Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro

The recombinant human hepatitis B virus-X protein (rhHBx) has been expresse d as inclusion bodies in Escherichia call and purified. By sequential dialy sis of urea, rhHBx was folded into the native structure, which was demonstr ated by both the efficacy of its transcriptional activation of the adenovir us major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the i ntrinsic fluorescence emission confirmed the ability of HBx to refold in lo wer concentrations of urea to produce the active protein. After purificatio n and renaturation, the rhHBx protein was found to be phosphorylated by pro tein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo ph osphorylation of HBx was also demonstrated. Although PKC and MAPK enhance t he HBx phosphorylation in vitro. neither protein kinase A nor caseine kinas e II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein. Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK. (C) 2001 Elsevier Science B,V. All rights reserved.