Figure 4.

Tyrosine phosphorylation of PKR by interferons. (A) Recombinant Jak2 and GST–PKRK296R were subjected to phosphorylation using non-radioactive ATP. The reactions were then subjected to immunoblotting with the indicated phosphospecific (panels a and b) and pan-specific (panel c) antibodies against PKR. (B–D) 2fTGH, U1A or U4A cells were stimulated with IFN-α2b (B,C) or IFN-γ (D) for the indicated time periods. Protein extracts were immunoprecipitated (IP) for PKR followed by immunoblotting with the indicated PKR phosphospecific antibodies (panel a). Total PKR in the immunoprecipitates was detected by immunoblotting (panel b). The ratio of phosphorylated to total PKR for each lane is indicated. ATP, adenosine triphosphate; GST, glutathione S-transferase; IFN, interferon; PKR, dsRNA-dependent protein kinase; PKRK296R, PKRLys296Arg.