Mechanism of interferon action. Characterization of sites of phosphorylation in the interferon-induced phosphoprotein P1 from mouse fibroblasts: evidence for two forms of P1.

The sites of phosphorylation of the interferon (IFN)induced protein designated Pi isolated from mouse L29 cells were characterized by partial proteolysis peptide mapping and by phosphoamino acid analysis. The Mr = 67,000 protein P1 phosphorylated in the presence of double-stranded RNA (dsRNA) was isolated from ribosomnal salt wash fractions of untreated and IFNtreated cells by poly(rI).poly(rC)-Sepharose affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IFN treatment increased the amount of dsRNA-activated P1 phosphorylation about 5-fold; the dsRNA-activated phosphorylation slightly altered the mobility of P1 on sodium dodecyl sulfate gels. The partial proteolysis phosphopeptide maps obtained with Staphylococcus aureus V8 protease and with a-chymotrypsin showed that P1 from untreated cells and P1 from cells treated with natural or cloned IFNs were very similar if not identical. A protein of Mr = 64,000 present in both untreated and IFN-treated cells was phosphorylated in the absence of dsRNA. This protein bound to poly(rI)-poly(rC)-Sepharose and was designated pre-P because it shared all major and minor phosphopeptides except for one with P1 . A major phosphopeptide designated Xds generated by digestion with V8 protease was present in P1 but was absent from pre-Pi. Phosphoserine was the major O-phosphoester linkage in both P1 and pre-Pi; no phosphotyrosine was detected in either P1 or pre-Pi. These results suggest that the Mr = 64,000 protein designated pre-PI phosphorylated in the absence of dsRNA and the Mr = 67,000 protein designated P1 phosphorylated in the presence of dsRNA are two structurally different forms of the same protein or very similar proteins and that the uninduced protein P1 of untreated cells and the IFN-induced protein P1 differ in amount but not in the pattern of phosphorylation.