Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E⅐4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70S6k , resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucineinduced effects. Thus, leucine acting through an mTORdependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.Certain amino acids, notably the essential amino acids, not only serve as precursors for protein synthesis, but also have important regulatory roles in the initiation phase of mRNA translation (1-3). Regulation of translation initiation is known to occur through modulation of two of the numerous steps in the pathway. The first regulated step is the binding of methionyl-tRNA i (Met-tRNA i ) to the 40 S ribosomal subunit to form the 43 S preinitiation complex (reviewed in Refs. 4 and 5). This step is mediated by eukaryotic initiation factor, eIF2, and involves formation of an eIF2⅐GTP⅐Met-tRNA i ternary complex followed by binding of the ternary complex to the 40 S ribosomal subunit. The overall process of Met-tRNA i binding is regulated through changes in the activity of the guanine nucleotide exchange factor for eIF2, termed eIF2B, and appears to involve changes in phosphorylation of either the ␣-subunit of eIF2 and/or the ⑀-subunit of eIF2B.The second regulated step in translation initiation is the binding of mRNA to the 43 S preinitiation complex (reviewed in Refs. 4 and 5). This step is mediated by a group of proteins collectively referred to as eIF4. During this step, eIF4E binds to the m 7 GTP cap structure present at the 5Ј-end of essentially all eukaryotic mRNAs and, through association with eIF4G, also binds to the 40 S ribosomal subunit. The mRNA binding step is regulated through changes in phosphorylation of eIF4E, with phosphorylation increasing the affinity of eIF4E for the cap structure (6) as well as by changes in the availability of eIF4E to form the active eIF4E⅐eIF4G c...

cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA i (Met-tRNAi) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2 ⅐ GTP ⅐ Met-tRNA i complex is thought to bind to the 40S ribosomal subunit before the eIF4G ⅐ eIF4E ⅐ mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the ␣-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes. eIF4E; eIF4G; eIF3; unfolded protein response; PERK ONE OF THE BEST-CHARACTERIZED mechanisms for regulating mRNA translation in eukaryotic cells involves phosphorylation of the ␣-subunit of eukaryotic initiation factor, eIF2 (reviewed in Ref. 16). During initiation, eIF2 forms a complex with GTP and initiator methionyl-tRNA i (met-tRNA i ), and this ternary complex subsequently binds to the 40S ribosomal subunit to form the 43S preinitiation complex (reviewed in Refs. 10 and 24). Through the action of a translation initiation factor complex referred to as eIF4F, which is comprised of eIF4A, eIF4E, and eIF4G, mRNA is bound to the 43S preinitiation complex, resulting in formation of the 48S preinitiation complex (reviewed in Ref. 20). During a later step in initiation, the GDP bound to eIF2 is hydrolyzed in an eIF5-mediated process and initiation factors are released from the ribosome. Before binding Met-tRNA i and reforming the ternary complex, the GDP bound to eIF2 must be exchanged for GTP, a reaction that is catalyzed by the guanine nucleotide exchange factor, eIF2B. One mechanism for regulating the activity of eIF2B involves phosphorylation of the ␣-subunit of eIF2 on Ser51, an event that converts eIF2 from a substrate into a competitive inhibitor of eIF2B (reviewed in Ref. 10). Thus, by inhibiting eIF2B, phosphorylation of eIF2␣ results in a global inhibition of protein synthesis.Hyperphosphorylation of eIF2␣ occurs under a variety of conditions that result in disruption of normal cell homeostasis. For example, conditions that impede correct folding of newly synthesized proteins in the lumen of the endoplasmic reticulum (ER), i.e., the so-called unfolded protein response, result in activation of the eIF2␣ kinase, PERK (also referred to as PEK) (28). PERK is a trans-ER membrane protein with a luminal domain...

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...