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

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 two distinct protein kinase activities, PCI and PC2. PCI, which .In lysates of heme-deficient rabbit reticulocytes, the rate of protein synthesis proceeds linearly for several minutes and then declines abruptly (1-3). The shut-off of protein synthesis results from an inhibition that occurs at the level of protein chain initiation (4-6); inhibition is due to the action of a translational inhibitor that is activated in the absence of heme (2, 3, 7-9); addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition similar to that observed in heme deficiency (8, 10, 11). We reported that a partially purified inhibitor preparation contained a protein kinase activity (12). This inhibitor preparation (designated DS2 in this report) has been resolved by chromatography on phosphocellulose into two distinct protein kinase activities, designated PC1 and PC2. We examined these activities utilizing as putative substrates 40S ribosomal subunits and the initiation factor (IF) that binds initiator Met-tRNAf in the ternary complex [IF, Met-tRNAf, GTP] (13).We present the following evidence here: (i) PC1 is a 3':5'-cyclic AMP(cAMP)-dependent protein kinase that does not inhibit protein synthesis and does not phosphorylate IF; however, PC1 does phosphorylate 40S subunits at one major site.(ii) PC2 inhibits protein synthesis and phosphorylates the 38,000-dalton subunit (14) of IF in a reaction that does not require cAMP; PC2 does not act on 40S subunits. MATERIALS AND METHODSRabbit reticulocyte lysates were prepared as described (8).Salt-washed (0.5 M KCl) 40S ribosomal subunits and the initiation factor that binds Met-tRNAf in the ternary complex [IF, Met-tRNAf, GTP] were prepared from reticulocyte polyribosomes as described for the corresponding components from mouse fibroblasts (13, 15); IF was further purified on phosphocellulose to approximately 50% purity. Reticulocyte protein kinases DS1 and DS2 are DEAE-Sephadex fractions that correspond to PK1 and PK2 previously described (12,16,17). Beef heart muscle protein kinase (18) was a gift of Dr.

Ca2+/calmodulin‐dependent phosphorylation of elongation factor 2

“…Phosphorylation of initiation factors (see, e.g. [1,2]), ribosomal proteins [31, aminoacyltRNA synthetases [3] and elongation factor 1 [4,5] has been reported. Phosphorylation of the initiation factor elF-2 ce-subunit has been studied most extensively and has been shown to play an important role in the regulation of protein biosynthesis (see, e.g.…”

Relationship between phosphorylation and activity of heme-regulated eukaryotic initiation factor 2 alpha kinase.

“…Protein synthesis in reticulocytes and reticulocyte lysates is inhibited by heme deficiency (1)(2)(3)(4)(5)(6). This inhibition is due to the rapid activation of a cyclic AMP-independent protein kinase that specifically phosphorylates the a subunit (Mr 38,000) of the reticulocyte eukaryotic initiation factor 2 (eIF-2) (6-10).…”

ACCUMULATION OF MET-tRNA-80S RIBOSOME COMPLEX IN A CELL-FREE SYSTEM OF GLOBIN SYNTHESIS

“…These findings, however, cannot be considered to be caused by the action of hemin-controlled repressor (Maxwell et al, 1971;Adamson et al, 1972;Gross and Rabinovitz, 1972) since the inhibition of globin synthesis under hemin-deficient conditions was shown to result from impaired binding of the initiator tRNA to 40S ribosomal subunits by hemin-controlled repressor (Legon et al, 1973;Balkow et al, 1973). By use of a highly purified preparation of the hemin-controlled repressor, more recently, it was further shown to be a 3':5'-cyclic AMP-independent protein kinase phosphorylating the small subunit of the initiation factor that forms a ternary complex with Met-tRNA1 and GTP (Kramer et al, 1976;Levin et al, 1976;Raunu and London, 1976). To clarify the mechanism whereby the ternary complex reported herein accumulates, further studies are required on the regulation of the protein synthesis in a cellfree system employing subcomponents from the ribosomal wash fraction.…”