INVITED REVIEW
4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

O. Jameel Shah, Joshua C. Anthony, Scot R. Kimball, and Leonard S. Jefferson

Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
OVERVIEW OF TRANSLATION...
PHYSIOLOGICAL SCENARIOS OF...
4E-BP1 AND S6K1: MECHANISMS...
CONCLUDING REMARKS
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Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.

eukaryotic initiation factor 4E binding protein; 70-kDa ribosomal protein S6 kinase; translation initiation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
OVERVIEW OF TRANSLATION...
PHYSIOLOGICAL SCENARIOS OF...
4E-BP1 AND S6K1: MECHANISMS...
CONCLUDING REMARKS
REFERENCES

IN THE SECTIONS THAT FOLLOW, our current ideas of translational control will be discussed largely in the context of skeletal muscle, because this tissue has been extensively characterized and because it is exquisitely responsive to changes in circulating concentrations of hormones and nutrients. Moreover, the individual and interdependent roles of amino acids, insulin, and glucocorticoids as determinants in translational control, particularly at the level of translation initiation, will be discussed. Finally, the mechanisms and pathways through which these regulatory molecules influence the translational machinery will be presented, with particular weight given to control of the availability of eukaryotic initiation factor 4E (eIF4E), as well as to the activation of the 70-kDa 40S ribosomal protein S6 kinase (S6K1).


    OVERVIEW OF TRANSLATION INITIATION
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The process of messenger RNA (mRNA) engagement by the ribosome is regulated by a number of protein factors functionally classified as eukaryotic initiation factors, or eIFs (Fig. 1). It is these translational effectors that collaboratively orchestrate the initial event in protein biosynthesis (reviewed in Refs. 59 and 81). As a prelude to the initiation event itself, the initiator methionyl tRNA (tRNAiMet) is recognized and coupled to eIF2, a heterotrimeric guanine nucleotide binding protein that serves to shuttle tRNAiMet to the 40S ribosomal subunit. However, this recognition is conditional: only in the GTP-liganded conformation can eIF2 productively interact with Met-tRNAiMet. The nucleotide binding state of eIF2 is, in turn, regulated by eIF2B, a complex, heteropentameric guanine nucleotide exchange factor that catalyzes replacement of a molecule of eIF2-bound GDP with GTP. Therefore, eIF2B's intrinsic exchange activity dictates the translational competence of eIF2. The activity of eIF2B is negatively regulated by phosphorylation of Ser51 on the alpha -subunit of eIF2, which converts eIF2 from a substrate into a competitive inhibitor of eIF2B (reviewed in 43). Several eIF2alpha kinases have been identified, including the heme-regulated translational inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), the mammalian homolog of yeast GCN2 (mGCN2), and pancreatic eIF2alpha kinase/PKR-like endoplasmic reticulum (ER) kinase (PEK/PERK), whose activation under conditions of cellular stress induces an abrupt translational arrest. The activity of eIF2B also appears to be subject to direct regulation through phosphorylation of its varepsilon -subunit (123) and perhaps allosterically by the redox state of pyridine dinucleotides (reviewed in 122).


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Fig. 1.   Diagrammatic representation of the initiation phase of mRNA translation. Eukaryotic initiation factors (eIFs) are depicted with respective alphanumeric designations, e.g., 1A for eIF1A, and the like. 4E-BP1, eIF4E binding protein 1; AA, amino acids; AUG, nucleotide triplet encoding the translational start codon.

A second, well characterized mechanism of translational control involves the 7-methyl-GTP cap-mediated recruitment of mRNA to the 40S ribosomal subunit. The efficiency of this mRNA binding event is determined by the availability of eIF4E, which recognizes the 7-methylguanosine modification at the 5' terminus of the vast majority of cellular mRNA. When the demand for translation is low, eIF4E is sequestered by a family of eIF4E binding proteins (4E-BPs; 4E-BP1 was formerly known as PHAS-I for phosphorylatable heat- and acid-stable inhibitor), thus precluding its interaction with other initation factors. 4E-BP1, however, is acutely phosphorylated in response to growth-promoting stimuli, which serves to perturb the otherwise stable eIF4E · 4E-BP1 complex. Once disengaged, eIF4E can productively associate with eIF4G, a 150-kDa protein scaffold that makes additional physical contacts with eIF4A. The complex comprised of eIF4E, eIF4G, and eIF4A is collectively referred to as eIF4F. eIF4A, in synergy with eIF4B, comprises an RNA helicase that uncoils secondary structure within the mRNA's 5'-untranslated region. eIF3 tethers the 40S ribosomal subunit to eIF4G, and the poly(A)-binding protein-eIF4G interaction brings both of the mRNA's 5' and 3' termini into proximity, potentially increasing the likelihood of reinitiation.

The translational machinery itself appears subject to a unique mode of translational regulation. The mRNAs encoding numerous ribosomal proteins and translation factors exhibit a distinctive structural feature: an oligonucleotide tract rich in pyrimidines juxtaposed to the 5'-cap. This nucleotide signature, designated TOP (terminal oligopyrimidine), confers selective translational induction of such messages in response to a range of mitogenic stimuli. There is a strong correlation between expression of TOP mRNAs and the phosphorylation status of the ribosomal phosphoprotein S6. As an integrant of the 40S ribosomal particle, S6 is localized to regions involved in mRNA and tRNA recognition (11, 112, 113, 116). Ribosomes phosphorylated on S6 display an augmented binding capacity and diminished dissociation rate for synthetic polyuridine oligomers (39). Thus phospho-S6, through mechanisms that are ill defined, recruits TOP-bearing mRNAs to the ribosome and thereby destines the expression of the proteins they encode. The growth factor-stimulated phosphorylation of S6 is effected by the S6Ks, a family of rapamycin-sensitive, serine/threonine protein kinases. In avidly dividing cells, S6K1 is necessary for progression beyond the G1 checkpoint (61) and mediates the transcriptional activation of E2F (12). Moreover, Drosophila S6K (dS6K) is involved in cell size determination (72), whereas embryonic deletion of S6K1 results in a small mouse phenotype (105). Although TOP mRNAs may be derived from fewer than 200 genes (68), this transcript family can represent as much as 20-30% of total cellular mRNA, highlighting the importance of S6K function.


    PHYSIOLOGICAL SCENARIOS OF TRANSLATION CONTROL
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Starvation/Nutrient Insufficiency

It has long been appreciated that starvation or nutrient deprivation profoundly modifies protein metabolism in skeletal muscle. Early work suggested that, in a fed and nutritionally sufficient state, the rate of protein synthesis was limited by elongation rather than initiation, because most ribosomal subunits were polysomal and thus actively engaged in translating mRNA (73). However, in animals subjected to a 48-h fast, the accumulation of free 40S and 60S ribosomal subunits indicated that the reduced rate of protein synthesis reflected a diminished rate of translation initiation (91). Interestingly, the fasting-induced attenuation of protein synthesis in skeletal muscle is somewhat biphasic: the initial 24-h period of food deprivation is associated with reduced translational efficiency secondary to hindered translation initiation, whereas greater periods of starvation additionally involve suppressed translational capacity; that is, RNA content in the muscle declines (65). The eIF4F system appears to be rapidly impacted by starvation, because the protein synthetic reduction observed in an 18-h fast is associated with an increase in the electrophoretic mobility of 4E-BP1 (132, 133), which is an index of 4E-BP1 dephosphorylation. Predictably, the stability of the inhibitory eIF4E · 4E-BP1 complex is favored, whereas that of the eIF4E · eIF4G complex is deterred. Consumption of a nutritionally complete meal, within 1 h, reactivates eIF4F and restores rates of protein synthesis (132). Such brief starvation, however, does not alter the activity of eIF2B (132), suggesting that the eIF4 regulatory element is the primary site of regulation during transient periods of nutrient deficiency.

When evaluated in perfused psoas muscle preparations, longer periods of starvation, i.e., 48 or 72 h, were associated with a precipitous reduction in [35S]Met-tRNAiMet bound by postribosomal supernatants, implying that "initiation factor activity" or "eIF2-like activity" was diminished (91). Although no data are currently available regarding the activation of either eIF2B or eIF4F under conditions of prolonged starvation, it is reasonable to predict an impairment in both processes.

Role of Insulin

Although alterations in both protein synthesis and translation initiation brought about by a meal have been well defined, the primary mediator of these changes in skeletal muscle has been difficult to identify. Several lines of evidence, however, suggest that insulin may regulate postprandial protein metabolism in skeletal muscle. First, insulin concentrations rise rapidly in the circulation in response to glucose and amino acids, which facilitates uptake of these fuels and protein substrates into relevant tissues. Furthermore, experimental and pathological states of insulin deficiency, such as diabetes, are characterized by compromised translational function, which can be corrected by provision of exogenous insulin (reviewed in Refs. 49 and 48). Finally, insulin is mitogenic in dividing cells and anabolic in complex, terminally differentiated tissues, provided the cell and tissue, respectively, express sufficient levels of the insulin receptor. It was initially believed that insulin's anabolic properties could be attributed to accelerated amino acid import and tRNA aminoacylation (57, 127). These findings were subsequently refuted, as studies in perfused skeletal muscle revealed that insulin enhanced rates of protein synthesis despite reducing the intracellular concentrations of all amino acids save glutamine, serine, and lysine (51). In perfused rat hindlimb preparations, it was found that extended perfusion periods of 2-3 h produced a restraint on translation initiation, as evidenced by an increase in size of free ribosomal subunit pools. Addition of insulin during these periods, however, restored rates of protein synthesis and ribosomal distribution to preperfusion levels (50). In rat hindlimb perfusions, addition of insulin augments both the protein synthetic rate and assembly of eIF4F in the gastrocnemius (56), whereas in alloxan-induced diabetes, the stability of the inhibitory eIF4E · 4E-BP1 complex is favored but reversed by insulin treatment (55). Despite these and several additional investigations demonstrating that addition of insulin or insulin-like growth factor I (IGF-I) stimulates the protein synthetic response to feeding (47, 53, 110, 119, 131), accumulating evidence suggests that the role of insulin in stimulation of postprandial protein synthesis may be only a permissive one. Indeed, both nonobese diabetic (NOD) and ob/ob mice, which exhibit characteristics of insulin-dependent (type 1) and insulin-independent (type 2) diabetes, respectively, respond to feeding in a fashion similar to normal mice: activation of both eIF4F and S6K1 are preserved, the respective defects in insulin action notwithstanding (109). Furthermore, leucine administered orally is as effective as a nutritionally complete meal in stimulating translation initiation and protein synthesis without increasing circulating concentrations of insulin (4). Although anti-insulin antibodies administered to fasted animals substantially reduce the anabolic response to feeding (88, 110, 131), one major distinction must be made between these and the aforementioned studies: the addition of anti-insulin antibodies renders circulating insulin concentrations undetectable (88, 110), whereas those values obtained from NOD mice are on the order of 25 µU/l (110). Collectively, the available data are consistent with a model in which the postprandial stimulation of translation initiation and overall protein synthesis are largely independent of the meal-induced increase in circulating insulin concentrations. This anabolic response does, however, require permissive concentrations of the hormone. It should be noted that such a paradigm does not invalidate the numerous reports of stimulation of mRNA translation by supraphysiological concentrations of insulin.

Role of Amino Acids

Perhaps the most crucial element involved in the postprandial acceleration of translation initiation and protein synthesis is the protein substrates themselves---that is, amino acids. The anabolic properties of amino acids have long been appreciated; however, only recently has significant progress been made in elucidation of mechanisms by which amino acids modulate the protein synthetic apparatus. In humans, oral administration of either the full complement of amino acids (10, 119) or simply the essential amino acids (114) enhances protein synthesis in skeletal muscle. It is apparent that translation initiation, as evidenced by the aggregation of polysomes, is related to the dietary quality of proteins (129) as well as the supplemental amount of individual essential amino acids (30, 107, 108, 130). In skeletal muscle of fasted rats, whereas a meal consisting of 20% protein stimulates assembly of eIF4F concomitant with global protein synthesis, a meal devoid of protein, but identical in all other respects, is ineffective in these regards (133). Furthermore, both protein-enriched and protein-free diets give rise to similar increases in the concentration of plasma insulin despite a disparity in protein metabolism (133), underscoring the necessity of amino acids in the postprandial enhancement of translation initiation. Of the essential amino acids, leucine appears to harbor the bulk of the regulatory influence on protein metabolism. In fact, leucine, administered orally to fasted rats, restores protein synthetic rates to the levels observed in animals fed ad libitum (4). Such increases in protein synthesis are accompanied by assembly of eIF4F and activation of S6K1, and they occur independently of changes in plasma insulin (4).

Role of Glucocorticoids

Glucocorticoids are among the most potent negative regulators of protein homeostasis and display vastly diverse functions across a broad range of tissues. An excess production of glucocorticoids, derived from either pathological (e.g., Cushing's syndrome) or exogenous (e.g., glucocorticoid therapy) sources, has been associated with weight loss (15, 36), atrophy of the musculature (15, 35, 36, 117), and diminished rates of protein synthesis (35, 69, 94, 101, 106). Early studies indicated that glucocorticoids acutely reduce translational efficiency, as revealed by the decline in ribosomal particles incorporated into polysomes (92, 94). This acute effect of glucocorticoids emerges within the initial 4 h interval and appears to be independent of the exchange activity of eIF2B (101). The acute reduction in the protein synthetic rate involves disassembly of productive eIF4F. In rats administered glucocorticoids for 4-h, skeletal muscle extracts display predominantly hypophosphorylated forms of 4E-BP1, an increase in the number of eIF4E · 4E-BP1 complexes, and reduced eIF4E-eIF4G interaction. Furthermore, S6K1 from similar extracts is dephosphorylated and thereby inactivated after exposure of the animal to glucocorticoids. Because S6K1 is known to control the translation of mRNAs encoding a portion of the translational apparatus, its inhibition by glucocorticoids may contribute to the reduced translational capacity observed in muscles from chronically treated animals. Studies conducted in L6 myoblasts have demonstrated that the regulation of translational effector pathways is due to a direct effect of glucocorticoids on the muscle cell itself rather than a result of some physiological perturbation that affects the muscle secondarily. Moreover, disassembly of eIF4F and dephosphorylation of 4E-BP1, S6K1, and the ribosomal protein S6 follow a temporally similar pattern of regulation by glucocorticoids, which manifests significantly within 2 h and is precipitously exacerbated in relation to the exposure period. Extended periods of glucocorticoid excess reduce translational capacity in muscle as total RNA declines, reflecting diminished ribosome biogenesis (92-94). Additionally, ternary complex formation is attenuated (92, 94) with prolonged exposure, suggesting an impairment in the activity of eIF2B.


    4E-BP1 AND S6K1: MECHANISMS OF REGULATION
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Although our understanding of the signaling events initiated through activation of various growth factor receptors has become increasingly refined in recent years, relatively little is known regarding points at which amino acid- and glucocorticoid-induced signals converge upon such relay networks, particularly those relay systems utilized by the insulin receptor. Because numerous insightful reviews on insulin signal transduction are currently available (6, 79, 111), this section will be strictly limited to a discussion of those insulin-activated pathways directly impacting the translational apparatus. Moreover, these translational effector pathways will serve as the context for discussion of mechanisms through which information generated by amino acids, insulin, and glucocorticoids is integrated (Fig. 2). For the sake of continuity, this section will remain tailored to the regulation of the phosphorylation state of 4E-BP1 and the activation of S6K1.


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Fig. 2.   Integration of insulin-, amino acid-, and glucocorticoid-generated information by translational effectors as supported by available data. In such a model, the liganded insulin receptor recruits insulin receptor substrates (IRSs), which provide a docking site for phosphatidylinositol (PI) 3-kinase. The latter interaction stimulates activity of the catalytic subunit (p110) of PI 3-kinase, thereby increasing the local concentration of membrane phosphoinositides (PIP2 and PIP3). Pleckstrin homology (PH) domain-harboring proteins such as phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB) are recruited to the membrane by virtue of their affinity for phosphoinositides; this facilitates activation of PKB, and indeed other substrates, e.g., ribosomal protein S6 kinase 1 (S6K1), PKCzeta /lambda , and PKCdelta , by PDK1. PDK1 phosphorylates PKCzeta /lambda , a component of a poorly understood effector complex that transduces activating stimuli to a secondary effector unit comprised of mammalian target of rapamycin (mTOR) and PKCdelta . PKB may also have a role in activation of the mTOR · PKCdelta complex, which in turn may regulate eIF4E binding protein 1 (4E-BP1) and S6K1 directly through phosphorylation, or alternatively, indirectly through restraint of a protein phosphatase (PPase), which would otherwise dephosphorylate and thereby inactivate 4E-BP1 and S6K1. Amino acids are sensed through an unknown mechanism; on recognition of amino acids, information is, in a cryptic manner, propagated to mTOR, which in turn modulates 4E-BP1 and S6K1. As with amino acids, the site of action of glucocorticoids is unknown (represented by gray box). However, both amino acids and glucocorticoids appear to regulate distal rather than proximal components of the insulin signal transduction pathway. RTK, receptor tyrosine kinase; PIP2 and PIP3, phosphatidylinositides.

Growth Factors, Such as Insulin

The insulin receptor, which is a prototype for the receptor tyrosine kinase (RTK) family of growth factor receptors, serves to organize multiple signaling molecules at the plasma membrane, allowing productive interaction with substrates and efficient transmission of information about the cellular environment into the cell. These effectors are recruited directly or indirectly [via adapter proteins, e.g., the insulin receptor substrate (IRS) and Shc] by the insulin receptor; two prominent signaling modules activated in this manner include the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway and the phosphatidylinositol (PI) 3-kinase/phosphoinositide dependent kinase 1 (PDK1)/ protein kinase B (PKB) pathway, both of which, to differing extents, appear to modulate the translational apparatus.

The Ras/Raf/MEK/ERK Pathway

The discovery of inhibitors of specific effector molecules has facilitated characterization of particular routes of signal relay, as well as ascription of their relevance in translational control. Because PD-098059, a specific inhibitor of MEK1/2, does not prevent the rapid, RTK-mediated phosphorylation of 4E-BP1 (5, 7, 66, 83, 120), neither MEK1/2 nor ERK1/2 appears to represent immediate upstream effectors. Moreover, in cells expressing dominant-interfering Ras (70) or partial loss-of-function platelet-derived growth factor (PDGF) receptors (21), S6K1 activation is maintained despite defects in the Ras/Raf/MEK/ERK pathway. However, it has been reported that insulin-stimulated S6K1 activation and 4E-BP1 phosphorylation are susceptible to inhibition by PD-098059 at insulin concentrations below the ERK activation threshold (100). These data imply that an insulin-stimulated and PD-098059-inhibitable S6K1 and 4E-BP1 effector that is not ERK1/2 may represent an authentic upstream input.

Although rapid, RTK-mediated activation of S6K1 and phosphorylation of 4E-BP1 occur independently of either MEK or ERK, prolonged activation of the Ras/Raf/MEK/ERK pathway may modulate S6K1 activation and 4E-BP1 phosphorylation. For instance, farnesyltransferase inhibitors, which interfere with Ras activation (Ki-Ras being a notable exception), suppress S6K1 activity and 4E-BP1 phosphorylation in dividing cells (62). Furthermore, stable expression of a conditionally active estrogen receptor-Raf-1 chimera (Delta Raf-1:ER) renders S6K1 responsive to estradiol (64). Finally, the activity of S6K1 in cells transfected with a constitutively active form of MEK1 is equivalent to the activity observed in quiescent cells stimulated with serum (64). However, it remains unclear whether artificial activation of the Ras/Raf/MEK/ERK pathway through such genetic manipulations affects S6K1 activation directly or rather circuitously, perhaps via an effect at the genomic level.

The PI 3-Kinase/PDK1/PKB Pathway

The findings that two structurally unrelated inhibitors of PI 3-kinase, e.g., wortmannin and LY-294002, greatly diminish the responsiveness of S6K1 and 4E-BP1 to various mitogenic stimuli indicated an essential role for PI 3-kinase-activated pathways in this process. PI 3-kinase phosphorylates phosphoinositides [e.g., PI, (4)-P, PI-(4,5)-P2] at the D3 position of the inositol ring, generating bioactive phospholipids [e.g., PI-(3)-P, PI-(3,4)-P2, PI-(3,4,5)-P3] within the plasma membrane. These integral membrane phospholipids, particularly PI-(3,4)-P2 and PI-(3,4,5)-P3, are recognized by the pleckstrin homology (PH) domains present in numerous proteins, a property that serves to recruit PH domain-harboring proteins to the plasma membrane during periods of phosphoinositide synthesis. Both the PDK1 and protein kinase B (PKB/Akt) possess PH domains and thus co-localize at the membrane in response to mitogens. This co-localization at the membrane allows efficient phosphorylation of PKB by PDK1 (3) [and perhaps by another kinase, i.e., the Ser473 kinase, whose identity remains controversial (8, 24, 115)]; PKB is then competent to phosphorylate both cytosolic and nuclear substrates (reviewed in 23, 54). The increased availability of eIF4E, which accompanies growth factor stimulation, appears to be dependent on pathways downstream of PKB. Whereas a constitutively membrane-targeted (and thus constitutively active) form of PKB promotes phosphorylation of 4E-BP1, a catalytically inert PKB variant blocks 4E-BP1 phosphorylation in response to insulin (33). Moreover, expression of a conditionally active PKB/estrogen receptor chimera confers 4-hydroxytamoxifen-inducible phosphorylation of 4E-BP1 with kinetics similar to those of activation of the chimeric protein itself (58). Also, activated PKB variants protect 4E-BP1 from inhibition by wortmannin (33), indicating that PKB operates downstream of PI 3-kinase in the regulation of eIF4E availability. Although expression of a constitutively membrane-anchored PKB variant induces the activation of S6K1, evidence exists that undermines the authenticity of PKB as an upstream activator of S6K1. Constitutive activity is bestowed on PKB by either of two modifications: a membrane-targeting sequence or acidic substitution of two key activating phosphorylation sites (T308 and S473), which artificially mimic their phosphorylation. Whereas either gain-of-function variant promotes phosphorylation of 4E-BP1, only the membrane-targeted form activates S6K1 (27). Furthermore, a dominant-interfering PKB mutant has no effect on insulin-induced S6K1 activation, although PKB activation and glycogen synthase kinase (GSK)-3beta inactivation are adversely affected (27). Because the activation of PKB and S6K1 are dissociable under various conditions (22, 102), cumulatively the data suggest that S6K1 activation by membrane-targeted forms of PKB is likely to be an artifact of membrane localization, which allows promiscuous activation of some S6K1 activator.

Mammalian Target of Rapamycin

How are signals propagated from PKB to the translational apparatus? Several lines of evidence suggest that the mammalian target of rapamycin (mTOR) may represent a critical intermediate in this pathway. First, an antibody (mTAB1) raised against an epitope within mTOR harboring a near-consensus PKB phosphorylation site displays reduced immunoreactivity both in cells treated with insulin and in cells expressing a conditionally activated form of PKB under activating conditions (98). These findings led Scott et al. (98) to speculate that activation of PKB leads to phosphorylation of mTOR within its PKB consensus sequence. Furthermore, this effect was sensitive to wortmannin but not to rapamycin, consistent with a role for PKB. In accord with these data, incubation of activated PKB with immunopurified mTOR leads to phosphorylation of mTOR on S2448, the presumptive PKB phosphorylation site (78). Furthermore, phosphorylation of mTOR at S2448 is stimulated by insulin, inhibited by wortmannin, and resistant to rapamycin (78). Because mTOR is enriched in membrane fractions, particularly in plasma (96) and microsomal (126) membranes, the possibility exists that the generation of phosphoinositides in such locales may serve to co-localize PKB and mTOR. The relevance of mTOR phosphorylation by PKB and the contribution of phospho-S2448 in mTOR's signaling properties, however, await characterization.

It is broadly appreciated that both the catalytic activity of S6K1 and the availability of eIF4E increase rapidly after stimulation of cells with insulin and other RTK-activating hormones. These events are absolutely dependent on S6K1 and 4E-BP1 phosphorylation, respectively, which are also enhanced by growth factors. The discovery that both the phosphorylation and function of S6K1 and 4E-BP1 are hindered by rapamycin suggested that mTOR plays an essential role in these processes. Rapamycin operates in this manner through association with one of its intracellular targets, the 12-kDa FK506-binding protein (FKBP12); it is the rapamycin · FKBP12 complex that physically and repressively interacts with mTOR. Indeed, expression of an mTOR point mutant defective in binding the rapamycin · FKBP12 complex protects both S6K1 and 4E-BP1 functions from inhibition by rapamycin (13, 14, 40, 85). Despite intense investigative efforts, mTOR and its role as a mitogenic effector continue to be imperspicuous.

mTOR is the mammalian counterpart of Tor1p, identified in yeast, and is most closely related to the PI 4-kinase family of lipid kinases. Immunopurified mTOR does indeed display PI 4-kinase activity; however, this activity is not affected by rapamycin (97), and because lipid kinase activity is detected in immunoprecipitates containing catalytically inert mTOR (13), this activity appears to be associated rather than intrinsic. The capacity of mTOR to signal downstream is not limited to lipid substrates, however. It is well documented that mTOR exhibits autokinase activity, which, although not appreciably stimulated under growth-promoting conditions (40, 85), is sensitive to wortmannin (85, 126) and at least partially sensitive to rapamycin (13, 85, 126). The function served by mTOR autophosphorylation, however, is currently unknown. The necessity of mTOR's intrinsic catalytic activity is underscored by the finding that overexpression of a kinase-dead variant functions in a dominant-negative fashion, i.e., the activity and phosphorylation, respectively, of S6K1 and 4E-BP1 are attenuated (40). Several laboratories have demonstrated that mTOR, or a co-purifying kinase, can directly phosphorylate both 4E-BP1 and S6K1. Regarding 4E-BP1, phosphorylation by mTOR is augmented in cells treated with serum (32) or insulin (14, 98, 99), although the sufficiency of this phosphorylation event in disinhibition, i.e., liberation, of eIF4E is contentious (14, 16, 32). Recently, two co-precipitating, yet separable 4E-BP1 kinase activities were detected in mTOR immunoprecipitates, the latter of which was sufficient to alleviate the eIF4E-4E-BP1 interaction (42). Significantly, both divisible kinase activities phosphorylated 4E-BP1 when complexed with eIF4E (42). The identity of the mTOR-associated kinase awaits description. With regard to S6K1, two distinct sets of phosphorylation sites are targets for mTOR: a cluster of proline-directed serine/threonine sites in the kinase's COOH terminus (46), and T389 (16, 46), an internal site essential for enzyme activity. Initially, the finding that T389 is one of the primary rapamycin-sensitive S6K1 phosphorylation sites (25, 84, 124) provided compelling evidence that mTOR may directly signal to S6K1 through phosphorylation of T389. This hypothesis is, however, called into question, because an amino and carboxy terminally deleted S6K1 variant exhibits wortmannin-sensitive, yet rapamycin-resistant phosphorylation of T389 (25, 41). The rapamycin insensitivity of T389 phosphorylation implies that mTOR, whose catalytic activity would be inhibited by rapamycin, is not S6K1's T389 kinase. Extending this logic, if mTOR can phosphorylate S6K1 at T389, and mTOR-catalyzed phosphorylation of T389 is not relevant, is the 4E-BP1 kinase activity observed in mTOR immunoprecipitates physiologically significant?

Perhaps an alternative mode of mTOR function may sufficiently explain these apparently discrepant findings. In Saccharomyces cerevisiae, Tor1p and Tor2p control protein synthesis in response to availability of nutrients; moreover, inhibition of the Tor proteins impairs translation initiation (9). In this system, Tor1p operates through restraint of Pph21/22 and Sit4, the yeast equivalents of the catalytic subunit of PP2A. The ability of Tor1p and Tor2p to control the activities of Pph21/22 and Sit4 depends on the rapamycin-inhibitable phosphorylation of an intermediary protein, Tap42. When phosphorylated, Tap42 competitively excludes Pph21/22 from its regulatory A and B subunits (Tpd3 and Cdc55, respectively), thereby reducing Pph21/22's inherent protein phosphatase activity (52). A similar mechanism of translational control in mammalian cells may be operational. In fact, alpha 4, the mammalian homolog of Tap42 (80), associates with the catalytic subunits of PP2A, PP4, and PP6 (18, 77), whereas PP2A dephosphorylates 4E-BP1, the association of alpha 4 with the catalytic subunit of PP2A inhibits this dephosphorylation (76). To date, however, a demonstration of alpha 4 phosphorylation by mTOR is lacking. In the analogous system in yeast, rapamycin induces the dissociation of Tap42 and PP2A (26), an effect that has not been consistently observed in mammalian cells (45, 76). If mTOR were acting to restrain a protein phosphatase, i.e., PP2A, rapamycin treatment would be predicted to enhance phosphatase activity. Such an effect has been demonstrated in both PP2A immunoprecipitates (45, 86) and in whole cell extracts (86). Moreover, PP2A interacts with wild-type S6K1 (86, 125) but not a rapamycin-resistant, double truncation variant (86), suggesting that the rapamycin insensitivity of the S6K1 mutant may reflect its inability to interact with PP2A. Because it has become increasingly apparent that PP2A function often involves specific interprotein associations that afford favorable phosphatase-substrate proximity, it will be essential to discriminate between the relevant pool of PP2A (or related phosphatases), which presumptively mediates the effects of mTOR, and irrelevant pools.

4E-BPs

The eIF4E binding protein family currently consists of three members (4E-BP1, 4E-BP2, and 4E-BP3) exhibiting distinct patterns of tissue-specific expression. Specifically, 4E-BP1 mRNA, although detected in most tissues, is robustly expressed in skeletal muscle, adipose tissue, pancreas, liver, heart, and kidney (44, 118); levels of 4E-BP1 protein generally parallel mRNA abundance (67). Although 4E-BP2 mRNA expression is somewhat ubiquitous (44, 118), the respective protein levels are highest in liver and kidney (66). Interestingly, the mRNA encoding 4E-BP3 displays a tissue distribution almost identical to that of 4E-BP1, i.e., it is highly expressed in skeletal muscle, pancreas, heart, and kidney (87). An obvious distinction, however, is that in contrast to 4E-BP1, 4E-BP3 mRNA is poorly expressed in liver. Both the 4E-BP (87, 118) and eIF4G (excluding p97) (74) families bear a common structural region that confers eIF4E recognition. As such, 4E-BPs compete with eIF4G for productive association with eIF4E, an interaction that is mutually exclusive. The dynamics of this competition is modulated by the phosphorylation status of multiple regulatory sites within 4E-BPs. With regard to 4E-BP1, six mitogen-stimulated phosphorylation sites have been described (T37, T46, S65, T70, S83, and S112); of these, five sites conform to a substrate preference for proline-directed kinases (29, 75). Despite this unique characteristic, the identity of relevant upstream kinases is almost entirely enigmatic. Proline-directed, serine/threonine protein kinases of the ERK, c-Jun-NH2-terminal kinase (JNK), and p38 families can phosphorylate these sites in vitro. However, the apparent insensitivity of RTK-mediated 4E-BP1 phosphorylation to inhibitors of the ERKs and p38s exonerates these kinases as physiological upstream effectors. The involvement of JNK, which is activated by RTKs in multiple cell types, as a candidate regulator remains to be determined. As mentioned earlier in this review, immunoisolated mTOR can phosphorylate 4E-BP1. However, it remains to be seen whether this phosphorylation of 4E-BP1 is a function of the mTOR molecule itself or involves a co-purifying kinase activity. 4E-BP1 phosphorylation at two NH2-terminal sites (T37 and T46) is both LY-294002 and rapamycin sensitive and is relatively constitutive; mutation of these two amino acids reduces serum-stimulated phosphorylation of the remaining sites (32). These findings led Gingras et al. (32) to conclude that mTOR-mediated phosphorylation of T37 and T46 serves to prime 4E-BP1 for subsequent mitogen-stimulated phosphorylation. In a separate investigation, Mothe-Satney et al. (75) observed that mutation of either putative priming site differentially affects the insulin-stimulated phosphorylation of additional mitogen-sensitive sites (i.e., S64 and T69). Nevertheless, the relative rapamycin insensitivity of several sites predicts that phosphorylation by an additional kinase(s) is necessary to generate the maximally phosphorylated 4E-BP1 species. As alluded to previously, such an effector has not been described.

S6Ks

The S6Ks are a family of mitogen-activated, serine/threonine protein kinases performing essential functions in translational and cell cycle control (reviewed in 28, 38, and 90). The S6Ks are members of the AGC (protein kinases A, G, and C) superfamily of serine/threonine protein kinases that is currently comprised of four members derived from the alternative mRNA processing and translation of distinct gene products. The S6K1/p70alpha gene encodes nuclear and cytoplasmic proteins referred to, respectively, as S6K1alpha /p85S6k/p70alpha I and S6KIbeta /p70S6k/p70alpha II, whereas the newly characterized S6K2/p70beta gene encodes S6K2beta I and S6K2beta II (37), which exhibit apparent molecular masses of 60 and 54 kDa, respectively (63). Interestingly, the S6K2 proteins may display a largely nuclear subcellular localization (63). Both S6K1 and S6K2 are phosphoproteins whose enzymatic activity is regulated by phosphorylation. With regard to S6K1, to date, 12 serum-stimulated phosphorylation sites have been described (S17, T229, T367, T371, T389, S404, S411, S418, T421, S424, S429, and T447). In the current model, S6K1 undergoes an ordered series of phosphorylations, which serves to increasingly unfold the protein, and in doing so exposes internal sites of phosphorylation (reviewed in Ref. 90). It is phosphorylation of these internal sites, namely T229, S371, and T389, that collectively confers optimal catalytic activity. The kinase responsible for phosphorylation of T229 has been identified as PDK1 (2, 89), which phosphorylates, among other substrates, PKB, PKCs, and PKA. In contrast to PDK1-mediated activation of PKB, which is strongly dependent on PI-(3,4,5)-P3, PDK1 phosphorylates S6K1 in a largely phosphoinositide-independent manner (2). Intriguingly, PDK1 is found complexed with S6K1 under basal and mitogen-stimulated conditions (95); thus the dependence on phosphoinositides, which serves to co-localize PDK1 with other substrates, e.g., PKB, may be circumvented through this constitutive association. Apart from PDK1, no true S6K1 kinases have been unequivocally described. As mentioned above, immuopurified mTOR has been shown to phosphorylate S6K1 (46), although whether this phosphorylation is direct or mediated by a mTOR-associated kinase is unknown. The atypical PKCs, PKClambda and PKCzeta , appear to play some role in the activation of S6K1. Overexpression of catalytically inert forms of PKClambda (1) or PKCzeta (95) interferes with S6K1 activation, whereas both forms co-purify with S6K1. Although PKCzeta synergizes with PDK1 in the activation of S6K1 (95), the role of atypical PKC types in S6K1 activation may be indirect, because neither PKClambda nor PKCzeta has been reported to directly phosphorylate S6K1. Curiously, activation of PKCdelta and PKCvarepsilon , members of the novel class of PKCs, has been shown to require rapamycin-sensitive phosphorylation of S662 and S729, respectively, hydrophobic sites analogous to T389 of S6K1 (82, 134). Moreover, PKCzeta appears to modulate the phosphorylation of the S662 site in PKCdelta (134). Taken together, these data indicate that S6K1, in addition to PKCdelta and PKCvarepsilon , is subject to regulation by both mTOR and PKCzeta as well as PDK1. Other upstream effectors for S6K1 include the small GTPases Cdc42 and Rac1, which interact with the catalytically inactive forms of S6K1 and do so with a dependence on GTP (19). Although the significance of such interaction is incompletely understood, it could serve to localize inactive S6K1 to particular subcellular regions, e.g., plasma membrane, in which activators are subsequently made available.

Amino Acids

The pathway(s) through which amino acids are sensed by the translational apparatus is incompletely understood. Generally, proximal events in the insulin signal transduction pathway do not appear to mediate amino acid-generated signals. In fact, in hepatocytes, amino acids antagonize the action of insulin with regard to tyrosine phosphorylation of IRS-1 and association of IRS-1 with either the p85 regulatory subunit of PI 3-kinase or Grb2 (83). In Chinese hamster ovary cells that overexpress the insulin receptor (CHO-IR), deprivation of amino acids affects tyrosine phosphorylation of neither the insulin receptor nor IRS-1; furthermore, the activities of PI 3-kinase and PKB are undiminished under similar conditions (41). Because the activity of ERK (41, 83), JNK (83, 121), or p38 (121) is not affected by the availability of amino acids, their respective upstream effector pathways are unlikely to be involved in propagation of amino acid-induced signals. Apart from inhibition of 4E-BP1 phosphorylation and S6K1 activity, amino acid deprivation has also been shown to induce dephosphorylation of eIF4E and phosphorylation of eukaryotic elongation factor 2 (eEF2) (121).

The finding that amino acid deprivation drastically inhibits S6K1 activity in cells expressing a constitutively active form of PI 3-kinase (41) supports one of two conclusions, which are not necessarily mutually exclusive. In the first, amino acid sufficiency affects a PI 3-kinase-independent pathway involved in the regulation of S6K1 (and 4E-BP1), whereas in the second, amino acids act downstream of PI 3-kinase yet upstream of S6K1 (and 4E-BP1). The signal for amino acid sufficiency primarily reflects the sufficiency of all amino acids, because deletion of single amino acids (except perhaps cysteine and glutamine) from the full complement is not sufficient to maintain S6K1 activity to the level of that obtained when cells are exposed to a complete amino acid mixture (41). Both the stimulation of S6K1 activity by insulin (41) and IGF-I-stimulated 4E-BP1 phosphorylation (128) are greatly impaired by amino acid deprivation. It appears that 4E-BP1 and S6K1 are unique among translational effectors in that their responsiveness to insulin depends only on the presence of amino acids, whereas eEF2 and eIF2B display an additional requirement for glucose (17). Among the essential amino acids, the branched-chain group, i.e., leucine, isoleucine, and valine, displays the greatest potency in stimulating 4E-BP1 phosphorylation (128), whereas leucine alone is unrivaled in its ability to induce the activation of S6K1 (104). Although the leucine metabolite alpha -ketoisocaproic acid (alpha -KIC) induces 4E-BP1 phosphorylation, this property is attenuated by (aminooxy)acetic acid (AOAA), an inhibitor of reversible leucine transamination (31). Thus the ability of leucine to enhance the phosphorylation status of 4E-BP1 appears to occur independently of downstream transamination metabolites. The apparently strict structural requirement for leucine in the stimulation of S6K1 (104) predicts that only an equally strict "leucine-sensing" molecule would be competent to generate the appropriate leucine-induced signal(s). Because the system L amino acid transporter is relatively nonselective in the amino acids it imports, e.g., leucine, isoleucine, valine, methionine, histidine, and phenylalanine (20), this import mechanism is unlikely to be directly involved in detecting leucine.

Although the site of amino acid detection (or perhaps just leucine detection) is currently unknown, considerable evidence supports a role for mTOR and PKCdelta in this pathway. First, both rapamycin and wortmannin markedly attenuate S6K1 activation (41) and 4E-BP1 phosphorylation by amino acids, as well as by leucine alone (104). Interestingly, Ziegler and co-workers (82, 134) have demonstrated rapamycin-sensitive phosphorylation of S662 in PKCdelta , a hydrophobic site equivalent to T389 of S6K1. The role of mTOR in this process was confirmed, because a rapamycin-immune mTOR variant protected PKCdelta from dephosphorylation at S662 in cells treated with rapamycin (82). Furthermore, S662 displays nominal phosphorylation in the absence of amino acids, whereas addition of either the full complement of amino acids or leucine alone rapidly induces its phosphorylation (82). Because of the rapamycin sensitivity of PKCdelta phosphorylation (and, indeed, activity), it may be reasonably inferred that PKCdelta mediates amino acid-generated signals downstream of mTOR. In support of this hypothesis, PKCdelta associates with mTOR, and both respective catalytic activities are necessary for phosphorylation of 4E-BP1 (60). In addition, 4E-BP1 phosphorylation induced by a constitutively active form of PKCdelta is resistant to inhibition by wortmannin, yet remains sensitive to rapamycin (60). In light of this differential sensitivity to PI 3-kinase and mTOR inhibitors, it may be inferred that PKCdelta is not the sole rapamycin-sensitive effector of 4E-BP1 phosphorylation.

The role of PKCdelta in the regulation of S6K1 by amino acids, or any other stimulus for that matter, remains to be determined. Nevertheless, an S6K1 double truncation variant that is resistant to rapamycin is also resistant to amino acid deprivation, yet remains sensitive to wortmannin (41). Collectively, the data are consistent with a model in which mTOR, which lies along an amino acid-sensing pathway, transmits modulative signals to S6K1. The insensitivity to both rapamycin and amino acid deprivation bestowed upon the double truncation S6K1 mutant reflects an inability of mTOR to transmit inhibitive signals to S6K1 (presumably because S6K1 lacks an NH2 terminus). As alluded to earlier, an interesting property of this S6K1 truncation variant is that, in contrast to wild-type S6K1, it is unable to interact with PP2A (86) (see also section on mTOR). Thus PP2A may represent an effector for amino sufficiency downstream of mTOR.

Glucocorticoids

The negative regulation of translation initiation by glucocorticoids is exerted primarily through 4E-BP1 and S6K1. Similar to amino acids, glucocorticoids do not appear to impact proximal insulin signaling events, at least in the short term. It should be noted, however, that prolonged exposure (72 h) to glucocorticoids gives rise to an isoform-specific increase in the expression of PI 3-kinase's regulatory subunit (p85alpha ) in L6 skeletal myoblasts (34). This increased expression of p85alpha is associated with a reduction in IRS-1-associated PI 3-kinase activity, prompting Giorgino et al. (34) to speculate that the increased abundance of noncatalytic p85alpha observed in glucocorticoid-treated cells competes with PI 3-kinase heterodimers (p85alpha · p110 and p85beta · p110) for coupling to IRS-1. In T cells, brief exposure to glucocorticoids attenuates both basal and interleukin-2-stimulated S6K1 activity, although PI 3-kinase activity remains unchanged (71). Moreover, IGF-I-induced phosphorylation of PKB at sites essential for activation, i.e., T308 and S473, is undiminished in cultured muscle cells (102), suggesting that glucocorticoids affect neither the activity of PI 3-kinase, on whose activity the phosphorylation of these sites strictly depends, nor that of PKB kinases, e.g., PDK1 and the S473 kinase.

Although the phosphorylation state of 4E-BP1 and the activity of S6K1 in response to glucocorticoids are downregulated in quiescent populations of cells (103), these two effector molecules exhibit differential sensitivities to inhibition by glucocorticoids after stimulation with IGF-I (102). Specifically, S6K1 remains susceptible to inhibition by glucocorticoids, whereas 4E-BP1 phosphorylation is refractory to such an effect. This disparity underscores the involvement of distinct regulatory pathways in the control of 4E-BP1 and S6K1, despite a shared rapamycin sensitivity. Moreover, this finding implies that glucocorticoids act on one or more unshared regulatory events. Because of the importance of S6K1 phosphorylation in its activation, and because the status of particular phosphorylation sites appears to be regulated by multiple upstream kinases, an understanding of how glucocorticoids affect S6K1 phosphorylation should facilitate identification of effector pathways selectively targeted by glucocorticoids.

Glucocorticoids operate through activation of ligand-inducible, cytosolic receptors that function as hormone-activated transcription factors. As such, the many described effects of glucocorticoids manifest largely at the genomic level. Any of a number of transcriptional mechanisms may be involved in the regulation of gene expression by glucocorticoids, but ultimately, one of two outcomes will result: gene induction or gene repression. Because actinomycin D, which interferes with the basal transcriptional machinery, abrogates glucocorticoid-induced inhibition of S6K1 (71), glucocorticoids appear to induce rather than repress an intermediating gene product. Whether such an effect entails induction of a single gene, multiple genes, or multiple rounds of gene induction at distinct genomic sites is currently unclear. What is also obscure is the function of such an intermediate. Conceivably, S6K1 phosphorylation/activation could be inhibited through a number of mechanisms, e.g., inactivation or activation, respectively, of an upstream kinase or phosphatase; a change in S6K1's subcellular location so as to hinder the efficiency of its activation; or a protein-protein interaction that physically or otherwise impedes its activation.


    CONCLUDING REMARKS
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ABSTRACT
INTRODUCTION
OVERVIEW OF TRANSLATION...
PHYSIOLOGICAL SCENARIOS OF...
4E-BP1 AND S6K1: MECHANISMS...
CONCLUDING REMARKS
REFERENCES

Recent years have been witness to the rapid expansion of virtually every frontier in translational control research. These advances have uncovered an elaborate routing network that, in response to myriad environmental cues, efficiently relays signals to the translational apparatus. This system is sufficient not only for the detection and transmission of information but also for the integration of multiple signals of distinct origin, e.g., amino acids, insulin, and glucocorticoids. At the heart of this integrative process lies S6K1 and 4E-BP1, which, respectively, harmonize the efforts of the ribosome and eIFs, as dictated by the tumultuous flux of information with which cellular environs are interfused. It is through such a strategy that translational control is achieved.

Despite numerous advances and the emergence of an increasingly refined picture of mRNA translation, several important issues remain unaddressed. For instance, what is the amino acid recognition site within the cell, and what are the immediate consequences of such recognition? Does mTOR operate through inhibition of a protein phosphatase, or does it represent a bona fide 4E-BP1 and/or S6K1 kinase? Which inducible gene products mediate the translational inhibition imposed by glucocorticoids? Do hormones and amino acids exert their respective effects on 4E-BP1 and S6K1 through regulation of a common effector? These are only a few of the many challenges that lie ahead. Nevertheless, the future of translational control promises to remind us of a universal, underlying certainty: the truth is more complex than we imagine.


    ACKNOWLEDGEMENTS

Work summarized in this review, which was performed in the laboratory of the authors, was supported in part by National Institute of Health Grants DK-15658 (L. S. Jefferson) and T32 GM-08619 (O. J. Shah and J. C. Anthony).


    FOOTNOTES

Address for reprint requests and other correspondence: L. S. Jefferson, Dept. of Cellular and Molecular Physiology, The Pennsylvania State Univ. College of Medicine, PO Box 850, Hershey, PA 17033 (E-mail: jjefferson{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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