Interaction between Hck and HIV-1 Nef negatively regulates cell surface expression of M-CSF receptor

Abstract

Nef is a multifunctional pathogenetic protein of HIV-1, the interaction of which with Hck, a Src tyrosine kinase highly expressed in macrophages, has been shown to be responsible for the development of AIDS. However, how the Nef-Hck interaction leads to the functional aberration of macrophages is poorly understood. We recently showed that Nef markedly inhibited the activity of macrophage colonystimulating factor (M-CSF), a primary cytokine for macrophages. Here IntroductionHIV-1 infections lead to the development of AIDS by causing progressive degeneration of the immune system. [1][2][3] The main cellular targets of HIV-1 are CD4 ϩ T cells and macrophages, and the depletion of CD4 ϩ T cells caused by an infection is suggested to account for many aspects of the pathogenesis of HIV-1. [1][2][3] Meanwhile, a number of studies have revealed the functional aberration of HIV-1-infected macrophages. 4,5 Infected macrophages showed an altered profile of the production of cytokine/chemokines 4 or migratory capacity, 5 which might contribute to the uncontrolled homeostasis of the immune system. Indeed, functional analyses of HIV-1 Nef protein have revealed that macrophages as well as CD4 ϩ T cells play an important role in the development of AIDS.Nef is a 25-to 30-kDa protein with no enzymatic activity encoded by the HIV-1 genome. 6,7 Studies of HIV-1-infected patients have clearly demonstrated Nef to be a critical determinant of the development of AIDS: HIV-1 strains without an intact Nef gene were frequently isolated from nonprogressive long-term survivors. 8,9 Subsequent study of HIV-1 transgenic mice confirmed the pathogenetic activity of Nef: targeted expression of the entire coding sequence of HIV-1 in CD4 ϩ T cells and macrophages caused a severe AIDS-like disease in mice, which was completely abolished by the disruption of the Nef gene. 10 Importantly, only an amino acid substitution in the proline-rich (PxxP) motifs of Nef was sufficient to protect mice from the development of AIDS-like disease. 11 A number of studies have revealed that Nef interacts with a subset of cellular Src family tyrosine kinases, via the PxxP motifs. 12-15 The Nef PxxP motifs had an affinity for the Src homology (SH3) domain of Hck, Lyn, and possibly c-Src, but not of Fgr, Fyn, Lck, In particular, the interaction between the Nef PxxP motifs and the Hck SH3 domain was likely to be important, because the interaction caused the activation of Hck. [13][14][15] Indeed, a study with HIV-1 transgenic mice clearly demonstrated the importance of the Nef-Hck interaction for the development of AIDS: the appearance of the AIDS-like disease was significantly delayed when the HIV-1 transgenic mice expressing an intact Nef gene were crossed with an hck Ϫ/Ϫ background. 11 Given that Hck is expressed in macrophages but not in CD4 ϩ T cells, 16 the finding indicates that the Nef-Hck interaction in macrophages is at least in part responsible for the development of AIDS. However, little is known of the molecular mechanisms by which the Nef-Hck interaction c...

IL-34 and M-CSF share the receptor Fms but are not identical in biological activity and signal activation

“…15 Because TF-1 cells require granulocyte-macrophage (GM)-CSF for their growth but lack the expression of Fms, 16 neither M-CSF nor IL-34 stimulated the growth of TF-1 cells (data not shown). In contrast, both cytokines stimulated the growth of TF-1-fms cells (Figure 2a), which was abolished by the Fms kinase inhibitor GW2580 17 ( Figure 2b).…”

“…The insert cDNAs were subcloned into pcDNA3.1 expression vector (Invitrogen), and the expression plasmids were transfected into 293T cells using Lipofectamine 2000 as described previously. 16,24 The supernatants collected after 2 days were analyzed for the expression of Flagtagged proteins by western blotting with anti-Flag antibody (clone M2; Sigma) and the activity to stimulate the growth of TF-1-fms cells, or subjected to the ligand binding analysis. The sequence alignment was performed using the ClustalW algorithm.…”

“…The purity of the day 6-macrophages prepared by this method was routinely more than 95% when assessed by the expression of CD14, as reported previously. 16 The number of macrophages was monitored using MTT reagent (Sigma, St Louis, MO, USA). In brief, MTT was added to each well at a final concentration of 0.5 mg/ml, and 0.01 N HCl-isopropanol was added after the incubation for 4 h at 371C.…”

HIV-1 Nef Triggers Macrophage Fusion in a p61Hck- and Protease-Dependent Manner

“…The binding affinity between Nef and Hck is the highest among the SH3-mediated interactions (22,23); however, very little is known about the functional consequences of Hck activation by Nef in macrophages. It has been shown to interfere with M-CSF signaling (24,25) and to activate Stat3 (26). Moreover, in a long-term HIV-positive nonprogressor, Trible et al (27) identified an unusual Nef variant that failed to activate Hck, indicating that Hck/ Nef interaction is important for AIDS progression.…”

Proteus in the World of Proteins: Conformational Changes in Protein Kinases

“…The SH2 and SH3 domains of ZAP70 achieve this by binding to the C-lobe and reducing the flexibility of the hinge region [56]. In Hck [57], Src [58], and Abl nase regulation by SH domains is also illustrated by the Human Immunodeficiency Virus (HIV) which uses its protein Nef to activate human tyrosine kinase Hck by binding to its SH3 domain [60] and thus triggers pathways favorable for HIV pathogenicity [61].…”