Regulation of protein synthesis in reticulocyte lysates: phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from hemedeficient lysates.

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Abstract
A previous study demonstrated that the translational inhibitor isolated from lysates of heme-deficient rabbit reticulocytes is associated with a protein kinase activity. Chromatography of this inhibitor preparation on phosphocellulose yields 2 distinct protein kinase activities, PC1 and PC2. PC1, which constitutes about 90% of the activity in the unresolved preparation, does not inhibit protein synthesis in lysates, but actively phosphorylates calf thymus histone II in a 3'':5''-cyclic[c]AMP-dependent reaction. PC2 contains the translational inhibitor, phosphorylates histone poorly and is not cAMP-dependent. With [.gamma.-32P]ATP as the phosphate donor, the 2 kinase fractions were analyzed with the putative substrates, salt-washed 40S ribosomal subunits and the initiation factor that mediates the binding of Met-tRNAf to the 40S subunit. PC1 is inactive with the initiation factor, but phosphorylates 40S subunits at a single major site that migrates as a 31,000-dalton band in sodium dodecyl sulfate-acrylamide gels; phosphorylation requires cAMP. Similar phosphorylation of the reticulocyte 40S site (31,000 daltons) can be demonstrated with other cAMP-dependent kinases from reticulocytes, rat liver and bovine heart muscle. PC2 phosphorylates the small subunit (38,000 daltons) but not the large subunit(s) of the initiation factor; the reaction does not require cAMP. PC2 does not phosphorylate 40S subunits. In the presence of 40S subunits, the initiation factor appears to be rapidly bound in a manner that effectively blocks phosphorylation of the initiation factor by PC2; under the same conditions phosphorylation of the 40S subunit by PC1 is not affected. The initiation factor may reverse the inhibitions of protein chain initiation induced in lysates by heme deficiency, double-stranded RNA, oxidized glutathione or the purified translational inhibitor. The observation that the Met-tRNAf binding factor is phosphorylated by PC2 supports the hypothesis that this initiation factor is a target for the action of the translational inhibitor activated in heme deficiency.

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