Induction of PKR tyrosine phosphorylation by interferons. The indicated cells were treated with IFN-γ for the indicated time periods. (A,B) Protein extracts were subjected to PKR immunoprecipitation (IP) followed by immunoblotting with the phosphotyrosine 4G10 monoclonal antibody (panel a). The total PKR protein levels are shown in panel b. The ratio of phosphorylated to total PKR for each lane is indicated. IFN, interferon; PKR, dsRNA-dependent protein kinase; TC-PTP, T-cell protein tyrosine phosphatase.

Tyrosine phosphorylation of PKR by Jaks. (A) 2fTGH and U4A cells were stimulated with IFN-α2b for the indicated time periods. Protein extracts were immunoprecipitated (IP) for PKR followed by immunoblotting with phosphotyrosine 4G10 antibody (panel a), PKR monoclonal antibody (panel b) and eIF2α antibody (panel c). The levels of PKR (panel d) and eIF2α (panel e) in WCE were detected by immunoblotting. (B) U4A cells were transfected with the catalytically inactive Flag–PKRK296R complementary DNA in the absence or presence of either wild-type (WT) or a kinase-dead (KD) K296R mutant of Jak1 cDNA. Protein extracts were immunoprecipitated for Flag followed by immunoblotting with phosphotyrosine 4G10 (panel b) or PKR antibody (panel c). Jak1 levels (panel a) were detected by immunoblotting of WCE. (C) Recombinant murine Jak2 and GST–PKRK296R were subjected to in vitro phosphorylation with [γ-³²P]ATP and autoradiography (panels a and b). The phosphorylated proteins were detected after an equal time of exposure. Total Jak2 (panel c) and GST–PKRK296R (panel d) levels were detected by Coomassie blue staining. IFN, interferon; PKR, dsRNA-dependent protein kinase; PKRK296R, PKRLys296Arg; WCE, whole-cell extracts.

Regulation of eIF2α phosphorylation and protein synthesis by interferons. 2fTGH, U4A (A, C) or U1A (B) cells were stimulated with either IFN-α2b or IFN-γ for the indicated time periods. Total protein extracts were subjected to immunoblotting with the indicated antibodies. (D) 2fTGH and U1A cells were left untreated or treated with IFN-α2b in the absence or presence of 0.5 μg ml⁻¹ actinomycin D (Act D) for 2 h. Cells were subjected to [³⁵S]-methionine labelling for 1 h. (E) PKR^+/+ and PKR^−/− MEFs were left untreated or treated with IFN-γ for 2 h followed by [³⁵S]-methionine labelling for an additional hour. (D,E) The graphs show the levels of incorporation of radioactivity into protein as percentages of the corresponding control (CON) values. The data are from three independent experiments performed in duplicate. eIF, eukaryotic initiation factor 2; IFN, interferon; MEF, mouse embryonic fibroblasts 2; PKR, dsRNA-dependent protein kinase; WCE, whole-cell extracts.

Physical interactions between Jaks and PKR. (A–D) The indicated cells were treated with either IFN-α2b or IFN-γ for the indicated time periods. Protein extracts were subjected to PKR immunoprecipitation (IP) followed by immunoblotting with the indicated antibodies. For the detection of tyrosine-phosphorylated Jak1, the phosphospecific pTyr1022/1023 antibody (A, panel a) or the phosphotyrosine 4G10 antibody (B, panel a) was used. The total levels of Jak1 or Tyk2 were detected by immunoblotting of whole-cell extracts (WCE; A,B, panel d; C,D, panel c). IFN, interferon; PKR, dsRNA-dependent protein kinase.

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.