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 expressed as inclusion bodies in Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein. After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo phosphorylation of HBx was also demonstrated. Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase 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.
Lee
Y I
YI
Liver Cell Signal Transduction Research Unit, Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, 305-606, Taejon, South Korea. yilee@kirbb4680.kribb.re.kr
Kim
S O
SO
Kwon
H J
HJ
Park
J G
JG
Sohn
M J
MJ
Jeong
S S
SS
D000595:Amino Acid Sequence; D001665:Binding Sites; D006515:Hepatitis B virus; D006801:Humans; D006868:Hydrolysis; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019606:Phosphoamino Acids / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D020539:Sequence Analysis, Protein; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D054334:Viral Regulatory and Accessory Proteins
D019606:Phosphoamino Acids; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases