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Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.

Davis CB, Dikic I, Unutmaz D, Hill CM, Arthos J, Siani MA, Thompson DA, Schlessinger J, Littman DR - J. Exp. Med. (1997)

Bottom Line: In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2.The response requires CXCR4 and CCR5 to be accessible on the cell surface.The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathogenesis, Skirball Institute for Biomolecular Medicine, NYU Medical Center 10016, USA. davis@saturn.med.nyu.edu

ABSTRACT
Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

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Tyrosine phosphorylation of Pyk2 in response to chemokines. (a and b) HL60 promyelocytic leukemia cells were incubated with 500 nM  RANTES (a) or SDF-1α (b) at 37°C for the indicated times before lysis. (+PTx) Cells were pretreated with pertussis toxin before incubation with  chemokines. Pyk2 was immunoprecipitated (IP) with rabbit antibodies against Pyk2 and blotted with antiphosphotyrosine (anti-PTyr) or anti-Pyk2 antibodies. (c) DU6 CD4+ T cells were incubated with RANTES, MIP-1β, or SDF-1α (500 nM each) for the indicated times before lysis and processing as  in a and b.
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Figure 1: Tyrosine phosphorylation of Pyk2 in response to chemokines. (a and b) HL60 promyelocytic leukemia cells were incubated with 500 nM RANTES (a) or SDF-1α (b) at 37°C for the indicated times before lysis. (+PTx) Cells were pretreated with pertussis toxin before incubation with chemokines. Pyk2 was immunoprecipitated (IP) with rabbit antibodies against Pyk2 and blotted with antiphosphotyrosine (anti-PTyr) or anti-Pyk2 antibodies. (c) DU6 CD4+ T cells were incubated with RANTES, MIP-1β, or SDF-1α (500 nM each) for the indicated times before lysis and processing as in a and b.

Mentions: Binding of chemokines to their cognate receptors is often assayed by measuring calcium mobilization (13). Treatment of several CD4+ cell lines with HIV-1 envelope failed to trigger a calcium response, even though such responses could be elicited by chemokines (Davis, C.B., unpublished data). Since chemokine receptor stimulation can have biological effects in the absence of measurable calcium mobilization (14), we searched for other signaling molecules that could be positioned downstream of the chemokine receptors. Pyk2 is a protein tyrosine kinase, related to p125-FAK, and is expressed primarily in cells of the neuronal and hematopoietic lineages (15). Pyk2 is phosphorylated on tyrosine after ligand binding to G protein–coupled receptors or treatment with agents that elevate intracellular Ca2+ concentration (9, 15). Tyrosine phosphorylation of Pyk2 in turn leads to stimulation of kinase activity and phosphorylation of exogenous substrates (9, 15). Since CXCR4 and CCR5 are G protein–coupled receptors, we examined the possibility that their ligands could activate Pyk2. Human promyelocytic leukemia cells (HL60) or CD4+ T cells (DU6) were treated with the following chemokines: RANTES, which binds primarily to CCR1, CCR4, and CCR5; MIP-1β, which binds to CCR5; and SDF-1α, which binds to CXCR4 (13). RANTES and SDF-1α, but not MIP-1β, rapidly induced Pyk2 phosphorylation in HL60 cells (Fig. 1, a and b, and Dikic, I., unpublished data). The response peaked at ∼30 s and had declined to basal levels by 5 min. Pretreatment of HL60 cells with pertussis toxin ablated the Pyk2 response, suggesting that Pyk2 phosphorylation is mediated via Gi proteins (Fig. 1, a and b) (16). The RANTES effect is probably mediated through CCR1 or CCR4, since HL60 cells failed to mobilize calcium to MIP-1β (Davis, C.B., unpublished data), consistent with the lack of CCR5. In contrast, the DU6 cell line did mobilize calcium in response to MIP-1β (Davis, C.B., unpublished data), indicating expression of CCR5. Indeed, MIP-1β, as well as RANTES and SDF-1α, induced Pyk2 phosphorylation in DU6 cells with kinetics comparable to those in HL60 cells (Fig. 1 c).


Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.

Davis CB, Dikic I, Unutmaz D, Hill CM, Arthos J, Siani MA, Thompson DA, Schlessinger J, Littman DR - J. Exp. Med. (1997)

Tyrosine phosphorylation of Pyk2 in response to chemokines. (a and b) HL60 promyelocytic leukemia cells were incubated with 500 nM  RANTES (a) or SDF-1α (b) at 37°C for the indicated times before lysis. (+PTx) Cells were pretreated with pertussis toxin before incubation with  chemokines. Pyk2 was immunoprecipitated (IP) with rabbit antibodies against Pyk2 and blotted with antiphosphotyrosine (anti-PTyr) or anti-Pyk2 antibodies. (c) DU6 CD4+ T cells were incubated with RANTES, MIP-1β, or SDF-1α (500 nM each) for the indicated times before lysis and processing as  in a and b.
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Figure 1: Tyrosine phosphorylation of Pyk2 in response to chemokines. (a and b) HL60 promyelocytic leukemia cells were incubated with 500 nM RANTES (a) or SDF-1α (b) at 37°C for the indicated times before lysis. (+PTx) Cells were pretreated with pertussis toxin before incubation with chemokines. Pyk2 was immunoprecipitated (IP) with rabbit antibodies against Pyk2 and blotted with antiphosphotyrosine (anti-PTyr) or anti-Pyk2 antibodies. (c) DU6 CD4+ T cells were incubated with RANTES, MIP-1β, or SDF-1α (500 nM each) for the indicated times before lysis and processing as in a and b.
Mentions: Binding of chemokines to their cognate receptors is often assayed by measuring calcium mobilization (13). Treatment of several CD4+ cell lines with HIV-1 envelope failed to trigger a calcium response, even though such responses could be elicited by chemokines (Davis, C.B., unpublished data). Since chemokine receptor stimulation can have biological effects in the absence of measurable calcium mobilization (14), we searched for other signaling molecules that could be positioned downstream of the chemokine receptors. Pyk2 is a protein tyrosine kinase, related to p125-FAK, and is expressed primarily in cells of the neuronal and hematopoietic lineages (15). Pyk2 is phosphorylated on tyrosine after ligand binding to G protein–coupled receptors or treatment with agents that elevate intracellular Ca2+ concentration (9, 15). Tyrosine phosphorylation of Pyk2 in turn leads to stimulation of kinase activity and phosphorylation of exogenous substrates (9, 15). Since CXCR4 and CCR5 are G protein–coupled receptors, we examined the possibility that their ligands could activate Pyk2. Human promyelocytic leukemia cells (HL60) or CD4+ T cells (DU6) were treated with the following chemokines: RANTES, which binds primarily to CCR1, CCR4, and CCR5; MIP-1β, which binds to CCR5; and SDF-1α, which binds to CXCR4 (13). RANTES and SDF-1α, but not MIP-1β, rapidly induced Pyk2 phosphorylation in HL60 cells (Fig. 1, a and b, and Dikic, I., unpublished data). The response peaked at ∼30 s and had declined to basal levels by 5 min. Pretreatment of HL60 cells with pertussis toxin ablated the Pyk2 response, suggesting that Pyk2 phosphorylation is mediated via Gi proteins (Fig. 1, a and b) (16). The RANTES effect is probably mediated through CCR1 or CCR4, since HL60 cells failed to mobilize calcium to MIP-1β (Davis, C.B., unpublished data), consistent with the lack of CCR5. In contrast, the DU6 cell line did mobilize calcium in response to MIP-1β (Davis, C.B., unpublished data), indicating expression of CCR5. Indeed, MIP-1β, as well as RANTES and SDF-1α, induced Pyk2 phosphorylation in DU6 cells with kinetics comparable to those in HL60 cells (Fig. 1 c).

Bottom Line: In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2.The response requires CXCR4 and CCR5 to be accessible on the cell surface.The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathogenesis, Skirball Institute for Biomolecular Medicine, NYU Medical Center 10016, USA. davis@saturn.med.nyu.edu

ABSTRACT
Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

Show MeSH
Related in: MedlinePlus