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...

M-CSF is a cytokine essential for both the proliferation and differentiation of monocytes/macrophages. In this study, we established a new M-CSF-mediated differentiation-inducing system, and examined how the level and duration of the activation of ERK preceded M-CSF-mediated differentiation. TF-1-fms human leukemia cells rapidly proliferated in response to M-CSF. However, in the presence of a phorbol ester, TPA, TF-1-fms cells definitely switched their responsiveness to M-CSF from proliferation to differentiation, as evidenced by a more drastic morphological change and the appearance of cells with a higher level of phagocytic activity. In TF-1-fms cells expressing HIV-1 Nef protein in a conditionally active-manner, both M-CSF-mediated proliferation and M-CSF/TPA-mediated differentiation were inhibited by the activation of Nef. The Nef-active cells showed perturbed patterns of ERK activation. Under the proliferation-inducing conditions (TPA-free), parental or Nef-inactive cells showed modest ERK activation following M-CSF stimulation, whereas Nef-active cells showed an earlier and transient ERK activation, despite a decrease in their proliferation rate. Under the differentiation-inducing conditions, parental or Nef-inactive cells showed increased and prolonged ERK activation following M-CSF stimulation, whereas Nef-active cells showed transient ERK activation. These results supported the idea that the increased and prolonged ERK activation led to M-CSF-mediated macrophage differentiation but not to proliferation.

The interaction between HIV-1 Nef and the Src kinase Hck in macrophages has been shown to accelerate the progression to AIDS. We previously showed that Nef disturbed the N-glycosylation/trafficking of Fms, a cytokine receptor essential for maintaining macrophages in an anti-inflammatory state, in an Hck-dependent manner. Here, we show the underlying molecular mechanism of this effect. Using various Hck isoforms and their mutants and Golgi-targeting Hck mutants, we confirmed that Hck activation at the Golgi causes the Nef-induced Fms N-glycosylation defect. Importantly, we found that both the co-expression of Nef and Hck and the expression of a Golgi-targeted active Hck mutant caused alterations in the distribution of GM130, a Golgi protein that was shown to be required for efficient protein glycosylation. Moreover, the activation of Hck at the Golgi caused strong serine phosphorylation of the GM130-interacting Golgi structural protein GRASP65, which is known to induce Golgi cisternal unstacking. Using pharmacological inhibitors, we also found that the activation of Hck at the Golgi followed by the activation of the MAP kinase ERK-GRASP65 cascade is involved in the Fms N-glycosylation defect. These results suggest that Nef perturbs the structure and signaling of the Golgi by activating Hck at the Golgi, and thereby, induces the N-glycosylation/trafficking defect of Fms, which is in line with the idea that Src family kinases are crucial Golgi regulators.

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