Sulfotyrosines of the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor promote tumorigenesis through autocrine activation

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) is a bona fide signaling molecule that is implicated in KSHV-associated malignancies. Whereas vGPCR activates specific cellular signaling pathways in a chemokine-independent fashion, vGPCR binds a broad spectrum of CC and CXC chemokines, and the roles of chemokines in vGPCR tumorigenesis remain poorly understood. We report here that vGPCR is posttranslationally modified by sulfate groups at tyrosine residues within its N-terminal extracellular domain. A chemokine-binding assay demonstrated that the tyrosine sulfate moieties were critical for vGPCR association with GRO-alpha (an agonist) but not with IP-10 (an inverse agonist). A sulfated peptide corresponding to residues 12 through 33 of vGPCR, but not the unsulfated equivalent, partially inhibited vGPCR association with GRO-alpha. Although the vGPCR variant lacking sulfotyrosines activated downstream signaling pathways, the ability of the unsulfated vGPCR variant to induce tumor growth in nude mice was significantly diminished. Furthermore, the unsulfated vGPCR variant was unable to induce the secretion of proliferative cytokines, some of which serve as vGPCR agonists. This implies that autocrine activation by agonist chemokines is critical for vGPCR tumorigenesis. Indeed, GRO-alpha increased vGPCR-mediated AKT phosphorylation and vGPCR tumorigenesis in a sulfotyrosine-dependent manner. Our findings support the conclusion that autocrine activation triggered by chemokine agonists via sulfotyrosines is necessary for vGPCR tumorigenesis, thereby providing a rationale for future therapeutic design targeting the tumorigenic vGPCR.

FIG. 1.

The KSHV vGPCR efficiently incorporates radiolabeled sulfate. (A) The KSHV vGPCR, human cytomegalovirus (HCMV) US28, and murine gamma herpesvirus 68 (γHV68) vGPCRs contain a putative tyrosine sulfation motif within their N-terminal extracellular domains. These putative tyrosine sulfation motifs are underlined. (B) Wild-type (WT) vGPCR efficiently incorporates radiolabeled sulfate. HEK293T cells were transfected with plasmids encoding wild-type vGPCR, a variant in which tyrosines 26 and 28 had been altered to aspartic acid (designated YYDD), or a plasmid encoding CCR5 that serves as a positive control. Cells were split, labeled with [³⁵S]methionine-cysteine (Met/Cys) or [³⁵S]sulfate (SO₄), and lysed. Cell lysates were incubated with anti-Flag or C9 anti-CCR5 antibody. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. (C) Experiment similar to panel B except that cells were cotransfected with plasmids encoding wild-type vGPCR or the YYDD variant and TPST2 as indicated. (D) Experiment similar to panel B except that cells were transfected with plasmids encoding wild-type vGPCR, YYDD, and Y26D variants as indicated. Numbers at the bottom indicate relative ratios of sulfated vGPCR normalized against [³⁵S]Met-Cys-labeled vGPCR.

FIG. 2.

Sulfotyrosines of vGPCR at residue 26 and 28 are necessary for vGPCR association with GRO-α but not for vGPCR association with IP-10. (A) Control NIH 3T3 (Vector) and NIH 3T3 cells stably expressing HA-tagged wild-type vGPCR (WT) or the YYDD variant were fixed, permeabilized (Perm) or not permeabilized (Non-Perm), incubated with anti-HA (green) and anti-calreticulin (red) (as a permeability control) antibodies, and analyzed by immunofluorescence microscopy. (B) NIH 3T3 cells stably expressing the HA-tagged wild-type vGPCR or the YYDD variant were incubated with anti-HA antibody, fixed, and analyzed by flow cytometry. (C) NIH 3T3 cells stably expressing wild-type vGPCR or the YYDD variant were incubated with 20 pM [¹²⁵I]GRO-α and the indicated amounts of unradiolabeled human GRO-α competitor. Cells were washed, and bound [¹²⁵I]GRO-α was quantitated by scintillation counting. (D) Experiment similar to panel C except that cells were incubated with 20 pM [¹²⁵I]IP-10 and the indicated amounts of unradiolabeled human IP-10. For both panels C and D, data represent the mean of at least three independent experiments, and error bars denote standard deviations. The binding of [¹²⁵I]GRO-α and [¹²⁵I]IP-10 to NIH 3T3/Vector cells (endogenous receptors) is approximately 5% ± 2% of that of NIH 3T3/vGPCR cells.

FIG. 3.

The sulfated peptide but not the unsulfated equivalent partially inhibits vGPCR association with GRO-α. (A) The sulfated peptide corresponding to residues 11 to 33 of the vGPCR N terminus is shown. The peptide carrying sulfate groups at positions 26 and 28 (denoted by *) and its unsulfated equivalent were synthesized and purified. (B) NIH 3T3 cells stably expressing wild-type vGPCR were incubated with 20 pM [¹²⁵I]GRO-α and the indicated amount of a peptide corresponding to residues 12 through 33 of the vGPCR with the sulfated tyrosines at position 26 and 28 (vG-N-S) or with unmodified tyrosines at these positions (vG-N), or cells were incubated with buffer only; cells were washed, and bound [¹²⁵I]GRO-α was quantitated by scintillation counting. Data represent the mean of four independent experiments, and error bars denote standard deviations. *, P < 0.01 relative to vG-N as calculated by a Student's t test.

FIG. 4.

Sulfotyrosines of vGPCR are not critical for its constitutive activity. (A) HEK293T cells were cotransfected with NFAT-luciferase reporter construct (50 ng), β-galactosidase reporter construct (50 ng), and plasmids (150 ng) containing wild-type vGPCR or the YYDD variant. Cells were harvested at 36 h posttransfection, and luciferase and β-galactosidase activities were determined. Data represent the mean of at least three independent experiments, and error bars denote standard deviation. (B) Experiment similar to panel A except that cells were transfected with an NF-κB-luciferase reporter construct. (C) NIH 3T3 cells stably expressing wild-type vGPCR or the YYDD variant were lysed with buffer containing 1% NP-40. Precipitated AKT was incubated with purified GST-GSK and [γ³²P]ATP, and radiolabeled GST-GSK was analyzed by SDS-PAGE and autoradiography. Precipitated AKT was also analyzed by immunoblotting with anti-AKT antibody (bottom panel). Numbers indicate the relative intensity of phosphorylated GST-GSKp normalized to total AKT. Data represent three independent experiments.

FIG. 5.

Sulfotyrosines are essential for vGPCR tumorigenesis. (A) Relative expression of wild-type vGPCR and the YYDD variant in stable NIH 3T3 (1 × 10⁷) cells was analyzed by immunoprecipitation and immunoblotting with anti-HA antibody. For loading controls, whole-cell lysates (WCL) were analyzed by immunoblotting with anti-actin antibody (bottom). (B) Exponentially proliferating NIH 3T3/vector (Vec), NIH 3T3/vGPCR (WT), and NIH 3T3/YYDD cells were counted every 48 h to calculate their doubling times. P values as indicated were calculated with Student's t test relative to NIH 3T3/Vector cells. (C and D) NIH 3T3 cells stably expressing wild-type vGPCR or the YYDD variant, together with NIH 3T3 cells (1:1), were inoculated into the flanks of 3- to 5-week-old nude mice. Nude mice were sacrificed at 3 weeks after inoculation and photographed (C), and the tumor weight was determined (D). (E) Total RNA extracted from tumors derived from NIH 3T3/vGPCR cells was used for reverse transcription-PCR (RT-PCR) analyses with primers specific for vGPCR and β-actin. N, negative control (total RNA of tumor tissue derived from NIH 3T3/hGRO-α); P, positive control with a plasmid containing vGPCR. (F) Amounts of secreted IL-6, MIP-2, and TNF-α in the supernatant of SVEC/vector (lane 1), SVEC/vGPCR (lane 2), or SEVC/YYDD (lane 3) cells were determined by a multiplex cytokine array. Data represent mean values, and error bars denote standard deviations, with P values relative to SVEC/vector cells as calculated by Student's t test. *, P < 0.04.

FIG. 6.

GRO-α promotes vGPCR signaling and tumorigenesis in a sulfotyrosine-dependent manner. (A and B) SVECs stably expressing wild-type vGPCR (WT) or the YYDD variant were starved in serum-free conditioned medium and left untreated or were treated with GRO-α for 15 min (A) or IP-10 for 30 min (B). Whole-cell lysates were analyzed by immunoblotting with antibodies to phosphorylated AKT (p-AKT), total AKT (AKT), or β-actin (as a loading control). Numbers indicate the relative intensity of p-AKT by densitometry. Data represent three independent experiments. (C) NIH 3T3 cells stably expressing wild-type vGPCR or the YYDD variant were mixed with NIH 3T3 cells stably expressing human GRO-α or control NIH 3T3 stable cells and inoculated into nude mice. Mice were sacrificed at 3 weeks postinoculation, and tumor size was determined. P values between any two groups were calculated by Student's t test. *, P < 0.05. (D) vGPCR mRNA in tumors derived from NIH 3T3/hGRO-α mixed with NIH 3T3/vGPCR cells (lanes 1 to 4) or NIH 3T3/YYDD cells (lanes 5 to 8) was determined by RT-PCR with total RNA and primers specific for vGPCR, hGRO-α, and β-actin. (E) A hypothetical model of sulfotyrosines and chemokine agonists in vGPCR signaling and tumorigenesis. Chemokine agonists (GRO-α) bind vGPCR in a sulfotyrosine-dependent manner. Chemokine association promotes vGPCR signaling, leading to further production of proinflammatory cytokines including vGPCR chemokine agonists. Secreted cytokines and growth factors stimulate the proliferation of vGPCR-expressing cells in an autocrine manner and bystander cells in a paracrine manner. The autocrine feedback by chemokine agonists is critical for vGPCR tumorigenesis in vivo.