Recombinant rat and murine immune interferons are phosphorylated at a single site, Ser132

Recombinant rat (Ra) and murine (Mu) immune interferons (IFN-gamma) were found to be phosphorylated by bovine heart muscle cAMP-dependent protein kinase at a single site, in contrast to human (Hu) IFN-gamma, which was reported to be phosphorylated at two different serine residues. Chromatography of a Staphylococcal aureus V8 protease digest of Ra or MuIFN-gamma indicated that the site of phosphorylation was in the carboxy-terminal undecamer fragment of the protein. Due to inherent problems in measuring both phenylthiohydantoin-serine (PTH-serine) and PTH-phosphoserine with an automated sequenator, a novel approach, involving partial Edman degradation of aliquots of the peptide followed by high performance liquid chromatography (HPLC) analysis, was developed. It was determined that Ser132 is the exclusive site of phosphorylation for both IFNs.