Kaposi's sarcoma-associated herpesvirus-encoded G protein-coupled receptor ORF74 constitutively activates p44/p42 MAPK and Akt via G(i) and phospholipase C-dependent signaling pathways

Abstract

The G protein-coupled receptor encoded by Kaposi's sarcoma-associated herpesvirus, also referred to as ORF74, has been shown to stimulate oncogenic and angiogenic signaling pathways in a constitutively active manner. The biochemical routes linking ORF74 to these signaling pathways are poorly defined. In this study, we show that ORF74 constitutively activates p44/p42 mitogen-activated protein kinase (MAPK) and Akt via G(i)- and phospholipase C (PLC)-mediated signaling pathways. Activation of Akt by ORF74 appears to be phosphatidylinositol 3-kinase (PI3-K) dependent but, interestingly, is also mediated by activation of protein kinase C (PKC) and p44/p42 MAPK. ORF74 may signal to Akt via p44/p42 MAPK, which can be activated by G(i), through activation of PI3-K or through PKC via the PLC pathway. Signaling of ORF74 to these proliferative and antiapoptotic signaling pathways can be further modulated positively by growth-related oncogene (GROalpha/CXCL1) and negatively by human gamma interferon-inducible protein 10 (IP-10/CXCL10), thus acting as an agonist and an inverse agonist, respectively. Despite the ability of the cytomegalovirus-encoded chemokine receptor US28 to constitutively activate PLC, this receptor does not increase phosphorylation of p44/p42 MAPK or Akt in COS-7 cells. Hence, ORF74 appears to signal through a larger diversity of G proteins than US28, allowing it to couple to proliferative and antiapoptotic signaling pathways. ORF74 can therefore be envisioned as an attractive target for novel treatment of Kaposi's sarcoma.

FIG. 1.

ORF74-mediated induction of inositol phosphate accumulation. (A) COS-7 cells (10⁶ cells) were transiently transfected with increasing amounts of cDNA encoding ORF74 or a single amount of cDNA encoding US28 (2 μg/10⁶ cells). At 48 h after transfection, inositol phosphate accumulation was measured. (Inset) Stimulation and inhibition of ORF74-induced inositol phosphate accumulation by the agonist GROα (100 nM, 2 h) and the inverse agonist IP-10 (100 nM, 2 h), respectively. (B) COS-7 cells (10⁶ cells) were transiently transfected with increasing amounts of cDNA encoding ORF74, and ¹²⁵I-IL-8 binding was performed as described in Materials and Methods. Data are presented as percentages of the values of control (mock-transfected) cells. Representative experiments performed in triplicate are shown. Each experiment was done at least three times.

FIG. 2.

ORF74-induced activation of p44/p42 MAPK. (A) Effect of ORF74 (0.5 μg/10⁶ cells) and US28 (2.0 μg/10⁶ cells) on the basal phosphorylation of p44/p42 MAPK in transiently transfected COS-7 cells as determined by Western blot analysis using specific anti-phosho-p44/p42 (P-p44/p42) antibodies. Phosphorylation was quantified by chemiluminescence and corrected for total MAPK (T-p44/p42) expression on stripped blots. Data are presented as fold control (mock-transfected) cell values. (B) Transiently transfected COS-7 cells were incubated with IP-10 (100 nM, 48 h) or with GROα (30 nM, 5 min) before the cells were lysed. (C) Involvement of the G_i signaling pathway in ORF74-induced p44/p42 MAPK phosphorylation. COS-7 cells transiently transfected with ORF74 alone (0.5 μg/10⁶ cells) or cotransfected with Gα_t (1 μg/10⁶ cells) were grown in serum-free medium in the presence or absence of PTX (100 ng/ml, 48 h) or the PI3-K inhibitor wortmannin (WM) (100 nM, 2 h) before lysis and Western blot analysis. Data are presented as the percentage of ORF74-induced p44/p42 MAPK phosphorylation in at least three independent experiments, and representative blots are shown. (D) Role of the PLC signaling pathway in constitutive phosphorylation of p44/p42 MAPK by ORF74. COS-7 cells transiently transfected with ORF74 (0.5 μg/10⁶ cells) were grown in serum-free medium in the presence or absence of the MEK1/2 inhibitor U0126 (10 μM, 2 h), the PKC inhibitor bisindolylmaleimide I (Bis; 1 μM, 2 h), or the phorbol ester PMA (100 nM, 48 h) before lysis and Western blot analysis. Data are presented as the percentage of ORF74-induced p44/p42 MAPK phosphorylation in at least two independent experiments, and representative blots are shown.

FIG.3.

ORF74-induced activation of Akt. (A) Effects of ORF74 (0.5 μg/10⁶ cells) and US28 (2.0 μg/10⁶ cells) on the basal phosphorylation of Akt in transiently transfected COS-7 cells as determined by Western blot analysis using specific anti-phosho-Akt (P-Akt) antibodies. Phosphorylation was quantified by chemiluminescence and corrected for total Akt (T-Akt) expression on stripped blots. (B) Regulation of the ORF74-induced constitutive phosphorylation of Akt by IP-10 (100 nM, 48 h) and GROα (30 nM, 5 min) in transiently transfected COS-7 cells. Data are presented as fold control (mock-transfected) cell values. Representative blots are shown. Each experiment was done at least three times. (C) Involvement of the G_i signaling pathway in ORF74-induced constitutive Akt phosphorylation. COS-7 cells transiently transfected with ORF74 alone (0.5 μg/10⁶ cells) or cotransfected with Gα_t (1 μg/10⁶ cells) were grown in serum-free medium in the presence or absence of PTX (100 ng/ml, 48 h) or the PI3-K inhibitor wortmannin (WM; 100 nM, 2 h) before lysis and Western blot analysis. Data are presented as the percentage of ORF74-induced Akt phosphorylation of at least two independent experiments, and representative blots are shown. (D) Akt phosphorylation by mutationally activated H-Ras and via activation of PKC. COS-7 cells were transiently transfected with mutationally activated H-Ras (1 μg/10⁶ cells) or empty vector and grown in serum-free medium for 48 h. PMA-treated cells were incubated with PMA (1 μM) 5 min before lysis. (E) Role of the PLC signaling pathway in constitutive phosphorylation of Akt by ORF74. COS-7 cells transiently transfected with ORF74 (0.5 μg/10⁶ cells) were grown in serum-free medium in the presence or absence of the MEK1/2 inhibitor U0126 (10 μM, 2 h) or the phorbol ester PMA (100 nM, 48 h) before lysis and Western blot analysis. Data are presented as the percentage of ORF74-induced Akt phosphorylation in at least two independent experiments, and a representative blot is shown.

FIG. 4.

Hypothetical scheme of signal transduction pathways activated by ORF74. Based on data from this study, activation of p44/p42 MAPK by ORF74 seems to be G_i mediated via G_βγ subunits involving PI3-K and PLC dependent via activation of PKC, presumably at the level of Raf-1 (39). Activation of Akt by ORF74 appears to be PI3-K dependent, likely via G_βγ from G_i, and seems also to be mediated by activation of the PKC and Ras-Raf pathways.