Purification and characterization of vaccinia virus protein kinases (1994)

The nucleotide sequence of the B1R open reading frame of vaccinia virus predicts a polypeptide that bears significant sequence similarity to the catalytic domains of known protein kinases. To investigate whether the protein product of the B1R gene has protein kinase activity, appropriate constructs were made to express B1R in bacteria in the form of a glutathione S-transferase chimera. Affinity-purified preparations of the fusion protein not only underwent autophosphorylation but also phosphorylated casein and histone H1 on serine and/or threonine residues. When lysine 41 was replaced by glutamine within the conserved kinase catalytic domain, all protein kinase activity was abolished, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide. Immunoblotting experiments with polyclonal anti-B1R IgG's showed that this protein was present within virion particles. Chromatography of virion extracts resulted in separation of B1R protein kinase from the other viral proteins that also had protein kinase activity. Evidently multiple protein kinases are present in the virion particle and B1R is distinct from the previously described vaccinia virion-associated protein kinase. No physiologically relevant substrate for B1R protein kinase has been identified. MBP from bovine brain was used as a model substrate for the analysis of the specificity of B1R kinase. Digestion of MBP with trypsin did not significantly alter the sites of phosphorylation, indicating that the immediate amino acid sequence environment of phosphorylated residues in MBP was the main substrate specificity determinant. These peptides were isolated by reverse phase HPLC, and their identities were determined by mass spectrometry. Sequence comparison of five peptide substrates revealed the common sequence (S/T/Y)-(G/A/L)-S-X$\sb{0-5}$-(K/R) around potential sites of phosphorylation. Phosphoamino acid analysis and Edman degradation demonstrated that B1R kinase phosphorylated one or both serines and threonines within the proposed consensus sequence. This information should be useful in the prediction of potential substrates for the B1R protein kinase. A virion-associated protein kinase, distinct from B1R, was purified 196-fold from the virion core extract. The specific activity of this material was about 200,000 pmol/min/mg protein using peptide DDDDVASLPGLRRR as substrate. Peak fractions from final step appeared to be homogenous by SDS-PAGE. The apparent molecular weight on calibrated SDS-PAGE and the sedimentation rate of this enzyme through a 15%-35% glycerol gradient indicated that it was a monomeric protein of 50 kDa. This kinase readily phosphorylated a casein kinase I-specific peptide substrate, and also underwent autophosphorylation in an intramolecular manner. Amino acid sequence analysis showed that this kinase is encoded by gene F10L of vaccinia virus. The F10L gene product, expressed in bacteria, also showed kinase activity toward the same substrates as the protein purified from virions, further confirming that the kinase activity was associated with F10L gene product. Vaccinia virus is the only DNA virus found to encode two distinct protein kinases.