Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.
We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.
Cooper
J
J
MRC Virology Unit, Institute of Virology, Glasgow, United Kingdom.
Conner
J
J
Clements
J B
JB
D000241:Adenosine / Q000031:analogs & derivatives; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D002250:Carbon Radioisotopes; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D018258:Herpesvirus 2, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D002250:Carbon Radioisotopes; D009838:Oligodeoxyribonucleotides; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D012264:Ribonucleotide Reductases; D011494:Protein Kinases; D000241:Adenosine