Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: Inhibition of interaction of Met-tRNA _f ^Met binding factor with another initiation factor in formation of Met-tRNA _f ^Met ·40S ribosomal subunit complexes

Abstract

Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme (1-3). In heme deficiency, a heme-regulated translational inhibitor (HRI) is activated (4-6), which blocks protein chain initiation (2, 3, 5-10). The inhibition of protein synthesis is associated with the disappearance of the complexes of Met-tRNAfMet (Met-tRNAf) and 40S ribosomal subunits (9). The purified HRI has functional properties that are similar to those of the in situ heme-regulated inhibitor (11,12). The inhibition induced in heme deficiency or by the addition of HRI is overcome or prevented by the Met-tRNAf binding factor (11,(13)(14)(15), which forms a ternary complex with Met-tRNAf and GTP (16)(17)(18)(19)(20)(21)(22). The Met-tRNAf binding factor (IF) is called EIF-1 in the nomenclature of Gupta and coworkers (23) and eIF-2 in the proposed new nomenclature (24); in this paper, it is called IF. The HRI has been shown to be an adenosine 3':5'-cyclic monophosphate (cAMP) independent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37), which specifically phosphorylates the 38,000-dalton subunit and not the 52,000-dalton and 50,000-dalton subunits of IF (12,(25)(26)(27)(28). These findings suggest that the inhibition by HRI involves the phosphorylation of IF. Significant inhibition of the binding of Met-tRNAf to 40S subunits has been shown to occur with high concentrations of HRI (13,29,30), and some inhibition of ternary complex formation has also been reported (31). However, the question of the molecular mechanism by which HRI exerts its inhibitory effect is not fully resolved.In the present studies we have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNAf to 40S subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF) of Majumdar et al. (23)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNAf to 40S subunits. This inhibitory effect of HRI requires ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of IF.The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also dissociate the ternary complex in the presence of 5 mM Mg2+ at 0°(23). At low concentrations of HRI and in the presence of ATP, this TDF-promoted dissociation of the ternary complex is inhibited. These results are consistent with the view that the phosphorylation of IF by HRI modifies its conformation and thus inhibits the protein-protein interaction of IF with one or more components of TDF in the formation of the Met-tRNAf-40S subunit initiation complex.Recently Das and Gupta (32) reported the isolation of a ternary complex dissociation inhibitor (TDI) from reticulocyte ribosome-free supernate. Like HRI, TDI inhibits ternary complex dissociation and the binding of Met-tRNAf to 40S subunits. In our experiments TDI and purified HRI behave similarly in all the results presented here. The apparent lack of requiremen...

Small molecule ISRIB suppresses the integrated stress response within a defined window of activation

“…In response to stress, eIF2 kinases are activated and subsequently phosphorylate a single, conserved Ser-51 residue in eIF2α. Four eIF2α kinases have been identified: protein kinase R (PKR), which is activated by recognition of “nonself” (e.g., viral) RNA (5, 6); PKR-like endoplasmic reticulum kinase (PERK), which responds to an accumulation of misfolded proteins in the ER (7); general control nonderepressible 2 (GCN2), which is activated by amino acid starvation and UV light (8, 9); and heme-regulated inhibitor (HRI), which is activated at low levels of heme and exposure to heavy metals (10). Phosphorylation of eIF2α inhibits eIF2B by stabilizing its association with eIF2•GDP (11).…”

Protein Kinase Activity in Purified Poliovirus Particles and Empty Viral Capsid Preparations

“…The activity of the initiation factor elF2 is also regulated by phosphorylation and dephosphorylation (Ranu et al, 1978;Rosen & Krebs, 1981 ;De Haro et al, 1982). It will be of great interest to see whether poliovirus infection results in increased phosphorylation of elF2 and of the cap-binding protein.…”

Role of Heme and Iron Metabolism in Controlling Protein Synthesis

“…In a reconstituted system with highly purified components, phosphorylated eIF‐2 is as efficient as unphosphorylated eIF‐2 in the formation of the 40S and 80S initiation complexes 22 . However, recent evidence indicates that the formation of the ternary complex (eIF‐2‐GTP‐met tRNA f ) and its subsequent binding to the 40S subunit involves other factors in addition to eIF‐2 23,24 and the phosphorylation of eIF‐2 may inhibit its interaction with these factors 24–26 . Work in this laboratory supports this view that HCR is not directly cAMP dependent.…”