HBx has been suggested as an important determinant mediating the pathological effects of HBV via interacting with various cellular proteins. To identify new HBx-interacting proteins and elucidate a possible mechanism associated with HBx and HBx-interacting proteins in hepatocellular carcinoma, yeast two-hybrid screening was performed. We identified a novel HBx-interacting protein, serine/threonine protein phosphatase PP2C alpha, and investigated the effects of PP2C alpha on HBx-mediated IL-6 regulation. The interaction between endogenous PP2Ca, and HBx was confirmed by co-immunoprecipitation. Recombinant HBx dose-dependently reduced enzyme activity of recombinant PP2C alpha in vitro. While ectopically expressed PP2C alpha in Cos-7 and Huh-7 cells reduced the expression of IL-6, overexpressed HBx with recombinant HBx-expressing adenovirus overcame PP2C alpha-mediated IL-6 downregulation. In the response of IL-6, HBx phosphorylated STAT3 and recovered PP2C alpha-mediated dephosphorylation of STAT3. These results supported that HBx might play a crucial role in HBV-associated hepatocarcinogenesis even in cases where cells express a negative regulator, PP2C alpha. (c) 2006 Elsevier Inc. All rights reserved.