shRNAs were used to stably knockdown Gq/11 or G12/13 in the control or vGPCR expressing HEK293A cells. intervention. system with either latent or lytic KSHV contamination. Human embryonic kidney cells (HEK293A) were latently infected with recombinant KSHV (rKSHV.219) and lytically induction was triggered by sodium butyrate treatment42. The expression of lytic KSHV genes upon induction was confirmed using quantitative PCR (Physique 2b, Physique S2a). In this system, we found that the latent contamination of KSHV only induced a minor YAP/TAZ activation. On the other hand, following expression of lytic KSHV genes, YAP/TAZ were strongly activated. YAP phosphorylation was dramatically decreased, as revealed by both immunoblotting using a phosphospecific (S127) YAP antibody or Phos-tag gels (Physique 2c). Consistently, TAZ protein level was increased upon lytic induction. Moreover, we observed a significant decrease of Lats1 Trigonelline Hydrochloride phosphorylation in its hydrophobic motif (threonine 1079, T1079), which positively correlates with Lats activity, RNF49 upon induction of lytic KSHV gene expression (Physique 2c), suggesting that Lats1 is usually inactivated by KSHV. Comparable results were observed when HEK293T cells were infected with KSHV (Physique S2b). Taken together, these results suggest that KSHV contamination, particularly the lytic KSHV gene expression, leads to Lats inhibition and therefore, activation of YAP/TAZ. The KSHV-encoded vGPCR activates YAP/TAZ Among the different lytic KSHV genes, the vGPCR is particularly interesting because it is a major factor contributing to KS pathogenesis10. Moreover, GPCR signaling has been shown to regulate the Hippo pathway43C45. KS is usually developed from lymphatic endothelium2,10,46. We established a SV40-immortalized murine endothelial cell line (SVEC) stably expressing HA-tagged vGPCR. Overexpression of vGPCR resulted in YAP dephosphorylation (Phos-tag) and increased YAP protein levels (Physique 3a). The overexpressed vGPCR resolved into multiple bands, which appeared to be due to protein glycosylation (Physique 3a, Physique S3a). vGPCR overexpression also increased TAZ protein levels. The effect of vGPCR on YAP/TAZ activation was further confirmed in additional cell lines, such as HEK293A and the human breast epithelial cells Trigonelline Hydrochloride (MCF10A) (Physique 3a). The YAP/TAZ protein elevation in response to vGPCR overexpression was not due to a change in mRNA levels (Physique S2b). However, when protein synthesis was inhibited in the presence of cycloheximide (CHX, an inhibitor for protein synthesis), YAP/TAZ protein stability was increased in vGPCR expressing cells compared to control cells (CHX; Physique 3b, Physique S3c). These results are consistent with previous findings that YAP/TAZ phosphorylation promotes ubiquitination and proteasome-mediated degradation37,39,47 and demonstrates that vGPCR increases YAP/TAZ protein levels by dephosphorylation and stabilization. Open in a separate window Physique 3 vGPCR activates YAP/TAZ. (a) vGPCR expression Trigonelline Hydrochloride increases YAP and TAZ protein levels. MCF10A, HEK293A, and SVEC cells stably expressing either the vector control or HA-vGPCR were serum-starved for 12 hours before immunoblotting analysis. (b) vGPCR stabilizes YAP/TAZ. HEK293A cells stably expressing vGPCR were treated with cycloheximide for the indicated time (hours: hr). (c) vGPCR induces YAP/TAZ nuclear localization. HEK293A cells overexpressing either control (CTL) or vGPCR were serum-starved for 12 hours. YAP/TAZ subcellular localization were determined by immunofluorescence staining for YAP/TAZ (red), TAZ (green), along with DAPI for DNA (blue). (d) vGPCR activates a YAP/TAZ reporter. Control (CTL), vGPCR, or positive control LPAR plasmids were co-transfected with a 5 X UAS-luciferase reporter, Renilla and Gal4-TEAD4 into HEK293A cell. 24 hours after transfection, cells were serum starved for 12 hours and luciferase activity was measured and quantified by Trigonelline Hydrochloride normalization to the co-transfected Renilla. (e) vGPCR induces expression of YAP/TAZ target genes. mRNA levels of indicated YAP/TAZ target genes was measured in Trigonelline Hydrochloride stably expressing control (CTL) and vGPCR cells following.