title: The association of eIF-2 with Met-tRNAi or eIF-2B alters the specificity of eIF-2 phosphatase.
abstract: In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities.
mesh_terms: D000818:Animals; D001798:Blood Proteins; D015852:Eukaryotic Initiation Factor-2; D007700:Kinetics; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors / Q000097:blood*; D010749:Phosphoprotein Phosphatases / Q000097:blood*; D010766:Phosphorylation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000097:blood*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001798:Blood Proteins; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; C019363:eIF-2 phosphatase; D010749:Phosphoprotein Phosphatases