Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates

• Published in: The Journal of biological chemistry 1992-06, Vol.267 (16), p.11500-11507
• Main Authors: MENDEZ, R, MORENO, A, DE HARO, C
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antibodies, Monoclonal; Biological and medical sciences; Blotting, Western; eIF-2 Kinase; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzymes and enzyme inhibitors; Ethylmaleimide - pharmacology; Fundamental and applied biological sciences. Psychology; Heme - metabolism; Hydrolysis; Mercaptoethanol - pharmacology; Phosphorylation; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Rabbits; Reticulocytes - enzymology; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)49938-9

We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.