Implication of eIF2B Rather Than eIF4E in the Regulation of Global Protein Synthesis by Amino Acids in L6 Myoblasts

Abstract

The present study was designed to investigate the mechanism through which leucine and histidine regulate translation initiation in L6 myoblasts. The results show that both amino acids stimulate initiation and coordinately regulate the activity of eukaryotic initiation factor eIF2B. The changes in eIF2B activity could be explained in part by modulation of the phosphorylation state of the ␣-subunit of eIF2. The activity changes might also be a result of modulation of the phosphorylation state of the eIF2B ⑀-subunit, because deprivation of either amino acid caused a decrease in eIF2B⑀ kinase activity. Leucine, but not histidine, additionally caused a redistribution of eIF4E from the inactive eIF4E⅐4E-BP1 complex to the active eIF4E⅐eIF4G complex. The redistribution was a result of increased phosphorylation of 4E-BP1. The changes in 4E-BP1 phosphorylation and eIF4E redistribution associated with leucine deprivation were not observed in the presence of insulin. However, the leucine-and histidine-induced alterations in global protein synthesis and eIF2B activity were maintained in the presence of the hormone. Overall, the results suggest that both leucine and histidine regulate global protein synthesis through modulation of eIF2B activity. Furthermore, under the conditions employed herein, alterations in eIF4E availability are not ratecontrolling for global protein synthesis but might be necessary for regulation of translation of specific mRNAs.Amino acids play important and multiple roles in regulating protein synthesis in skeletal muscle (1). An obvious role is to act as precursors for protein synthesis. A less obvious but equally important role involves the regulation of translation initiation. The initiation of mRNA translation is a complicated process involving over a dozen proteins, referred to as eukaryotic initiation factors (eIFs) 1 (reviewed in Refs. 2 and 3). Of all the steps in the initiation pathway, only two have been identified that are subject to regulation in vivo; the binding of tRNA iMet to the 40 S ribosomal subunit and the binding of mRNA to the 43 S preinitiation complex. In the first step in initiation, tRNA i Met binds to the 40 S ribosomal subunit as a ternary complex with eIF2 and GTP. Subsequently, the GTP bound to eIF2 is hydrolyzed to GDP, and eIF2 is released from the ribosomal subunit as a complex with GDP. Formation of the ternary complex is regulated by modulation of the activity of a second initiation factor, eIF2B, which mediates guanine nucleotide exchange on eIF2. It is regulated by phosphorylation of the ␣-subunit of eIF2, where phosphorylation converts eIF2 from a substrate into a competitive inhibitor of eIF2B.The binding of mRNA to the 43 S preinitiation complex involves a group of proteins collectively referred to as eIF4 (reviewed in Refs. 2 and 4). The protein that binds to the m 7 GTP cap present at the 5Ј-end of most eukaryotic mRNAs is termed eIF4E. The eIF4E⅐mRNA complex binds to the 40 S ribosomal subunit through the association of eIF4E with eIF4G. An important mechani...

Role of p70S6K1-mediated Phosphorylation of eIF4B and PDCD4 Proteins in the Regulation of Protein Synthesis

“…Preparation of the 4E-BP1 and eIF4E antibodies has been described previously (13,14). Anti-S6K1, and goat antirabbit IgG horseradish peroxidase-conjugated antibodies were purchased from Bethyl Laboratories.…”

Assessment of biomarkers of protein anabolism in skeletal muscle during the life span of the rat: sarcopenia despite elevated protein synthesis

“…The amount of eIF4G present in a complex with eIF4E was quantitated as described previously (24). Briefly, eIF4E was immunoprecipitated from the muscle homogenate using a monoclonal anti-eIF4E antibody.…”

Nck-1 Antagonizes the Endoplasmic Reticulum Stress-induced Inhibition of Translation

“…Phosphorylated eIF2␣ has a higher affinity for eIF2B and forms with it a stable complex where the bound GDP cannot be exchanged for GTP (14). Since eIF2 is normally in excess of eIF2B, phosphorylated eIF2␣ in the eIF2 complex essentially sequesters the cellular eIF2B activity, and this leads to a general inhibition of translation (14).…”

“…In order to efficiently initiate protein translation, the trimeric eIF2 complex composed of the ␣, ␤, and ␥ subunits needs to recycle from eIF2-GDP (inactive form) to eIF2-GTP (active form), and this is regulated by the guanine nucleotide exchange factor (GEF), eIF2B (13). Phosphorylated eIF2␣ has a higher affinity for eIF2B and forms with it a stable complex where the bound GDP cannot be exchanged for GTP (14). Since eIF2 is normally in excess of eIF2B, phosphorylated eIF2␣ in the eIF2 complex essentially sequesters the cellular eIF2B activity, and this leads to a general inhibition of translation (14).…”