Autophosphorylation of the protein kinase dependent on double-stranded RNA.
The double-stranded RNA (dsRNA)-dependent protein kinase (p68 kinase) from interferon-treated human cell is a Mr 68,000 protein induced by interferon. By the use of a specific monoclonal antibody, we have been able to study the two distinct protein kinase activities characteristic of purified p68 kinase. The first activity is functional for endogenous phosphorylation of the enzyme (p68 kinase), whereas the second one is responsible for the phosphorylation of exogenous substrates such as eukaryotic initiation factor 2 and histone. When activated by dsRNA in the presence of Mn2+ and ATP, p68 kinase is autophosphorylated and is then capable of catalyzing phosphorylation of histone in the absence of dsRNA. Whereas binding of 8-azido-[alpha-32P] ATP (8-N3ATP) to p68 kinase is dependent on both dsRNA and Mn2+, phosphorylated p68 kinase binds 8-N3ATP independent of dsRNA. This is consistent with a dsRNA requirement for the autophosphorylation of p68 kinase, but not for the phosphorylation of exogenous substrates. p68 kinase is mainly associated with the ribosomal pellet. It could be recovered efficiently by a buffer containing both high salt and a nonionic detergent. Synthesis of p68 kinase is induced several-fold by interferon in different types of human cells. Partial proteolysis of [35S]methionine and an 8-N3ATP-labeled p68 kinase preparation by Staphylococcus aureus V8 protease indicated the presence of a major Mr 48,000 polypeptide (p48) with a specific ATP-binding site. p48 probably contains the catalytic unit of p68 kinase and is analogous to a similar protein which we have previously described as a distinct protein present in a complexed form with p68 kinase. We now believe that the presence of p48 in previously purified kinase preparations was due to partial degradation of p68 kinase.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D002460:Cell Line; D006657:Histones / Q000378:metabolism; D006801:Humans; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism
D001386:Azides; D006657:Histones; D012330:RNA, Double-Stranded; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D008715:Methionine; D011494:Protein Kinases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase