Rabbit Anti-EIF-2ALPHA monoclonal antibody, clone TA12-17

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2alpha chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2alpha may bind to RNA.

Mouse erythroleukaemia (MEL) cells, which have not been induced into erythroid development, contain a protein kinase (MK(u)) which phosphorylates the alpha subunit of protein-synthesis-initiation factor 2 (eIF-2alpha). In this paper, we show that this kinase phosphorylates both eIF-2alpha and a synthetic peptide based on the phosphorylation site in eIF-2alpha at Ser51, the target residue for other eIF-2alpha kinases. Consistent with this, prior treatment of eIF-2 with MK(u) impaired the exchange of bound GDP for GTP which is catalysed by the exchange factor eIF-2B. Using a modified cell-free translation system, we have shown that Mk(u) inhibits translation, consistent with the above observations concerning the site of phosphorylation and the effect of phosphorylation on eIF-2B-mediated guanine-nucleotide exchange. MK(u) has been purified and its properties have been compared with those of the haem-controlled repressor eIF-2alpha kinase (HCR) from rabbit reticulocytes. Its behaviour on gel filtration is similar to that of HCR, while its behaviour on anion exchange resembles that of certain phosphorylated species of HCR. Highly purified preparations of MK(u) contain a protein with an apparent molecular mass of 98 kDa which comigrates with HCR on SDS/PAGE. This protein undergoes phosphorylation when incubated in the presence of Mg2+-ATP, and both this apparent autophosphorylation and the activity of the kinase against eIF-2alpha are inhibited by the same, low, (10 muM) concentrations of haemin. Phosphorylation of the 98-kDa components present in the MEL-cell kinase preparation and in purified rabbit reticulocyte HCR occurs on serine and threonine residues. Analysis of these phosphoproteins by peptide mapping reveals significant differences in their structures, indicating that they may be closely related, but are certainly not identical.