Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia 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 ribosomes or 40S ribosomal subunits as a substrate. Although the preparation was not homogeneous, a 34K component was identified, the chromatographic behaviour of which correlated with enzyme activity. During its purification the ribosomal protein S2 kinase was resolved from a less abundant ribosomal protein S13 kinase, demonstrating the two to be different entities. A second protein kinase activity against a 43K ribosomal protein comigrated with the ribosomal protein S2 kinase activity during 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 acidic ribosomal protein Sa, of isoelectric point approximately 5.2, previously shown to be phosphorylated during infection with vaccinia virus. Another substrate for the ribosomal protein S2/Sa kinase in vitro was the 36K viral ssDNA-binding protein, of isoelectric point approximately 5.0, which is also known to be phosphorylated in vivo. The 34K protein correlating with the catalytic activity in the most purified preparations of the ribosomal protein S2/Sa kinase was recognized by an antibody specific for a protein expressed in Escherichia coli from vaccinia virus gene B1R. This and other evidence suggest strongly that the ribosomal protein S2/Sa kinase is the product of this gene.
Beaud
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G
Institut Jacques Monod du CNRS, Université de Paris, France.
Sharif
A
A
Topa-Massé
A
A
Leader
D P
DP
D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D012269:Ribosomal Proteins / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000302:isolation & purification*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C047013:RPS13 protein, human; D012269:Ribosomal Proteins; D014764:Viral Proteins; C030004:ribosomal protein S2; C512838:rpsM protein, E coli; D011494:Protein Kinases