Initiation factor protein modifications and inhibition of protein synthesis.

Abstract

The protein covalent modification state of eucaryotic initiation factors eIF-2 and eIF-4B in HeLa cells was examined after they were exposed to a variety of conditions or treatments that regulate protein synthesis. A few factors (e.g., variant pH and sodium fluoride) altered the phosphorylation state of the initiation factor proteins, but the majority (hypertonic medium, ethanol, dimethyl sulfoxide, sodium selenite, sodium azide, and colchicine) had no effect on either protein. While initiation factor phosphorylation may regulate protein synthesis in response to many physiological situations, other pathways can regulate protein synthesis under nonphysiological circumstances.The rate of protein synthesis in mammalian cells can be regulated by a variety of physiological (4,8,12,15) and nonphysiological (28) treatments. Inhibition principally occurs at the initiation phase (16,22), wherein Met-tRNA, ribosomal subunits, and mRNA associate to form the translationally active ribosome. Each of the initiation steps requires or is promoted by one or more protein cofactors, called eucaryotic initiation factors, which transiently associate with the translational machinery during the initiation process (20,22).These protein factors are, a priori, likely candidates to serve as regulatory molecules. Substantial evidence indicates that phosphorylation of 20 to 30% of the eIF-2a molecules leads to a protein synthesis inhibition of greater than 75% in rabbit reticulocytes (11, 16) and probably in other eucaryotic cells as well (6,8,27). We and others have shown that covalent modification or cleavage of initiation factor proteins correlates with an inhibition of protein synthesis in a variety of instances including hemin deprivation (11), nutrient deprivation (8), heat shock (3, 6, 9), and poliovirus infection (10). Although it now seems clear that initiation factors play a role in translational control in some situations, it remains to be determined how frequently modulations of initiation rate are mediated by eIF-2a phosphorylation and how many of the factors can serve as controlling elements.Using rapid, sensitive qualitative and quantitative techniques for examining covalent modification of the initiation factor proteins (5), we have shown that eIF-2a, eIF-2p, eIF-4B, and eIF-4Fp28 (CBP-I) are covalently altered when translation is inhibited in HeLa cells by either heat shock (6, 8a) or serum depletion (8). Both treatments induced parallel initiation factor changes, suggesting the existence of a common response pathway that could be linked to many dissimilar stimuli. We initiated the present study to extend our survey of initiation factor status during translational inhibitions.The initiation process is inhibited by increasing the tonicity of the growth medium (24). Excess NaCl (125 mM) inhibits by >95% the incorporation of amino acids into protein by HeLa cells (data not shown). In these and subsequent experiments, cytoplasmic lysates prepared from inhibited cells were analyzed by isoelectric focusing/sodium * Corresponding ...

Phosphorylation of Eukaryotic Initiation Factor (eIF) 4E Is Not Required for de Novo Protein Synthesis following Recovery from Hypertonic Stress in Human Kidney Cells

“…2B). In agreement with published data for HeLa cells (40), over the times used in this study there was little change in the phosphorylation status of eIF2␣ in response to hypertonic stress (Fig. 2C).…”

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.…”

Endotoxin and cisplatin synergistically stimulate TNF-α production by renal epithelial cells

“…In studies of skeletal and cardiac muscle, sepsis interfered with translation initiation (18,19). eIF4E is the rate-limiting component of eukaryotic translation initiation (9). Since both ERK and p38 MAP kinase pathways, acting through Mnk1, can result in the phosphorylation of eIF4E (53, 54), we examined the effects of cisplatin and LPS on the phosphorylation states of eIF4E and Mnk1.…”