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.
D000818:Animals; D002918:Chymotrypsin; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D014357:Trypsin
D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D010748:Phosphopeptides; D011506:Proteins; D011494:Protein Kinases; D002918:Chymotrypsin; D014357:Trypsin