Regulation of initiation factors during translational repression caused by serum depletion. Covalent modification.
One to 2 h after transfer of HeLa cells into fresh serum-containing medium, when translation rates are maximal, the initiation factor proteins were examined on immunoblots of two-dimensional gels. Eukaryotic initiation factor (eIF)-2 alpha, eIF-2 beta, and eIF-4A each formed a single immunoreactive spot; eIF-2 gamma formed 2 spots; and eIF-4B formed a complex array of 12-20 spots. After 4 days of growth in unreplenished medium, when translation rates have dropped 4-6-fold, several alterations in the isoelectric forms were observed: eIF-2 alpha now occurred in 2 forms, eIF-2 beta was present in 3-4 forms, and the most acidic cluster of eIF-4B variants was decreased or absent while a new isoelectric variant appeared at the basic end of the array. No changes were observed for eIF-2 gamma or eIF-4A. The 35-50-kDa subunits of the multiprotein initiation factor eIF-3 also showed no changes when the aforementioned growth states were compared. Resolution of 32P-labeled lysates by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the eIF-2 alpha modification and the loss of eIF-4B variants reflected changes in phosphorylation states. Stimulation of 4-day grown cells with fresh serum-containing medium caused a reversal of the initiation factor modifications back to the forms prevailing shortly after replating. This analysis indicates that covalent modifications appear concurrently with decreasing initiation rates and suggests that they may be causative.
D001769:Blood; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D039601:Eukaryotic Initiation Factor-4A; D006801:Humans; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism
D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D010448:Peptide Initiation Factors; D011506:Proteins; D039601:Eukaryotic Initiation Factor-4A