The vaccinia virus B1R gene product is a serine/threonine protein kinase.
The nucleotide sequence of the vaccinia virus open reading frame B1 predicts a polypeptide with significant sequence similarity to the catalytic domain of known protein kinases. To determine whether the B1R polypeptide is a protein kinase, we have expressed it in bacteria as a fusion with glutathione S-transferase. Affinity-purified preparations of the fusion protein were found to undergo autophosphorylation and also phosphorylated the exogenous substrates casein and histone H1. Mutation of lysine 41 to glutamine within the conserved kinase catalytic domain II abrogated protein kinase activity on all three protein substrates, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide. Casein and histone H1 were phosphorylated on serine and threonine residues. The B1R fusion protein was phosphorylated on a threonine residue(s) by an apparently intramolecular mechanism. The autophosphorylation reaction resulted in phosphorylation of the glutathione S-transferase portion of the fusion and not the protein kinase domain. The protein kinase activity of B1R was specific for ATP as the phosphate donor; GTP was not utilized to a detectable extent. Immunoblotting experiments with anti-B1R antiserum showed that the protein kinase is located in the virion particle. Chromatography of virion extracts resulted in separation of the B1R protein kinase from the bulk of the total protein kinase activity, indicating that multiple protein kinases are present in the virion particle and that B1R is distinct from the previously described vaccinia virus-associated protein kinase.
Lin
S
S
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-6799.
Chen
W
W
Broyles
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005981:Glutathione Synthase / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013604:T-Phages / Q000235:genetics; D013912:Threonine / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000201:enzymology
D002364:Caseins; D006657:Histones; C074058:VB1R protein, Vaccinia virus; D014764:Viral Proteins; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D005981:Glutathione Synthase