Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 88; no. 2; pp. 315 - 319
• Main Authors: Chen, J J, Pal, J K, Petryshyn, R, Kuo, I, Yang, J M, Throop, M S, Gehrke, L, London, I M
• Format: Journal Article
• Language: English
• Published: United States National Acad Sciences 15.01.1991
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Blotting, Northern; eIF-2 Kinase; Electrophoresis, Polyacrylamide Gel; Heme - pharmacology; Kinetics; Molecular Sequence Data; Peptide Fragments - isolation & purification; Protein Binding; Protein Kinases - genetics; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Reticulocytes - metabolism; RNA, Messenger - genetics; Sequence Homology, Nucleic Acid; Trypsin
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.2.315

We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases.