. Recent studies have identified a new src family protein tyrosine kinase (hck) that is preferentially expressed in myeloid cells. To examine the hypothesis that this kinase may regulate myeloid cell activity, antisera were generated that define the 59-kD product of the hck gene. Functional activation of human cultured macrophages with LPS augmented the expression of hck transcripts and of p59hck, but decreased the level of transcripts encoded by the closely related c-fgr protooncogene. Thus these two structurally similar src family kinases almost certainly subserve distinct functions. Reasoning from the known properties of the src family protein tyrosine kinases, it is likely that the products of these two protooncogenes assist in regulating the behavior of activated phagocytes.