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...

The phosphorylation states of three proteins implicated in the action of insulin on translation were investigated, i.e., 70-kDa ribosomal protein S6 kinase (p70 S6k ), eukaryotic initiation factor (eIF) 4E, and the eIF-4E binding protein 4E-BP1. Addition of insulin caused a stimulation of protein synthesis in L6 myoblasts in culture, an effect that was blocked by inhibitors of phosphatidylinositide-3-OH kinase (wortmannin), p70 S6k (rapamycin), and mitogen-activated protein kinase (MAP kinase) kinase (PD-98059). The stimulation of protein synthesis was accompanied by increased phosphorylation of p70 S6k , an effect that was blocked by rapamycin and wortmannin but not PD-98059. Insulin caused dephosphorylation of eIF-4E, an effect that appeared to be mediated by the p70 S6k pathway. Insulin also stimulated phosphorylation of 4E-BP1 as well as dissociation of the 4E-BP1 ⋅ eIF-4E complex. Both rapamycin and wortmannin completely blocked the insulin-induced changes in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4E; PD-98059 had no effect on either parameter. Finally, insulin stimulated formation of the active eIF-4G ⋅ eIF-4E complex, an effect that was not prevented by any of the inhibitors. Overall, the results suggest that insulin stimulates protein synthesis in L6 myoblasts in part through utilization of both the p70 S6k and MAP kinase signal transduction pathways.

Until recently, transport of tRNA was presumed to be unidirectional, from the nucleus to the cytoplasm. Our published findings, however, revealed that cytoplasmic tRNAs move retrograde to the nucleus in Saccharomyces cerevisiae and that nuclear accumulation of cytoplasmic tRNAs occurs when cells are nutrient deprived. The findings led us to examine whether retrograde nuclear accumulation of cytoplasmic tRNAs occurs in higher eukaryotes. Using RNA FISH and Northern and Western analyses we show that tRNAs accumulate in nuclei of a hepatoma cell line in response to amino acid deprivation. To discern whether tRNA nuclear accumulation results from nuclear import of cytoplasmic tRNAs, transcription of new RNAs was inhibited, and the location of ''old'' tRNAs in response to nutrient stress was determined. Even in the absence of new RNA synthesis, there were significant tRNA nuclear pools after amino acid depletion, providing strong evidence that retrograde traffic is responsible for the tRNA nuclear pools. Further analyses showed that retrograde tRNA nuclear accumulation in hepatoma cells is a reversible and energy-dependent process. The data provide evidence for retrograde tRNA nuclear accumulation in intact mammalian cells and support the hypothesis that nuclear accumulation of cytoplasmic tRNA and tRNA re-export to the cytoplasm may constitute a universal mechanism for posttranscriptional regulation of global gene expression in response to nutrient availability.nucleus ͉ nutrient deprivation ͉ retrograde traffic I n eukaryotes the nuclear envelope separates mRNA transcription from cytoplasmic translation. The nuclear and cytoplasmic compartments interface at the nuclear pores, which regulate transport of molecules in and out of the nucleus. The majority of nuclear/cytoplasmic exchange occurs via a process that requires the small GTPase Ran and members of the ␤-importin protein family.With the exception of small nuclear RNA, the transport of RNA transcripts across the nuclear envelope was presumed to be unidirectional, from the nucleus to the cytoplasm (for review see ref. 1). This dogma has now been challenged. Yoshihisa and coworkers (2) showed that tRNA splicing in yeast occurs in the cytoplasm. Yet spliced tRNA can accumulate in the nucleus. Accumulation of spliced tRNA in the nucleus prompted examination of whether cytoplasmic tRNA is imported into the nucleus. Our work (3) and the work of Takano et al. (4) showed that tRNA moves retrograde from the cytoplasm to the nucleus in Saccharomyces cerevisiae.Retrograde tRNA nuclear import might serve to proofread tRNAs after splicing to separate improperly spliced tRNAs from the translation machinery. If so, then the retrograde process could be restricted to organisms with cytoplasmic pre-tRNA splicing. According to this scenario, vertebrate cells that splice pre-tRNA in the nucleus (5-7) may not import cytoplasmic tRNAs into their nuclei. However, because some tRNA modifications occur in the cytoplasm (for review see ref. 8) and the long-lived tRNAs could be damaged ...

Modulation of protein/protein interaction is an important mechanism involved in regulation of translation initiation. Specifically, regulation of the interaction of eIF2 with the guanine nucleotide exchange factor, eIF2B, is a key mechanism for controlling translation under a variety of conditions. Phosphorylation of the ␣-subunit of eIF2 converts the protein into a competitive inhibitor of eIF2B by causing an increase in the binding affinity of eIF2B for eIF2. Consequently, it has been assumed that the ␣-subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was found to bind only to the ␦-and ⑀-subunits of eIF2B, and eIF2B was shown to bind only to the ␤-subunit of eIF2 by far-Western blot analysis. The binding site on eIF2␤ for either the eIF2B holoprotein, or the isolated ␦-or ⑀-subunits of eIF2B was shown to be located within approximately 70 amino acids of the C terminus of the protein. Phosphorylation of the ␣-subunit of eIF2 did not promote binding of eIF2B to the isolated subunit. However, it did cause an increase in the affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase A of the ␤-subunit of eIF2 in the C-terminal portion of the protein increased the guanine nucleotide exchange activity of eIF2B, whereas phosphorylation by casein kinase II or protein kinase C was without effect.The translation initiation phase of protein synthesis is a complex process mediated by a family of at least 12 proteins collectively referred to as eukaryotic initiation factors (reviewed in Refs. 1-3). Regulation of translation initiation plays a crucial role in maintaining protein homeostasis within cells in response to a variety of hormonal, nutritional, and environmental stimuli. One of the most tightly regulated steps in translation initiation involves two multisubunit proteins referred to as eukaryotic initiation factors eIF2 and eIF2B. During initiation, eIF2 binds GTP and initiator methionyl-tRNA i (Met-tRNA i ) 1 , and the eIF2⅐GTP⅐Met-tRNA i ternary complex then binds to a 40 S ribosomal subunit. Upon joining of the 60 S ribosomal subunit, the GTP bound to eIF2 is hydrolyzed to GDP, and eIF2 is released from the ribosome as an eIF2⅐GDP binary complex. Before eIF2 can participate in another cycle of initiation, the GDP bound to the protein must be exchanged for GTP. This guanine nucleotide exchange reaction is mediated by the heteropentameric protein, eIF2B.One of the ways through which the activity of eIF2B is regulated occurs through an unusual mechanism involving phosphorylation of one of the subunits of its substrate eIF2 (reviewed in Ref.3). The smallest, or ␣-subunit, of eIF2 is phosphorylated in response to a variety of cellular stresses including viral infection, heat shock, heavy metals, and deprivation of amino acids or serum. Previous studies have reported that the affinity of eIF2B for eIF2(␣P) is increased either 2-fold (4) or 150-fold (5), depending on the method used to measure the interaction. An obvious assumption from these studies is that the ␣-subunit...

When primary cultures of rat hepatocytes were placed in a chemically defined serum-free medium containing a combination of insulin, glucagon, and dexamethasone, the synthesis of albumin and total protein and the cellular content of RNA and DNA were maintained at constant values for 8 days. Despite the constant rate of albumin synthesis, secretion of the protein increased more than twofold during the initial 4 days in culture and was then maintained at a value similar to that observed in vivo through day 8. This observation suggested an initial defect in albumin secretion that was corrected with time in culture. Deprivation of insulin between days 2 and 5 resulted in a decline in albumin secretion to approximately 40% of the control value. The decline in albumin secretion was accompanied by proportional decreases in albumin synthesis, albumin mRNA, and albumin gene transcription. Return of insulin-deprived cells to complete medium on day 5 restored albumin synthesis and secretion as well as albumin mRNA to control values by day 8. Deprivation of either glucagon or dexamethasone also resulted in reduced albumin synthesis and secretion accompanied by proportional decreases in albumin mRNA and gene transcription. However, the magnitude of the changes in these parameters was less with glucagon or dexamethasone deprivation compared with insulin deprivation. Return of glucagon- or dexamethasone-deprived cells to complete medium on day 5 restored albumin synthesis and secretion as well as albumin mRNA to control values by day 8.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonic anhydrase (CA) was examined in two adipocyte cell lines, 3T3-L1 and 3T3-F442A. Both CA III and non-CA III activities, measured by 18O mass spectrometry, were present in 3T3-L1 and 3T3-F442A adipocytes; however, no CA activity was detected in 3T3 preadipocytes of either line. These observations were supported by immunoblot experiments employing CA III and CA II isoform-specific antisera. CA III, a major protein in rodent and murine adipocytes, and CA II, another isoform known to be present in adipose tissue, were observed only in the differentiated 3T3 adipocytes. The differentiation-dependent expression of these isozymes may imply an adipocyte-related role for CA. Compared with cultures maintained in the absence of insulin, 3T3 adipocytes maintained in the presence of insulin exhibited 65-90% lower concentrations of CA III. CA II was unaffected. This negative effect of insulin on CA III may explain the metabolic regulation of adipose CA III observed in vivo. After media changes, 3T3 adipocyte cultures rapidly lower media pH, which in turn lowers the bicarbonate/CO2 of bicarbonate/CO2-buffered media. Cultures maintained at low pH displayed 50-90% lower concentrations of CA II and CA III. Similarly, cultures maintained in a low bicarbonate/CO2 media (GibCO2-I medium containing 1 mM bicarbonate under an atmosphere of 100% humidified air) displayed 30-50% lower CA II and CA III concentrations. Thus CA II and CA III concentrations are influenced by pH and bicarbonate/CO2. Neither effect, the pH or the GibCO2-I media effect, was associated with changes in the concentration of pyruvate carboxylase or ATP citrate lyase (2 markers of adipocyte differentiation). Because the regulation by pH and bicarbonate/CO2 may be relatively selective for CA in adipocytes, a simple method for reducing the concentration/activity of CA in 3T3 adipocytes is described that may be a useful tool for studies on the physiological role of the enzyme.