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.
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.
Mehta
H B
HB
Dholakia
J N
JN
Roth
W W
WW
Parekh
B S
BS
Montelaro
R C
RC
Woodley
C L
CL
Wahba
A J
AJ
D000818:Animals; D001156:Artemia / Q000196:embryology / Q000201:enzymology*; D047388:Casein Kinases; D004625:Embryo, Nonmammalian / Q000201:enzymology; D015852:Eukaryotic Initiation Factor-2; D006418:Heme / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012097:Repressor Proteins / Q000378:metabolism*; D012156:Reticulocytes / Q000201:enzymology*; D014157:Transcription Factors / Q000378:metabolism*; D014357:Trypsin
D015852:Eukaryotic Initiation Factor-2; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D011506:Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D006418:Heme; D011494:Protein Kinases; D047388:Casein Kinases; D014357:Trypsin