The cellular kinase binding motifs (PxxP and RR) in human immunodeficiency virus type 1 Nef protein are dispensable for producer-cell-dependent enhancement of viral entry.
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We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) Nef is required for enhancing viral infectivity by increasing the efficiency of viral entry in a producer-cell-dependent manner, suggesting the possible involvement of a cellular factor(s) in the enhancement of viral entry. Moreover, it has been reported that a proline-rich (PxxP) motif and an Arg-Arg (RR) motif in HIV-1 Nef bind to the SH3 domain of the Src-family tyrosine kinase Hck and to a serine/threonine kinase, respectively. To address whether these cellular kinase binding motifs, PxxP and RR, could be involved in virus producer-cell-dependent enhancement of viral entry, we constructed two nef mutant proviral clones in which these motifs were mutated. The results show that the HIV-1 Nef PxxP motif, which significantly influenced viral infectivity, and the RR motif, which modestly affected viral infectivity, were both dispensable for enhanced viral entry, thus suggesting that another interaction of Nef with a cellular factor(s) is involved in the efficiency of viral entry. (+info)
Urokinase receptor-dependent and -independent p56/59(hck) activation state is a molecular switch between myelomonocytic cell motility and adherence.
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Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo. (+info)
Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor.
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The crystal structure of the autoinhibited form of Hck has been determined at 2.0 A resolution, in complex with a specific pyrazolo pyrimidine-type inhibitor, PP1. The activation segment, a key regulatory component of the catalytic domain, is unphosphorylated and is visualized in its entirety. Tyr-416, the site of activating autophosphorylation in the Src family kinases, is positioned such that access to the catalytic machinery is blocked. PP1 is bound at the ATP-binding site of the kinase, and a methylphenyl group on PP1 is inserted into an adjacent hydrophobic pocket. The enlargement of this pocket in autoinhibited Src kinases suggests a route toward the development of inhibitors that are specific for the inactive forms of these proteins. (+info)
Simian immunodeficiency virus and human immunodeficiency virus type 1 nef proteins show distinct patterns and mechanisms of Src kinase activation.
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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. (+info)
Activation of src tyrosine kinases by peroxynitrite.
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In this study, we demonstrate that the phosphorylation activity of five tyrosine kinases of the src family from both human erythrocytes (lyn, hck and c-fgr) and bovine synaptosomes (lyn and fyn) was stimulated by treatment with 30-250 microM peroxynitrite. This effect was not observed with syk, a non-src family tyrosine kinase. Treatment of kinase immunoprecipitates with 0.01-10 microM peroxynitrite showed that the interaction of these enzymes with the oxidant also activated the src kinases. Higher concentrations of peroxynitrite inhibited the activity of all kinases, indicating enzyme inactivation. The addition of bicarbonate (1.3 mM CO2) did not modify the upregulation of src kinases but significantly protected the kinases against peroxynitrite-mediated inhibition. Upregulation of src kinase activity by 1 microM peroxynitrite was 3.5-5-fold in erythrocytes and 1.2-2-fold in synaptosomes, but this could be the result, at least in part, of the higher basal level of src kinase activity in synaptosomes. Our results indicate that peroxynitrite can upregulate the tyrosine phosphorylation signal through the activation of src kinases. (+info)
SH2-kinase linker mutations release Hck tyrosine kinase and transforming activities in Rat-2 fibroblasts.
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Biochemical and structural studies of Src and related kinases demonstrate that two intramolecular interactions suppress kinase activity. These interactions involve binding of the SH2 domain to a phosphotyrosine residue in the C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by amino acids linking the SH2 and kinase domains. Recent studies have shown that high affinity interaction of the SH3 domain of Hck with the human immunodeficiency virus type I Nef protein activates Hck tyrosine kinase and biological activities, suggesting a mechanism that involves disruption of the SH3-linker interaction. To test the role of this interaction in the regulation of Hck kinase activity in living cells, we substituted alanines for prolines 225 and 228 in the linker region and observed that the resulting mutant (Hck-2PA) demonstrated strong transforming activity in a Rat-2 fibroblast focus-forming assay. Hck-2PA also exhibited elevated tyrosine kinase activity in terms of autophosphorylation, endogenous substrate phosphorylation, and in an in vitro kinase assay. The transforming and kinase activities of Hck-2PA were remarkably similar to those observed with a Hck mutant activated by Phe substitution of the conserved tail Tyr residue and with wild-type Hck following co-expression with human immunodeficiency virus Nef. Introduction of the 2PA and tail mutations into a single Hck expression construct did not increase kinase or transforming activity relative to the individual mutations. These data provide new evidence that SH3-linker interaction may represent the dominant mechanism controlling Hck tyrosine kinase activity in vivo. (+info)
Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages.
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Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages. (+info)
Activation of the PAK-related kinase by human immunodeficiency virus type 1 Nef in primary human peripheral blood lymphocytes and macrophages leads to phosphorylation of a PIX-p95 complex.
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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. (+info)