Structural studies on the eukaryotic chain initiation factor 2 from rabbit reticulocytes and brine shrimp Artemia embryos. Phosphorylation by the heme-controlled repressor and casein kinase II

• Published in: The Journal of biological chemistry 1986-05, Vol.261 (15), p.6705-6711
• Main Authors: Mehta, H B, Dholakia, J N, Roth, W W, Parekh, B S, Montelaro, R C, Woodley, C L, Wahba, A J
• Format: Article
• Language: English
• Subjects: Animals; Artemia - embryology; Artemia - enzymology; Biological and medical sciences; Casein Kinases; Embryo, Nonmammalian - enzymology; Eukaryotic Initiation Factor-2; Fundamental and applied biological sciences. Psychology; Heme - pharmacology; Kinetics; Molecular and cellular biology; Molecular genetics; Molecular Weight; Peptide Fragments - analysis; Peptide Initiation Factors - metabolism; Phosphorylation; Protein Kinases - metabolism; Proteins - metabolism; Rabbits; Repressor Proteins - metabolism; Reticulocytes - enzymology; Transcription Factors - metabolism; Translation. Translation factors. Protein processing; Trypsin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)62673-6

In contrast to reticulocyte polypeptide chain initiation factor 2 (eIF-2), the Artemia factor retains activity in the presence of Mg2+ or after phosphorylation of its alpha-subunit by rabbit reticulocyte heme-controlled repressor (Mehta, H. B., Woodley, C. L., and Wahba, A. J. (1983) J. Biol. Chem. 258, 3438-3441). Furthermore, we have so far been unable to demonstrate a requirement for a GDP/GTP nucleotide exchange factor with Artemia eIF-2. In order to explain these differences we compared the structure of eIF-2 from Artemia and rabbit reticulocytes by using one- and two-dimensional phosphopeptide and iodopeptide maps. Partial trypsin digestion of the alpha-subunit of Artemia eIF-2 after phosphorylation by the heme-controlled repressor generates a 4000 Mr phosphopeptide. Upon extensive trypsin digestion, the two-dimensional phosphopeptide maps of the alpha-subunits for the reticulocyte and Artemia factors are indistinguishable, whereas the iodopeptide maps are different. In addition, immunoblotting indicates that there is no consistent cross-reactivity of the reticulocyte subunits with antibodies prepared in rabbits against the Artemia eIF-2 subunits. A casein kinase II activity was isolated from Artemia embryos that phosphorylates the beta-subunit of reticulocyte eIF-2, but specifically phosphorylates the alpha-subunit of eIF-2 preparations from several non-mammalian sources, including Artemia, yeast, and wheat germ embryos. Since this kinase phosphorylates a site distinct from that recognized by the heme-controlled repressor, and this phosphorylation does not alter the ability of Artemia eIF-2 to undergo nucleotide exchange, caution must be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in non-mammalian cells.