The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase.
The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.
Galabru
J
J
Institut Pasteur, Paris, France.
Katze
M G
MG
Robert
N
N
Hovanessian
A G
AG
D000260:Adenoviruses, Human / Q000235:genetics*; D002460:Cell Line; D004790:Enzyme Induction; D006801:Humans; D007370:Interferon Type I / Q000502:physiology*; D007700:Kinetics; D010766:Phosphorylation; D011066:Poly C / Q000378:metabolism; D011069:Poly I / Q000378:metabolism; D011070:Poly I-C / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*
D007370:Interferon Type I; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011069:Poly I; D011066:Poly C; D011494:Protein Kinases; D011070:Poly I-C