The R1 subunit of herpes simplex virus ribonucleotide reductase is a good substrate for host cell protein kinases but is not itself a protein kinase.
The N terminus of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is believed to be a protein kinase domain mainly because the R1 protein was phosphorylated in a protein kinase assay on blot. Using Escherichia coli and adenovirus expression vectors to produce R1, we found that, whereas the reductase activity of both recombinant proteins was similar, efficient phosphorylation of R1 and casein in the presence of Mg2+ was obtained only with the R1 purified from eukaryotic cells. Phosphorylation of this R1, in solution or on blot, results mainly from the activity of casein kinase II (CKII), a co-purifying protein kinase. Labeling on blot occurs from CKII leakage off the membrane and its subsequent high affinity binding to in vivo CKII-phosphorylated R1. CKII target sites were mapped to an acidic serine-rich segment of the R1 N terminus. Improvement in purification of the R1 expressed in eukaryotic cells nearly completely abolished its phosphorylation potential. An extremely low level of phosphorylation observed in the presence of Mn2+ with the R1 produced in E. coli was probably due to an unidentified prokaryotic protein kinase. These results provide evidence that the herpes simplex virus type 2 R1 does not possess an intrinsic protein kinase activity.
Langelier
Y
Y
Institut du Cancer de Montréal, Québec, Canada. langeliy@ere.umontreal.ca
Champoux
L
L
Hamel
M
M
Guilbault
C
C
Lamarche
N
N
Gaudreau
P
P
Massie
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D047390:Casein Kinase II; D002417:Cattle; D004926:Escherichia coli; D006801:Humans; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology*
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C100671:ICP10 protein, herpes simplex virus type 2; D012264:Ribonucleotide Reductases; D011494:Protein Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases