Site-specific phosphorylation if initiation factor 2 by three cyclic nucleotide-independent protein kinases.

The site-specific phosphorylation of initiation factor 2 (eIF-2) by three different cyclic nucleotide-independent protein kinases from rabbit reticulocytes was examined. The hemin-controlled repressor modified serine in the alpha subunit (Mr = 38,000), while casein kinase II and protease-activated kinase II phosphorylated serine residues of the beta subunit (Mr = 53,000). Under conditions of maximal phosphorylation, 1 mol of phosphate was incorporated into eIF-2 by each of the protein kinases. However, following treatment of eIF-2 with alkaline phosphatase, 2 mol of phosphate were added by casein kinase II, indicating residual phosphate was present on the beta subunit of purified eIF-2. The tryptic and chymotryptic peptides of the phosphorylated subunits were analyzed by two-dimensional peptide mapping involving thin layer electrophoresis followed by ascending chromatography. When eIF-2 was phosphorylated by the hemin-controlled repressor, three major chymotryptic and four tryptic phosphopeptides with molecular weights ranging from approximately 600 to 3000 were identified. When phosphorylation of eIF-2 beta was examined, two tryptic and two chymotryptic phosphopeptides were obtained after phosphorylation with casein kinase II and were different from the phosphopeptides observed following phosphorylation with protease-activated kinase II. This indicates that three distinct sites in the beta subunit were phosphorylated, two by casein kinase II and one by protease-activated kinase II.