PIN1 interacts with a specific serine-proline motif of hepatitis B virus X protein to enhance hepatocarcinogenesis

Abstract

Background We previously reported overexpression of the peptidyl prolyl isomerase PIN1 in over 50% of hepatocellular carcinoma (HCC) cases related to hepatitis B virus (HBV) and its functional role in hepatocarcinogenesis. HBV X protein (HBx), a potent transactivator implicated in the pathogenesis of HBV-related HCC, has a potential PIN1 binding motif. We conducted a study to test the hypothesis that PIN1 may interact with HBx to exert a synergistic effect in HCC carcinogenesis. Methodology Sections of HCC specimens were immunostained for co-expression of PIN1 and HBx. The physical interaction between PIN1 and HBx was studied by co-immunoprecipitation (co-IP) and glutathione-S-transferase (GST) pull-down assays. Site-directed mutagenesis was performed to mutate either 1 or both pSer-Pro sites at amino-acid position 39 to 42 (-SerProSerPro-) of HBx. A total of 7 HBx mutants were generated, and the PIN1 binding site was determined by repeating co-IP and GST pull-down assays with the various HBx mutants. Subcellular localization of the two proteins was examined by immunofluorescence microscopy. The luciferase activity of an NF-AT driven reporter transactivated by HBx expression alone was compared with that transactivated by co-expression of HBx and PIN1 (wild type or mutants) to demonstrate their synergistic effect. Oncogenicity of a non-tumorigenic human liver cell line (MIHA) doubly transfected with PIN1 and HBx (wild type or mutants) was evaluated by soft agar assay and subcutaneous injection into nude mice, and compared with MIHA transfected with either PIN1 or HBx alone. Results PIN1 positively correlated with HBx expression (p=0.024) by immunohistochemistry in HBV-related HCCs. Immunofluorescence microscopy confirmed co-localization of HBx and PIN1 in the nucleus, and co-IP showed that PIN1 bound to ectopically expressed flag-tagged HBx in Hela cells. GST pull-down assay further validated the binding between the two proteins. Mutation of either Ser or Pro in the pSer41-Pro motif of HBx abolished its binding with PIN1. Co-transfection of PIN1 and HBx transactivated the NF-AT driven luciferase reporter, with two-fold increase in luciferase activity compared with transfection of HBx alone, or HBx mutants with abolition of Ser/Pro residues at the Ser41-Pro motif. Both colony formation and growth in soft agar were increased with stable PIN1-HBx transfectants compared with PIN1-HBx mutants at Ser41-Pro, PIN1 or HBx transfectants alone. Injection of PIN1-HBx transfectants in nude mice resulted in larger tumors compared with PIN1 or HBx transfectants alone. Conclusion Our study demonstrated that PIN1 interacts with a specific Ser-Pro motif of HBx and augments its transactivating activity and oncogenicity, implicating PIN1 expression as an important step in HBV-related hepatocarcinogenesis. PIN1 thus represents a novel molecular target for inhibition of hepatocarcinogenesis.