RIBOSOMAL-PROTEIN S2 SA KINASE PURIFIED FROM HELA-CELLS INFECTED WITHVACCINIA VIRUS CORRESPONDS TO THE B1R PROTEIN-KINASE AND PHOSPHORYLATES IN-VITRO THE VIRAL SSDNA-BINDING PROTEIN/

A ribosomal protein S2 kinase was purified 6000-fold from cytoplasmic extracts of HeLa cells infected with vaccinia virus, using 80S ribosom es or 40S ribosomal subunits as a substrate. Although the preparation was not homogeneous, a 34K component was identified, the chromatograph ic behaviour of which correlated with enzyme activity. During its puri fication the ribosomal protein S2 kinase was resolved from a less abun dant ribosomal. protein S13 kinase, demonstrating the two to be differ ent entities. A second protein kinase activity against a 43K ribosomal protein comigrated with the ribosomal protein S2 kinase activity duri ng all five chromatographic procedures employed, and we conclude that the two activities are properties of a single species. Two-dimensional gel electrophoresis demonstrated that this second substrate was the a cidic ribosomal protein Sa, of isoelectric point approximately 5.2, pr eviously shown to be phosphorylated during infection with vaccinia vir us. Another substrate for the ribosomal protein S2/Sa kinase in vitro was the 36K viral ssDNA-binding protein, of isoelectric point approxim ately 5.0, which is also known to be phosphorylated in vivo. The 34K p rotein correlating with the catalytic activity in the most purified pr eparations of the ribosomal protein S2/Sa kinase was recognized by an antibody specific for a protein expressed in Escherichia coli from vac cinia virus gene B1R. This and other evidence suggest strongly that th e ribosomal protein S2/Sa kinase is the product of this gene.