PURIFICATION AND CHARACTERIZATION OF AN INITIATION-FACTOR-2 KINASE FROM UNINDUCED MOUSE ERYTHROLEUKEMIA-CELLS

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.