Serine 48 in initiation factor 2 alpha (eIF2 alpha) is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.
Phosphorylation of the serine 51 residue in the alpha-subunit of translational initiation factor 2 in eukaryotes (eIF2 alpha) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2 alpha phosphorylation in the interaction between eIF2 alpha(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2 alpha in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 alpha monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2 alpha kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 alpha subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2 alpha of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2 alpha(P)-eIF2B complex that occurs due to eIF2 alpha phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2 alpha compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.
Sudhakar
A
A
Department of Biochemistry, University of Hyderabad, Andhra Pradesh, India.
Krishnamoorthy
T
T
Jain
A
A
Chatterjee
U
U
Hasnain
S E
SE
Kaufman
R J
RJ
Ramaiah
K V
KV
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002458:Cell Fractionation; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020717:Eukaryotic Initiation Factor-2B / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D046911:Macromolecular Substances; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010766:Phosphorylation; D011070:Poly I-C / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism / Q000821:virology; D014162:Transfection
D000911:Antibodies, Monoclonal; D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012694:Serine; D011070:Poly I-C