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

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... he 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 i Met 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 mechanism for regulating the binding of mRNA to the 40 S ribosomal subunit occurs through sequestration of eIF4E into an inactive complex with the eIF4E binding protein, 4E-BP1 (5, 6). The eIF4E⅐4E-BP1 complex can still bind to the m 7 GTP cap structure but not to eIF4G (7, 8). Thus, binding of eIF4E to 4E-BP1 prevents the binding of mRNA to the ribosome. The binding of 4E-BP1 to eIF4E is regulated by phosphorylation of 4E-BP1 with increased phosphorylation of the protein causing a decrease in the affinity of 4E-BP1 for eIF4E. In skeletal muscle in vivo as well as in perfused muscle preparations, protein synthesis is stimulated by amino acids through a mechanism involving an increase in the rate of translation initiation (reviewed in Ref. 1). The stimulation of initiation caused by a complete mixture of amino acids can be reproduced by provision of only the branched chain amino acids (9, 10). Furthermore, of the branched chain amino acids, leucine has been shown to stimulate initiation in the absence of other amino acids (11), suggesting that it plays a key role in the regulation of protein synthesis. However, although leucine has been known to stimulate protein synthesis in muscle for years, the mechanism through which it acts on initiation is still unknown. Leucine also stimulates initiation in tissues other than skeletal muscle (1). However, in tissues such as liver, leucine is not the only amino acid that can regulate initiation. In particular, in rat liver, histidine stimulates initiation through a mechanism involving changes in the phosphorylation state of the ␣-subunit of eIF2 (eIF2␣) (12). The decreased phosphorylation of eIF2 caused by histidine is associated with a stimulation of eIF2B activity. Whether leucine has a similar effect on eIF2 phosphorylation in liver or muscle is unknown. In the present study, the mechanism through which leucine and histidine regulate translation initiation was examined in L6 myoblasts.