Chapter 3 Ribosomal Protein S6 Kinase

Ribosomal Protein S6 Kinase: From TOP mRNAs to Cell Size Oded Meyuhas and Avigail Dreazen Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew UniversityHadassah Medical School, Jerusalem 91120, Israel

I. S6 Kinases ................................................................................... A. Signaling to S6K ....................................................................... II. S6K Substrates and Interactors ......................................................... A. Protein Synthesis Machinery-Associated Proteins.............................. B. Cell Survival Mitochondrial Proteins ............................................. C. Nuclear Function-Associated Proteins............................................ D. Insulin Receptor Substrate .......................................................... III. Does S6K Regulate the Translation Efficiency of TOP mRNAs? ............... A. The Translational cis-Regulatory Element of TOP mRNAs.................. B. Members of the TOP mRNA Family ............................................. C. Translational Control of TOP mRNAs ............................................ D. Candidate Trans-Acting Factors ................................................... E. S6K and TOP mRNAs: The Rise and Fall of a Dogma ....................... IV. Physiological Roles of S6K ............................................................... A. Cell Size ................................................................................. B. Myoblast Size and Energy Charge................................................. C. Global Protein Synthesis............................................................. D. Glucose Homeostasis and Insulin Resistance ................................... E. S6K and LTP and Memory.......................................................... V. Concluding Remarks ...................................................................... References...................................................................................

110 110 116 116 120 120 121 121 122 123 127 133 134 135 135 137 137 138 140 140 141

Ribosomal protein S6 kinase (S6K) has been implicated in the phosphorylation of multiple substrates and is subject to activation by a wide variety of signals that converge at mammalian target of rapamycin (mTOR). In the course of the search for its physiological role, it was proposed that S6K activation and ribosomal protein S6 (rpS6) phosphorylation account for the translational activation of a subgroup of transcripts, the TOP mRNAs. The structural hallmark of these mRNAs is an oligopyrimidine tract at their 50 -terminus, known as the 50 -TOP motif. TOP mRNAs consists of about 90 members that encode multiple components of the translational machinery, such as ribosomal proteins and translation factors. The translation efficiency of TOP mRNAs indeed correlates with S6K activation and rpS6 phosphorylation, yet recent biochemical and genetic studies Progress in Molecular Biology and Translational Science, Vol. 90 DOI: 10.1016/S1877-1173(09)90003-5

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Copyright 2009, Elsevier Inc. All rights reserved. 1877-1173/09 $35.00

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have established that, although S6K and TOP mRNAs respond to similar signals and are regulated by mTOR, they maintain no cause and effect relationship. Instead, S6K is primarily involved in regulation of cell size, and affects glucose homeostasis, but is dispensable for global protein synthesis, whereas translational efficiency of TOP mRNAs is a determinant of the cellular protein synthesis capacity. Despite extensive studies of their function and mode of regulation, the mechanism underlying the effect of S6K on the cell size, as well as the transacting factor that mediates the translational control of TOP mRNAs, still await their identification.

I. S6 Kinases A search for a kinase that phosphorylates ribosomal protein S6 (rpS6) yielded initially a 90-kDa polypeptide from Xenopus oocytes that was later termed p90 ribosomal protein S6 kinase (RSK, also known as p90RSK).1 However, purification of the avian and mammalian major rpS6 kinase recovered 65- to 70-kDa polypeptides2,3 that are currently referred to as S6K. The discovery that S6K is the predominant rpS6 kinase in somatic cells4,5 has led to a widely accepted belief that RSK, despite its name, is physiologically irrelevant for rpS6 phosphorylation. This notion, however, has recently been challenged, as detailed in Section II.A.1. Mammalian cells contain two isoforms of S6K, S6K1 and S6K2, which are encoded by two different genes and share a very high level of overall sequence homology. S6K1 has cytosolic and nuclear isoforms (p70 S6K1 and p85 S6K1, respectively), whereas both S6K2 isoforms (p54 S6K2 and p56 S6K2) are primarily nuclear (Ref. 6 and references therein) and partly associated with the centrosome.7 In addition to these isoforms, overexpression of the splicing factor SF2/ASF promotes the expression of an alternatively spliced isoform, S6K1 isoform-2, whose mRNA is identical to that encoding p70 S6K1 and p85 S6K1 up to exon 6, but encodes a protein with a different C-terminus. Cells overexpressing SF2/ASF or even just isoform-2 undergo transformation, whereas knockdown of either SF2/ASF or isoform-2 of S6K1 is sufficient to reverse transformation caused by the overexpression of SF2/ASF in vitro and in vivo.8

A. Signaling to S6K 1. MITOGENIC STIMULI Mitogenic signaling to mammalian S6K by growth factors is initiated by activation of the respective receptor tyrosine kinase (RTK, Fig. 1). This in turn, leads to activation of class I phosphatidylinositol 3-kinase (PI3K),

S6K: FROM TOP mRNAS TO CELL SIZE

111 Insulin Insulin receptor

Growth factor

Hypoxia

RTK P

Amino acids

PIP3 P

PTEN

P

PIP2 P

PI3K IRS1

P

LKB1

Low energy (AMP/ATP) HIF-1

AMPK

REDD1

PDK-1

mTOR mLST8 Rictor SIN1 PRR5

Akt

mTORC2 TSC-2 TSC-1

?

? Rheb

GDP

RhebGTP

? mTORC1

mTORC3? Free mTOR? mTOR

mTOR Raptor

?

mLST8

CCUUUUCC

S6K 4E-BP

eEF2K

eIF-4E eEF2

TOP mRNAs

? eIF-4B

Ribosomal proteins, elongation factors, etc.

rpS6

? Translational apparatus

FIG. 1. Pathways transducing signals emanating from growth factors, amino acids, and oxygen deficiency to S6K and TOP mRNAs. Arrows, activation; bars, inhibition; dashed lines, putative pathways; question marks, unknown intermediates.

either through direct binding to the phosphorylated receptor or through tyrosine phosphorylation of scaffolding adaptors, such as insulin receptor substrate (IRS), which then binds and activates PI3K.9 PI3K converts the lipid phosphatidylinositol-4,5-P2 (PIP2) into phosphatidylinositol-3,4,5-P3 (PIP3), in a reaction that can be reversed by the PIP3 phosphatase PTEN (phosphatase and tensin homolog deleted from chromosome 10).10 PIP3 recruits both 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt (also known as protein kinase B (PKB)) to the plasma membrane,11 and PDK1 phosphorylates and activates Akt at T308.12 PDK1 also phosphorylates S6K1 at T412 and T252, however, in a PIP3-independent fashion.13,14 Activated Akt phosphorylates at multiple sites tuberous sclerosis complex 2 (TSC2), within the TSC1–TSC2 tumor suppressor dimer. Notably, the TSC1 and TSC2 genes were identified as the genetic loci mutated in the autosomal dominant disorder, that is characterized by the development of numerous benign tumors (e.g., hamartomas) most commonly affecting the brain, kidney, skin, heart, and lungs (reviewed in Ref. 15). The phosphorylation of TSC2

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blocks its ability to act as a GTPase-activating protein (GAP) for Rheb (Ras-homolog enriched in brain), thereby allowing Rheb-GTP to accumulate and operate as an activator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1).16 Notably, the mechanism by which Rheb activates mTORC1 is yet to be established. Many of the effects of mTORC1 are abolished by rapamycin, which exerts its inhibitory effect when complexed with its intracellular receptor, the immunophilin FKBP12 (FK506-binding protein).17 mTORC1 consists of mTOR, Raptor (regulatory-associated protein of TOR), and LST8 (also known as GbL).18 The precise role of two additional mTORC1 components, proline-rich Akt substrate of 40 kDa (PRAS40) and FKBP38 has been subjected to conflicting results. Thus, PRAS40 has been described as either an mTORC1 inhibitor or mTORC1 substrate (Ref. 19 and references therein). Likewise, FKBP38 has been implicated in inhibition of mTORC1 activity,20 while others have failed to detect such an effect.21 Once mTORC1 is activated it phosphorylates two well-characterized proteins that are involved in protein synthesis. S6K is phosphorylated at T389, and thereby is fully activated,22 and eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) are phosphorylated at multiple sites, which results in their dissociation from and derepression of eIF4E23 (Fig. 1). mTOR is also involved in a second multiprotein complex, mTORC2, which is composed of mTOR, rictor, LST8, SIN1, and proline-rich protein 5 (PRR5), an interactor protein of unknown function [also known as protor1 (protein observed with Rictor-1)] (Ref. 24 and references therein) and is involved in Akt activation.25 mTORC2, unlike mTORC1, is inhibited by FKBP12-rapamycin only after a prolonged exposure and in a cell type-specific manner.26 2. GROWTH SIGNALS Proliferation (increase in cell number) reflects two processes: cell growth (increase in cell size) and cell division, which are normally intermingled, to the extent that cells must attain a minimal size to progress in the cell cycle. However, under some physiological or pathological conditions cellular growth and cell division are separable, and therefore, appear as distinct processes (reviewed in Ref. 27). Indeed, overexpression of S6K1 resulted in increased cell size, due to augmented cell growth and not from delayed cell-cycle progression.28 Several growth stimuli have been shown to activate S6K1. Thus, nerve growth factor (NGF) induces S6K1 activation in quiescent rat pheochromocytoma PC1229 and subsequently an increase in their size through neurite outgrowth.30 Similarly, induced cardiac hypertrophy, by a variety of mechanical and pharmacological agents, is associated with elevated activity of S6K1.31 This correlative evidence, together with the observation that inhibition of S6K by

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rapamycin eliminates the hypertrophic effect, led to a model that S6K plays a key role in determining heart size through regulating the size of cardiomyocytes (Ref. 32 and references therein). It should be pointed out, however, that any role assigned to S6K solely through correlative evidence derived from indirect inhibition by rapamycin, might be misleading. Indeed, mice deficient of both S6K1 and S6K2 have disclosed that S6Ks are dispensable for cardiac hypertrophy in response to pathological or physiological stresses.33 Nevertheless, cells derived from these double mutant mice have established S6K1 as a growth regulator of several other cell types (see a detailed discussion in Section IV.A). 3. AMINO ACID SUFFICIENCY When mammalian cultured cells are deprived of essential amino acids (i.e., amino acids that cannot be synthesized by the organism and must be provided in the diet) the rate of their global protein synthesis is decreased.34,35 Of all amino acids, leucine appears to play a central role in the anabolic effect of amino acids.36 However, in addition to their obvious role as immediate precursors for protein synthesis, signals emanating from amino acid deficiency, or even just from leucine, are transduced into translational repression through at least two major pathways. The first involves the phosphorylation of the a-subunit of the eukaryotic initiation factor 2 (eIF2), thereby inhibiting the binding of the initiator form of methionyltRNA to the 40S ribosomal subunit to form the 43S preinitiation complex.37 The second pathway involves the inhibition of mTORC1 that leads to a variety of inhibitory effect at the initiation and elongation steps of translation.38–40 Amino acid starvation, unlike serum starvation, fails to downregulate PI3K or PKB,35,41 yet it results in a rapid dephosphorylation of S6K1, which is restored upon readdition of amino acids in an mTORC1-dependent (rapamycin-sensitive) fashion (Ref. 42 and references therein). Nevertheless, the involvement of the TSC1–TSC2 complex in mTORC1 activation by amino acids is subject to conflicting results,43 which suggest that at least some of the amino acid signal to mTORC1 and thereby to S6K occurs independently of the TSC1–TSC2 complex (Fig. 1). Multiple cellular pathways have recently been proposed to mediate amino acids signaling to mTORC1. (a) Glutamine-dependent leucine-uptake system. The uptake of leucine depends on two amino acid transporter systems: solute carrier family 1 member 5 (SLC1A5), a high-affinity l-glutamine transporter; and SLC7A5/SLC3A2, a heterodimeric bidirectional antiporter that imports leucine and other branched chain amino acids in exchange for the efflux of intracellular amino acids, such as l-glutamine.44 Pharmacological inhibition of each of these transporters or silencing the expression of the respective genes causes a marked decrease in mTORC1 activity, implying that they should be

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considered as physiologically relevant activators of mTORC1.45 (b) Rag proteins. Expression of constitutively active mutant forms of RagA or B (members of the Rag subfamily of Ras small GTPases) in mammalian cells substitutes the need for amino acids, but not the input of insulin, in activating mTORC1. Conversely, dominant-negative RagA or B or knockdown of Rag gene expression inhibits mTORC1 in the presence of amino acids.46,47 (c) Phospholipase D1 (PLD1). An essential role of PLD1 and its product, phosphatidic acid (PA), in activation of mTORC1 has been established by demonstrating that mTORC1 activation, in response to Rheb overexpression, is reduced upon PLD1 knockdown, and is rescued by the provision of PA.48 The fact that amino acid withdrawal inhibits PLD1 activation by serum or TSC2 knockdown suggests that this phospholipase is regulated by amino acid sufficiency and may partly mediate amino acid sensing in the mTORC1 pathway.48 Nonetheless, the mechanism by which amino acid sufficiency controls PLD activity is not yet clear. (d) Vacuolar protein sorting 34 (hVPS34). This class III PI3K (converts phosphatidylinositol to phosphatidylinositol-3-phosphate) has been shown to transduce the signal of amino acid sufficiency to mTORC1 independently of the TSC1–TSC2/Rheb axis.49,50 However, two contradictory observations have questioned Vps34’s role upstream of mTORC1. First, amino acids inhibit, rather than activate, Vps34 in mammalian C2C12 myotubes,51 and second, Drosophila Vps34 is dispensable for signaling to dTORC1.52 (e) RalA and RalGD. The RalA GTPase and its activator RalGD have recently been implicated as critical mediators of amino acid-induced activation of mTORC1, as their knockdown abolishes this activation. Furthermore, the ability of a hyperactive mutant of RalA to partially activate mTORC1 in cells, whose Rheb was knocked down, implies that RalA might function downstream of Rheb in amino acids signaling to mTORC1.53 (f) MAP4K3. Amino acid-induced activation of S6K is strongly suppressed by knockdown of MAP4K3, and this effect is not mediated by inhibition of TSC1–TSC2. Consistent with these results, overexpression of MAP4K3 delays S6K1 inactivation by amino acid starvation. Moreover, MAP4K3 activity is regulated by amino acids, but is not itself stimulated by insulin or inhibited by rapamycin.54 Clearly, resolving ambiguities concerning the role of some of these mediators, as well as establishing their hierarchical relationship and the relative contribution of the bona fide regulators are a prerequisite for delineating the pathway that transduces amino acids sufficiency to TORC1 and S6K activation. 4. ENERGY BALANCE Energy depletion from mammalian cells by glucose starvation, as well as inhibition of glycolysis or oxidative phosphorylation leads to decreased mTORC1 activity.55–57 The prevailing model assumes that energy levels are coupled to downregulation of mTORC1 activity through the AMP-activated

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protein kinase (AMPK) (Fig. 1). AMPK acts as a sensor of cellular energy status and is activated by an increase in the cellular AMP:ATP ratio caused by metabolic stresses that either interfere with ATP production (e.g., deprivation for glucose or oxygen) or that accelerate ATP consumption (e.g., muscle contraction, activation of ion pumps). Activation in response to increases in AMP levels involves phosphorylation by an upstream kinase, the tumor suppressor LKB1,58 as AMPK activation in response to low-energy conditions is blocked in LKB1 null cells.59 Furthermore, LKB1 mutant cells exhibit hyperactive TORC1 signaling.59,60 Activation of AMPK by 5-aminoimidazole4-carboxyamide (AICAR), an AMP analog, inhibits TORC1-dependent phosphorylation of S6K1.61 Likewise, expression of an activated form of AMPK decreases S6K1 phosphorylation, whereas a dominant-negative form of AMPK increases S6K1 phosphorylation.62 AMPK phosphorylates several targets to enhance catabolism and suppress anabolism in response to low energy, and exerts this effect by directly phosphorylating and activating TSC2, and thereby downregulates mTORC1.56 Thus, the phosphorylation of S6K1 is more resistant to glucose deprivation in TSC2/ cells or cells whose mutant TSC2 cannot be phosphorylated by AMPK.56 In addition, AMPK directly inhibits mTORC1 by phosphorylating raptor.63 It appears, therefore, that energy depletion is sensed by AMPK and relayed to mTORC1 either directly or through the TSC1–TSC2 complex (Fig. 1). 5. OXYGEN SUPPLY The delicate balance between the requirement for O2 as an energy substrate and the inherent risk of oxidative damage to cellular macromolecules requires a precise maintenance of oxygen homeostasis. This homeostasis is mediated to a large extent by the transcription factor hypoxia-inducible factor-1 (HIF-1).64 Hypoxia-induced upregulation of HIF-1, leads to transcriptional activation of the REDD1 gene (regulated in development and damage responses 1 also known as RTP801 gene),65 which in turn binds 14-3-3 and thereby alleviates the 14-3-3mediated inhibition of the TSC1–TSC2 complex.66 Indeed, mouse cells deficient in REDD1, TSC1, or TSC2 are defective in hypoxia-mediated inhibition of S6K activation (Fig. 1 and Ref. 67). However, hypoxia can also inhibit mTORC1 independently of REDD1 via the induction of energy stress, possibly due to reduced oxidative phosphorylation. AMPK is upregulated under these conditions, thereby activates TSC2 and inhibits mTORC1.68 6. OSMOLARITY An increase in the concentration of solutes outside the cell relative to that inside is termed a hyperosmotic stress. Such a stress causes water to diffuse out of the cell, resulting in cell shrinkage, which can lead to DNA and protein

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damage, cell-cycle arrest, and ultimately cell death.69 Hyperosmotic stress that is induced by treating cells with sorbitol elicits reversible inactivation of S6K1, in a phosphatase-dependent manner, since calyculin A, a phosphatase inhibitor, is able to prevent sorbitol-induced suppression of S6K.70 7. LONG-TERM POTENTIATION AND MEMORY Synaptic plasticity refers to long-lasting changes in synaptic strength that reflects the ability of neurons to alter communication with each other via synaptic connections in response to specific patterns of electrical stimulation and/or neurotrophic factors. The most studied forms of long-lasting synaptic plasticity in mammals are long-term potentiation (LTP) and long-term depression (LTD), which refer to long-lasting increases or decreases, respectively, in synaptic strength.71 LTP fulfils many of the criteria for a neural correlate of memory, as both require a very similar complex cascade of molecular and cellular events. Thus, LTP, like memory, occurs in two temporally distinct phases: early LTP depends on modification of preexisting proteins, whereas late LTP requires de novo protein synthesis.72 Interestingly, induction of LTP by high-frequency stimulation or glutamatergic stimulation of neurons from mouse fetuses results in S6K1 activation.73,74

II. S6K Substrates and Interactors The wide variety of signals that stimulate S6K activation suggests that if S6K is involved in the manifestation of at least part of the numerous different cellular responses it is likely that it operates through multiple substrates. Indeed, after more than a decade during which rpS6 was perceived as the only S6K substrate, new substrates are being identified. Presently, a total of 13 such substrates have been described, of which five are implicated in the translational machinery (Table I). However, the effect of the S6K-dependent phosphorylation on the biological activity of some of these substrates is still unclear, as discussed below.

A. Protein Synthesis Machinery-Associated Proteins 1. RIBOSOMAL PROTEIN S6 Of all 79 ribosomal proteins in the mammalian ribosome, it is rpS6 that has attracted most attention, since it undergoes phosphorylation in response to numerous physiological, pathological, and pharmacological stimuli (reviewed in Ref. 75). The phosphorylation sites in this protein have been mapped to five clustered residues, S235, S236, S240, S244, and S247,76 whose location at the

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TABLE I S6K1 SUBSTRATES AND THE CONSEQUENCE OF THEIR PHOSPHORYLATION Phosphorylation Proteina

Function

Sitesb

Outcome of phosphorylation

Referencesc

rpS6

Ribosomal protein

S235,

Increased cell size

82

236, 240,

244, 247

eIF4B

Initiation factor

S422

Increased interaction with eIF3 complex and translation

39, 87

FMRPd

RNA-binding protein

S499

Repressed expression of SAPAP3

91

PDCD4

Tumor suppressor

S67

Accelerated degradation of PDCD4 and enhanced protein synthesis

94

eEF2K

Elongation factor 2 kinase

S366

Inactivated, likely to derepress translation elongation

95

URI

Chaperone

S371

Release of PP1g from URI and downregulation of S6K1

99

BAD

Proapoptotic protein

S136

Downregulated BAD proapoptotic activity

98

IRS-1

Insulin receptor substrate 1

S270, 307,

Inactivated IRS-1 and induced insulin resistance

106, 107, 109

Transcription factor

S167

Activation of ER-mediated transcription

100

Protein kinase

S2448

Unknown

96, 97

Unknown

102

7

Unknown

105

Unknown

101

ERa mTOR d

SKAR

636, 1101

Cell growth regulator S383

CBP80

Nuclear cap-binding

S

CREMt

Transcription factor

S117

a

See the text for full names. The sites refer to the human protein. c The references are of papers describing the outcome of the S6K-dependent phosphorylation. d SKAR and FMRP are the only protein that has been examined and shown to be preferentially phosphorylated by S6K1 and not S6K2. b

carboxy terminus of higher eukaryotes is evolutionarily conserved.75 It has been proposed that phosphorylation progresses in an ordered fashion, with S236 as the primary phosphorylation site.77,78 Analysis of rpS6 phosphorylation in mouse cells deficient in either S6K1 or S6K2 suggests that both are required for full S6 phosphorylation, with the predominance of S6K2.79 However, phosphorylation of rpS6 is not confined to S6Ks. Thus, phosphorylation at S235 and S236 is still detectable in cells lacking

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both S6K1 and S6K2, albeit at a much lower level.79 Indeed, these sites can also be phosphorylated by RSK in a response to serum, growth factors, tumorpromoting phorbol esters, and oncogenic Ras80 and cAMP-dependent kinase (PKA).81 The establishment of a knockin mouse (rpS6P/), whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues,82 has enabled the reassessment of the validity of previously assigned roles and the establishment of rpS6 phosphorylation as a determinant of cell size, cell proliferation, and glucose homeostasis (see Section IV).

2. EUKARYOTIC TRANSLATION INITIATION FACTOR 4B Eukaryotic translation initiation factor 4B (eIF4B) lacks catalytic activity but interacts with eIF383 and serves as a cofactor of the RNA helicase, eIF4A and thereby increases its processivity.84 eIF4B is phosphorylated by S6K185 and accordingly, muscle that is deficient in this kinase displays impaired eIF4B phosphorylation.86 Interestingly, eIF4B is still phosphorylated at S422 in liver or hepatocytes from S6K-deficient animals or in rapamycin-treated HeLa cells, as this phosphorylation is also carried out by RSK.86,87 It has been proposed that S422 phosphorylation is involved in the recruitment of eIF4B to eIF4A at the translation initiation complex,39 and in addition, it enhances the interaction between eIF4B and eIF3.87 Importantly, the expression in cells of phosphomimetic S422D and S422E mutants of eIF4B resulted in increased translation and a constitutive high level of interaction between eIF4B and eIF3, respectively.39,87 These data indicate, therefore, that the interaction between eIF4B and eIF3 is regulated through S422 phosphorylation in eIF4B.87 Notably, it has recently been reported that S422 is phosphorylated in response to amino acid stimulation in an mTOR-dependent fashion, whereas mitogenic stimulation induces Akt-dependent eIF4B phosphorylation at both S422 and S406.88

3. FRAGILE X MENTAL RETARDATION PROTEIN Fragile X syndrome is the most common form of inherited mental retardation and is caused by a functional absence of the RNA-binding protein, fragile X mental retardation protein (FMRP).89 FMRP is known to associate with approximately 3% of the mammalian brain mRNAs, repressing their translation and its deficiency induces the translation of many of its target mRNAs.90 One such target is SAPAP3 mRNA that encodes a postsynaptic scaffolding protein.91 The ability of S6K1, but not S6K2, to phosphorylate S499 and the presence of phospho-FMRP in hippocampal lysates from wild type, but not from S6K1/ mice, strongly suggest that S6K1 is an FMRP kinase in the

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mouse hippocampus. This notion is further supported by the induced expression of SAPAP3 upon loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP, as in the case of FMRP deficiency.92

4. PROGRAMMED CELL DEATH PROTEIN 4 Programmed cell death protein 4 (PDCD4) is a tumor suppressor that displaces eIF4G and RNA from eIF4A and thereby inhibits translation initiation (Refs. 93,94 and references therein). PDCD4 is rapidly phosphorylated on S67 by S6K1 in response to mitogens, as knocking down of S6K1 inhibits this phosphorylation. This event, in turn, promotes the phosphorylation of S71 and S76 (by S6K1 or another kinase), which allows its binding to the E3 ubiquitin ligase bTRCP and thereby targeting PDCD4 for degradation.95 It appears, therefore, that mitogen-stimulated cells utilize PDCD4 degradation for enhancing protein translation efficiency, a prerequisite for cell growth and proliferation.

5. EUKARYOTIC ELONGATION FACTOR 2 KINASE This kinase negatively regulates translation elongation by phosphorylating and inhibiting eukaryotic elongation factor-2 (eEF2). This inhibition is relieved, however, when eEF2K is phosphorylated at S366 by S6K1 and thereby is inactivated.96 This mechanism could have partially explained the enhanced protein synthesis that occurs in response to serum or amino acid stimulation. However, this model still needs experimental validation by examining the consequences of a mutation at S366 on global protein synthesis. Moreover, S6K deficiency in muscle and liver, although mimicking the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation, lacks a repressive effect on eEF2 phosphorylation, implying that eEF2 kinase (eEF2K) activity is under the control of an mTOR-dependent but S6K-independent mechanism.86

6. mTOR It has recently been shown that mTOR, in addition to its role as an activator of S6K, is a bona fide substrate of the latter.97,98 It is tempting to speculate that this modification constitutes a positive or a negative regulatory loop. However, the functional significance of S6K-mediated phosphorylation of S2448 with regard to mTOR signaling is elusive, as no apparent differences in mTOR kinase activity were observed in vitro, when this site was mutated to alanine or glutamate.97

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B. Cell Survival Mitochondrial Proteins 1. BAD AND UNCONVENTIONAL PREFOLDIN RPB5 INTERACTOR The survival function of S6K1 is mediated, at least in part, by two mitochondrial substrates, unconventional prefoldin RPB5 interactor (URI) and BAD. BAD is a proapoptotic protein that remains hypophosphorylated at the S6K recognition motif (S136), when cells are S6K1-deficient. Moreover, cells are rescued from the apoptotic effect of BAD overexpression by coexpression of wild-type S6K1, but not if the exogenous BAD is mutated at the S6K phosphorylation site.99 URI, an unconventional member of the prefoldin (PFD) family of chaperones, associates with protein phosphatase (PP) 1g at mitochondria and undergoes S6K1-dependent phosphorylation at S371 in response to insulin-like growth factor 1 (IGF1).100 This phosphorylation, proven both in vitro and in vivo, leads to disassembly of the URI/PP1g complexes and the released PP1g is involved in downregulation of S6K1 activity in vivo. Consequently, phosphorylation of BAD at S136 is diminished, and thereby cells become more susceptible to apoptosis.100 The mitochondrial signaling network involving S6K1, URI, PP1g, and BAD seems to operate as a homeostatic mechanism that protects cells from the consequences of sustained S6K1 survival signaling to BAD.

C. Nuclear Function-Associated Proteins 1. ESTROGEN RECEPTOR-a Estrogen receptor-a (ERa) is a DNA-binding transcription factor, which is regulated by its ligand, 17b-estradiol. Addition of the ligand and growth factors leads to hyperphosphorylation and DNA binding of ERa, and consequently to transcriptional activation. One of the phosphorylation sites, S167 is phosphorylated by S6K1, as knockdown of the latter results in a pronounced decrease in insulin-stimulated phosphorylation of this site. In contrast, overexpression of S6K1 increases rapamycin-sensitive phospho-S167 levels. The phosphorylation of S176 is required for the transcriptional activity of ERa and thus might contribute to proliferation of ER-positive breast cancer cells.101 2. CAMP-RESPONSIVE ELEMENT MODULATOR t The transcription factor cAMP-responsive element modulator t (CREMt) is activated by the adenylate cyclase signaling pathway and is involved in modulation of gene expression by binding to the cAMP-responsive element. CREMt was assigned as an S6K1 substrate more than two decades ago,102 yet, the role of its phosphorylation in transcriptional regulation of any endogenous cAMP-responsive gene is still unknown.

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3. S6K1 ALY/REF-LIKE TARGET AND CAP-BINDING COMPLEX 80 S6K1 Aly/REF-like target (SKAR) is a nuclear protein with sequence homology with members of the Aly/REF family of RNA-binding proteins, which couple transcription, pre-mRNA splicing, and nucleocytoplasmic mRNA transport (Ref. 103 and references therein). SKAR has been implicated in regulation of cell growth, as knockdown of its expression reduced the cell size.103 It has recently been shown that SKAR is deposited at the exon junction complex (EJC) during splicing, and that S6K1 upon its activation, is recruited by SKAR to cap-binding complex 80 (CBP80)-bound mRNA ribonucleoprotein (mRNP) on a newly synthesized mRNA.104 CBP80 is a subunit of the nuclear cap-binding complex (CBC) that has been implicated in nonsensemediated mRNA decay (reviewed in Ref. 105). The SKAR-mediated recruitment of S6K1 to the CBP80-bound mRNP appears to be involved in the splicing-dependent increase in translation efficiency, yet the underlying mechanism is unclear.104 Interestingly, both SKAR and CBP80 have previously been described as S6K1 substrates.103,106 However, no role for the phosphorylation of either of these two proteins has been related to any of their assigned functions.

D. Insulin Receptor Substrate IRS plays a pivotal role in insulin signaling, and its function is often impaired in subjects with insulin resistance. This protein is subject to S6K1dependent phosphorylation of S302 in mouse IRS-1 (S307 in human) and inactivation, as exemplified by constitutive activation of S6K.107,108 The role of S6K1 in the regulation of IRS-1 activity is further supported by the observation that S6K1/ mice show decreased phosphorylation of IRS-1 on both S302 and S632 (S636 in human) and enhanced insulin sensitivity.109 In addition, S1097 (S1101 in human) has recently been shown to be directly phosphorylated by S6K1 in vitro and in the liver of obese db/db and wild type, but not S6K1/ mice.110 Finally, S6K directly phosphorylates mouse IRS-1 on S265 (S270 in human) in response to TNF-a and phosphorylation of this site is essential for efficient phosphorylation of S302, S632, and S1097 following TNF-a administration.111

III. Does S6K Regulate the Translation Efficiency of TOP mRNAs? Two lines of correlative evidence laid the ground for the hypothesis that S6K might be involved in the translational control of a subset of mRNAs: (a) rpS6 is located near the mRNA/tRNA-binding site at the interface between the small and large ribosomal subunits and potentially, this location enables

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rpS6 to alter translation efficiency112 and (b) inhibition of mTORC1 by rapamycin, and thereby of rpS6 phosphorylation, leads to partial repression of protein synthesis (Ref. 113 and references therein). Indeed, the translation efficiency of a subclass of mRNAs, known as TOP mRNAs, closely correlates, at least under some physiological conditions, with S6K activity.114 In the present section, we describe the structural hallmark of TOP mRNAs, the function of known proteins that are encoded by these mRNAs, the pathway that transduces various signals to their translation efficiency, and finally the evidence that disproves the role of S6K in this mode of regulation.

A. The Translational cis-Regulatory Element of TOP mRNAs Human TOP mRNAs possess a 50 -UTR, which varies in length from 12 (rpL23a) to 505 (poly(A)-binding protein, PABP) nucleotides and is devoid of upstream AUGs.115 However, a comparative analysis of sequences in the vicinity of the cap site of these TOP mRNAs indicates the following structural features: (a) an invariable C residue at the cap site, followed by an uninterrupted stretch of 4–15 pyrimidines (Table II and Ref. 115); (b) the composition of the pyrimidine stretch, although varying among TOP mRNAs even within a species, generally maintains a similar proportion of C and U residues; and (c) a CG-rich region immediately downstream of the 50 -TOP motif. Initiation of transcription at a C residue is rare among eukaryotic genes, which normally start at a purine residue (Ref. 116 and references therein), whereas the percentage of mammalian transcripts with a C residue at the cap site is only 17%.117 Evidently, the 50 -TOP motif comprises the core of the translational cis-regulatory element of TOP mRNAs and its function is fully reliant on its integrity and location at the 50 -terminus, to the extent that it is abolished if the C at the cap site is replaced, or even just preceded by an A residue.118,119 Moreover, full manifestation of the translational control of TOP mRNAs, at least in some cell lines, appears to require both the 50 -TOP motif and the CG-rich region.118 Initially, it was shown that the first 27–35 nt of TOP mRNAs are sufficient to confer translational control on a reporter mRNA in a mitosis-dependent manner.118–121 However, a closer look at the 30 -UTR disclosed a role for its length and composition in the translational behavior of TOP mRNAs (Ref. 122 and J. Kasir and O. Meyuhas, unpublished data). The involvement of downstream sequences in the translational control of TOP mRNAs is further supported by the fact that the first 29 nt of eEF2 mRNA can confer mitosisdependent translational control on a reporter mRNA, even in cells where the

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TABLE II HUMAN TOP mRNAS ENCODING NONRIBOSOMAL PROTEINS Function in protein synthesis machinery

50 -sequencea

Protein name

Symbol

Nocleophosmin (B23)

NPM1

Pre-rRNA processing, nuclear export of ribosomal subunits

CUUUCCCUGGUGUGAUU

Rack1

GNB2L1

Association of 40S and 60S ribosomal subunits

CUCUCUUUCACUGCAAG

eIF3E

EIF3E

Subunit of initiation factor 3

CUUUUCUUUGGCAAGAU

eIF3F

EIF3F

Subunit of initiation factor 3

CUUCUUUCUCGACAAGA

eIF3H

EIF3H

Subunit of initiation factor 3

CUCUUUCUUCCUGUCUG

Poly(A)-binding protein

PABPC1

Initiation factor

CCCCUUCUCCCCGGCGG

eEF1A

EEF1A1

Elongation factor

CUUUUUCGCAACGGGUU

eEF1Bb

EEF1B2

Subunit of elongation factor 1B

CUUUUUCCUCUCUUCAG

eEF1Bg

EEF1D

Subunit of elongation factor 1B

CCCUUUCAUCAGUCUUC

eEF1Bd

EEF1G

Subunit of elongation factor 1B

CCUUUCUUUGCGGAAUC

eEF2

EEF2

Elongation factor

CUCUUCCGCCGUCGUCG

TCTP (P21)

TPT1

Guanine nucleotide dissociation inhibitor for eEF1Bb

CUUUUCCGCCCGCUCCC

hnRNP A1

HNRNP1

Regulation of IRES-mediated translation of specific mRNAs

CCUUUCUGCCCGUGGAG

a

The first 17 nucleotides in the mRNA. The 50 -TOP motif appears in bold face letters.

endogenous mRNA is refractory to this mode of regulation,120 implying that downstream sequences in this mRNA can override the regulatory features of the 50 -TOP in a cell type-specific manner.

B. Members of the TOP mRNA Family 1. RIBOSOME STRUCTURE AND MATURATION a. Ribosomal Proteins. mRNAs encoding vertebrate ribosomal protein mRNAs were the first TOP mRNAs, whose translation was shown to be controlled by mitogenic stimulation.123 Moreover, each mRNA of the 79 different species of this class starts with a 50 -TOP motif (Fig. 2 and Refs. 115, 124, 125). It should be mentioned, however, that in addition to their canonical role within the ribosome, an increasing number of rps has been implicated in distinct extraribosomal functions, such as apoptosis, DNA repair, translational control of specific mRNAs, and even transcriptional regulation.126

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Human

Mouse

Xenopus

Drosophila 1 2 3 4 5 6 7 8 9 10111213141516 FIG. 2. The 50 -TOP motif of rp mRNAs. The consensus sequence of the first 16 nucleotides within TOP mRNAs from fruit fly, frog, mouse, and human (72, 9, 79, and 80 mRNAs, respectively) are compared and the proportion of each nucleotide is depicted by the relative height of the letters A, C, G, and U.

b. Nucleophospmin. Nucleophosmin (NPM1, also known as B23, numatrin or NO38), is a very abundant and highly conserved nucleolar phosphoprotein.127 NPM1 has been implicated in numerous cellular processes, including ribosome biogenesis, nucleocytoplasmic transport, centrosome and DNA duplication, transcriptional regulation, chromatin assembly and disassembly as a histone chaperone, binding and folding of denatured proteins, nucleic acid binding, and antiapoptotic activity (reviewed in Ref. 128). Given the multifunctionality of the NPM1 protein, it is not surprising that disruption of the corresponding gene leads to embryonic lethality at mid-gestation.129,130 The nucleolar localization and nuclear-cytoplasmic shuttling activity of NPM1,131 as well as its ability to bind nucleic acids132 and to cleave the second internal transcribed spacer in the pre-rRNA,133 are all elements that implicate NPM1 in the processing, assembly, or export of ribosomes. Indeed, it has recently been demonstrated that NPM1 is primarily rate limiting in nuclear export of both the 40S and 60S ribosomal subunits.134 Nevertheless, the ability of NPM1-deficient embryo to survive to mid-gestation129,130 indicates that either this protein is not essential for ribosome biogenesis, or that ribosome stores derived from the oocyte might compensate for NPM1 loss until midgestation. Notably, this protein is encoded by a TOP mRNA (Table II) and is translationally repressed upon serum starvation (Table II and 135).

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2. TRANSLATION INITIATION a. Initiation Factors. The initiation of translation consists of a series of steps, each of which is promoted by one or more initiation factors (eIFs). There are at least 12 well-characterized eIFs comprising 35 polypeptides.136 Recent bioinformatic surveys for a 50 -TOP motif have identified about one-third of these polypeptides to be encoded by 50 -TOP-containing mRNAs,124,137 yet only three of these have indeed been shown to be translationally activated by mitogenic stimulation (Table II and Ref. 137). Clearly, these results underscore the uncertainty in defining a TOP mRNA merely on the basis of a structural analysis, without a definitive functional assay for its translational behavior.

b. Poly(A)-Binding Protein. PABP (also referred to as PABP1, PAB1, PAB, and PABPC1) is a major eukaryotic RNA-binding protein that exhibits a preferential affinity for poly(A) stretches and is considered to be a canonical translation initiation factor.138 The role of PABP in the initiation process has been suggested on the basis of its interaction, while on the poly(A) tail, with factors located at the 50 -end that bring the ends of the mRNA into close proximity, forming a ‘‘closed loop.’’139 Moreover, it has been implicated in key steps of the translation initiation pathway.138 In addition, PABP has been implicated in mRNA stability, regulation of poly(A) tail length during the polyadenylation reaction, or poly(A) shortening (reviewed in Ref. 140). Study of PABP gene expression in various vertebrates has established the respective mRNA as a subject of mitogenic or developmental translational regulation, similar to that of rp mRNAs, and bearing a 50 -TOP motif (Table II and Refs. 120,121).

c. RACK1, Receptor for Activated Protein Kinase C1. RACK1, receptor for activated protein kinase C (PKC), was initially characterized by its homology to the guanine nucleotide-binding protein b-subunit and other proteins containing Trp–Asp (WD) repeat domains.141 Later on, it was identified as an anchoring protein for PKCbII142 and multiple other proteins (Ref. 143 and references therein). Recently, it has been assigned as an integral ribosomal protein.144 Thus, mass-spectroscopy studies identified RACK1 among the human ribosomal proteins.145 A cryo-EM map of the eukaryotic ribosome showed that RACK1 is located in proximity of the mRNA exit channel, in close contact with the binding surface of the eIF3 complex, next to mRNAbinding proteins.146 The presence of RACK1 at a 1:1 ratio with other ribosomal proteins147 and its persistence on ribosomes under high salt conditions,148 suggest that most ribosomes in the cell contain RACK1.

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The mammalian RACK1 mediates the recruitment of activated PKC to the ribosome, and thereby leads to the phosphorylation and release of eukaryotic initiation factor 6 (eIF6) from the 60S subunit, thus promoting the formation of a functional 80S complex.149 Notably, in addition to its effect on global protein synthesis, RACK1 has also been proposed to regulate the translation of specific transcripts.150 Finally, mammalian RACK1 mRNA contains a 50 -TOP motif and its translational control is coordinated with that of rp mRNAs (Table II and Ref. 151). 3. TRANSLATION ELONGATION FACTORS a. Elongation Factors. The structure of eukaryotic elongation factors (eEFs) and their role in peptide-chain elongation have been extensively reviewed.152 Briefly, eEF1A binds guanine nucleotides and when loaded with GTP can interact with aminoacyl-tRNA to bring it to the A-site of the ribosome. Following hydrolysis of the GTP, eEF1AGDP is released from the ribosome and cannot bind aminoacyl-tRNA. It recycles to the active GTP-bound form by eEF1B, which consists of three subunits, b, g and d. eEF1B thus acts as a guanine nucleotide exchange factor (GEF) for eIF1A. The second step of elongation, the translocation, in which the ribosome moves relative to the mRNA by the equivalent of one codon, is mediated by eEF2. The latter binds guanine nucleotides and is active when bound to GTP. The GTP is hydrolyzed late in the translocation process, and the energy released may be coupled to translocation. The first hint that the synthesis of eEF1A is subject to translational control, came from the observation that growth stimulation of resting Swiss 3T3 cells leads to an increase in the synthesis rate of this factor without a concomitant increase in the abundance of its mRNAs.153 Subsequently, it was demonstrated that this mRNA, as well as those encoding eEF1Bb, eEF1Bg, eEF1Bd, and eEF2, are recruited into polysomes upon mitogenic stimulation and are all equipped with a 50 -TOP motif (Table II and Refs. 118,120,137,154). It appears, therefore, that unlike the case of initiation and release factors, all known proteins that mediate peptide-chain elongation are encoded by bona fide TOP mRNAs.

b. Translationally Controlled Tumor Protein. Translationally controlled tumor protein (TCTP),155 also known as P21, p23, Q23, fortilin, and histaminereleasing factor (HRF), is ubiquitously expressed, despite its name. TCTP was originally discovered in a search for translationally regulated mRNAs,156,157 and like most other TOP mRNA-encoded proteins, TCTP has been implicated in the protein synthesis machinery. Thus, its direct interaction with elongation factor,

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eEF1A, stabilizes the latter in its GDP form and impairs the GDP exchange reaction promoted by its GEF eEF1Bb, which suggests that TCTP is involved in the elongation step of protein synthesis.158 Based on its interacting partners and functional assays, TCTP has also been implicated as HRF,159 microtubule stabilizer,160 tumor reversion,161 and antiapoptotic protein.162 In keeping with the latter function, TCTP knockout (TCTP–/–) embryos suffer from high incidence of apoptosis in the epiblast and die around mid-gestation.163,164 However, TCTP–/– and TCTPþ/þ mouse embryonic fibroblasts manifest similar proliferation activities and apoptotic sensitivities to various death stimuli, suggesting that TCTP regulates cell proliferation and survival in a tissue- or cell type-specific manner.163 Genetic manipulations have implicated that Drosophila TCTP (dTCTP) resides within the TSC–dTOR pathway and directly regulates Rheb.165 However, this mode of regulation does not seem applicable to the mammalian TCTP.21 TCTP (P21) mRNA is translationally activated upon mitogenic stimulation157 and contains a bona fide 50 -TOP motif (Table II and Ref. 166). 4. hnRNP A1 hnRNP A1 is an RNA-binding protein that shuttles continuously between nucleus and cytoplasm167 and is associated with poly(A)þ mRNA in the cytoplasm.168 It has been shown to function at various stages along the gene expression pathway, such as alternative splicing,169 mRNA export,170 inhibition of cap-independent translation,171 internal ribosome entry site (IRES)mediated translation,172,173 mRNA stability,174 and as an auxiliary factor for the processing of a specific microRNA (miRNA) substrate.175 Human hnRNP A1 mRNA, unlike mRNAs encoding hnRNP C1/C2 or A2/B1, is translationally regulated in response to mitogenic signals and contains a 50 -TOP motif, and therefore should be considered as a typical TOP mRNA (Table II and Ref. 176).

C. Translational Control of TOP mRNAs 1. MITOTIC STIMULI Mitotic arrest leads to dephosphorylation and inactivation of S6K (see Section I.A.1), as well as to translational repression of TOP mRNAs. Thus, when cells are arrested at G0 by a wide variety of treatments including serum starvation, induction for terminal differentiation, or contact inhibition, their TOP mRNAs undergo a selective shift from polysomes into mRNP particles (Fig. 3 and Refs. 177,178). Likewise, translational repression of TOP mRNAs has been observed when cells were arrested at the S-phase by hydroxyurea, an inhibitor of ribonucleotide reductase, or even when arrested at the M-phase by inhibiting the assembly of the mitotic spindle by nocodazole (Fig. 3 and

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Nocodazole HeLa rpL32 Actin P S P S

Serum starvation

M G2

G0

PC12 rpL32 Actin P S P S

Control

Control Nocodazole

- Serum

S G1

Hydroxyurea NIH 3T3 rpL32 Actin P S P S Control Hydroxyurea FIG. 3. Arrest of the cell-cycle progression results in translational repression of TOP mRNAs cytoplasmic extracts were prepared from rat pheochromocytoma (PC12), mouse NIH 3T3, and human HeLa cells that were either untreated (control) or arrested by serum starvation for 72 h, treated by 10 mM hydroxyurea for 24 h, or 15 mM nocodazole for 24 h, respectively. These extracts were centrifuged through sucrose gradients and separated into polysomal (P) and subpolysomal (S) fractions. RNA from equivalent aliquots of these fractions was analyzed by Northern blot hybridization with a TOP (rpL32) or non-TOP (actin) probe.

Ref. 178). Furthermore, a direct relationship between mitogenic activity and translational efficiency of TOP mRNAs is applicable also for whole animals, as translation of these mRNAs is repressed upon transition from the rapidly growing state in fetal liver to the quiescent state in adult liver. Likewise, resumption of translation can be observed in the regenerating liver.121,179 Notably, the distribution of TOP mRNAs between polysomes and mRNPs appears as an ‘‘all-or-none’’ phenomenon, that is, these mRNAs alternate between repressed and active states, and, when in the active state, they are translated at near maximum efficiency (Ref. 180 and references therein). This bimodal distribution clearly indicates that the translational repression results from a blockage at the translational initiation step. It appears, therefore, that the translation of TOP mRNAs is efficiently repressed when progression of the cell cycle is blocked by any means, at any phase, and in cells of any lineage. Biogenesis of the protein synthesis machinery, and particularly of ribosomes, is a highly resource-consuming process.181 Not surprisingly, therefore, cells that cease to proliferate and do not need to double their ribosome content during the cell cycle, as do dividing cells, operate a tight control mechanism to

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attenuate the production of the respective constituents. Nevertheless, given the fact that this attenuation is attained at the level of translation, rather than transcription, cells can resume the biogenesis of the translational apparatus more readily and thereby respond faster to changing conditions. 2. RIBOSOMAL STRESS Ribosomal stress (also known as ‘‘nucleolar stress’’), caused by perturbations that interfere with ribosome biogenesis, often results in p53 activation and cell arrest. Several ribosomal proteins have been shown to interact with MDM2, the E3 ubiquitin ligase of P53, and thereby impair its activity toward P53, which consequently is stabilized and accumulates (reviewed in Ref. 182). Strikingly, it has recently been shown that, regardless of whether biogenesis of the 60S or the 40S subunit is impaired, it is rpL11 that mediates p53 stabilization.183 Surprisingly, impaired biogenesis of the 40S ribosomal subunit, by knocking down one of its resident proteins, alleviates the translational repression of TOP mRNA, despite a twofold decrease in the rate of DNA replication. This unexpected translational activation is contrasted with the prominent unloading of TOP mRNAs from polysomes, when the biogenesis of the 60S, rather than the 40S subunit, is the one that is damaged. Efficient translation of TOP mRNAs under these circumstances seems to enable the accumulation of rpL11 (together with other TOP mRNA-encoded proteins) that ignites the p53 checkpoint response.183 One plausible explanation for the exceptional behavior of TOP mRNAs in cells that are unable to produce 40S subunits is, that at the time cells were harvested for polysomal analysis, they were still mitotically active due to the presence of preexisting subunits. Nonetheless, the differential response of TOP mRNAs under conditions of impaired biogenesis of the 40S or 60S subunit is unclear. 3. GROWTH SIGNALS The translation of TOP mRNAs, like S6K, is activated also by growth stimuli. Thus, the addition of NGF to quiescent (serum starved) PC12 cells, although causing no detectable increase in cell number, elicits a fast increase in the cellular mass due to neurite outgrowth, reflecting their terminal differentiation, as well as translational activation of their TOP mRNAs.30,184,185 Insulin, an important regulator of cell growth, survival, and proliferation, in addition to its role in metabolism, rapidly upregulates protein synthesis by activating translation factors (reviewed in Ref. 186). However, insulin can also induce an elevation in the protein synthesis capacity by inducing the biogenesis of the translational apparatus. It exerts this latter effect by inducing the transcription of the ribosomal RNA gene187 and the translation of TOP

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mRNAs.188 Interestingly, insulin upregulates the translation efficiency of TOP mRNAs concomitantly with enhancing cell proliferation, and even just the cell size, as exemplified with terminally differentiated adipocytes.188 The cardiac myocytes stop dividing after birth and undergo terminal differentiation, and therefore, irreversibly withdraw from the cell cycle. Consequently, the hypertrophic growth of the heart, apparently as a response to sustained increase in blood pressure and hormonal imbalances, occurs in the absence of significant cell division. Although such hypertrophic growth is generally considered as an adaptive response of the organism in an attempt to increase pump function, sustained and uncontrolled growth ultimately leads to diminished cardiac performance, resulting finally in the onset of heart failure.189 The primary determinant for the cardiomyocyte hypertrophy is the upregulation of the overall rate of protein synthesis, which reflects an increase in both translation efficiency and the capacity to synthesize protein (i.e., an increase in the translational machinery).190 Indeed, partial occlusion of the right pulmonary artery in male cats elicits right ventricular pressure overload, which selectively activates the translation of TOP mRNAs within 48 h.191 Moreover, treatment of adult feline cardiomyocytes with endothelin, which is involved in the cardiac hypertrophic response to mechanical stress,192 selectively stimulates the translation efficiency of TOP mRNAs.191 Taken together, these observations indicate that the requirement for increased capacity of protein synthesis when cells are induced to grow is satisfied, at least partly, by elevating the translation efficiency of TOP mRNAs.

4. AMINO ACID SUFFICIENCY The same physiological rationale that underlies the repressed translation of TOP mRNAs, when progression through the cell cycle is halted, is also applicable when cells are deprived of amino acids. Thus, it seems likely that deficiency of substrates for protein synthesis should signal for diminution of further wasteful biogenesis of the translational apparatus. Indeed, the translation of TOP mRNAs is selectively repressed in amino acid-starved cells.42 Moreover, cells subjected to starvation for both serum and amino acid display an additive repressive effect on the translation of TOP mRNAs, compared with deprivation of just one of these components. It should be pointed out, however, that amino acids have a much more pronounced effect than serum in both deprivation and replenishment experiments.193 These results imply that serum and amino acids signal to TOP mRNA translation, at least partially, through independent mechanisms.

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5. OXYGEN SUPPLY We have assumed that if the biogenesis of the translational machinery relies on the ability of cells to proliferate or grow, and on the availability of amino acids, it is conceivable that it should also reflect the cellular energy balance, as it is a highly energy-consuming process. Indeed, deprivation of HEK293 cells of oxygen (anoxia) for 6 h results in inactivation of S6K1, as well as translational repression of TOP mRNAs that can be alleviated by resumption of oxygen supply for 2 h (Y. Iluz, O. Cheshin, and O. Meyuhas, unpublished results). It appears, therefore, that adequate supplies of amino acids and energy, as well as the ability to divide or grow, are a prerequisite for efficient translation of TOP mRNAs. However, human lymphoblastoid cell lines comprise an exception, as the translation of their TOP mRNAs is constitutively repressed even when they are proliferating, well nourished and provided with 20% oxygen.178 This observation implies that efficient translation of TOP mRNAs is not a determinant of the cell cycle and is not a prerequisite for cell division. Nevertheless, we cannot exclude the possibility that the synthesis of TOP mRNAencoded proteins is still as efficient in lymphoblastoids as in any other dividing cells, because of a compensatory increase in the abundance of the respective mRNAs, for example. 6. LTP AND MEMORY LTP-related translation is controlled by coordinated mechanisms that regulate local protein synthesis and is thought to play an important role in establishing specific synaptic connections during late LTP.194 Moreover, an LTP-related increase in TOP mRNA-encoded proteins has led to the hypothesis that LTP induces translational activation of TOP mRNAs.195 Indeed, it has recently been shown that induction of late LTP in hippocampal slices by forskolin, leads to translational activation in dendrites of a 50 -TOP motif-containing reporter mRNA, but not an mRNA with a mutated motif.196 It should be pointed out, however, that in the absence of a pharmacological or genetic means for selective repression of TOP mRNA translation, we are presently unable to establish a causal relationship between the translational activation of these mRNAs and synaptic plasticity. 7. SIGNALING PATHWAYS TO mRNA TRANSLATION Translational activation of TOP mRNAs by serum- or insulin-stimulated cell proliferation as well as amino acids is strictly dependent on the integrity of the PI3K/PKB pathway. Thus, inhibition of PI3K by LY294002, a PI3K-specific inhibitor, completely abolishes the recruitment of TOP mRNAs into polysomes in refed cells.42,185,188 A similar translational repression can be attained by overexpression of (a) PTEN, a tumor suppressor with lipid phosphatase activity

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that antagonizes the activity of PI3K in vivo;197 (b) a dominant-negative mutant of p85, the regulatory subunit of PI3K (Fig. 1); and (c) a dominant-negative mutant of PKB.42,185 Likewise, overexpression of a constitutively active mutant of p110, the catalytic subunit of PI3K, or of PKB renders TOP mRNA translation resistant to serum starvation.42,185 Moreover, induction of growth, without cell division, by NGF or insulin leads to translational activation of TOP mRNAs in serum-starved PC12 or adipocytes, respectively,185,188 and also this activation is LY294002-sensitive. The translational control of TOP mRNAs requires TSC1, TSC2, and their target, Rheb, as deletion of either of the two TSC proteins or overexpression of Rheb, renders TOP mRNAs refractory to serum deprivation.188,198 The signal emanating from mitogenic stimuli through the PI3K/PKB pathway does not seem to be conveyed solely through inactivation of the TSC1–TSC2 complex. Thus, if this would have been the underlying mechanism, then deficiency of TSC1 or TSC2 should have rendered the translation of TOP mRNAs refractory to inhibition of PI3K. However, the deficiency of either of these proteins only partially relieved the LY294002-dependent translational repression of TOP mRNAs.188 This latter observation might reflect signaling of PI3K, or another LY294002 target, to TOP mRNAs through an as yet unknown route (Fig. 1). Notably, TOP mRNA translation is rescued from amino acid starvation in cells deficient of either TSC1 or TSC2, or overexpressing Rheb (I. Patursky-Polischuk and O. Meyuhas, unpublished results), even though the involvement of the TSC1–TSC2 complex in mTORC1 activation by amino acids is subject to conflicting results.43 This observation indicates that the TSC1–TSC2 complex is involved in transduction of the amino acid signal to translation efficiency of TOP mRNAs. Moreover, the same complex mediates also the oxygen signal, as anoxia-induced translational repression of TOP mRNAs can be relieved by TSC1 or TSC2 deficiency (Y. Iluz, O. Cheshin, and O. Meyuhas, unpublished results). It appears, therefore, that signals emanating from mitogens, amino acids, and oxygen all converge at the TSC1–TSC2 complex along their individual pathways to TOP mRNAs (Fig. 1). The establishment of the role of mTOR, a downstream effector of Rheb, in the transduction of signals emanating from insulin or amino acids signals to translation efficiency of TOP mRNAs, is based on both loss-of-function and gain-of-function genetic manipulations. Thus, mTOR knockdown suppresses translational activation of TOP mRNAs upon readdition of serum or amino acids to respectively starved cells (Ref. 188 and unpublished results). In contrast, expression of a rapamycin-resistant mutant of mTOR relieves the translational repression in amino acid-starved cells (I. Patursky-Polischuk and O. Meyuhas, unpublished results). Interestingly, when serum-starved HeLa cells are refed, the translational activation of their TOP mRNAs is rapamycinsensitive, whereas rapamycin blocks only about half the amino acid-induced activation of TOP mRNAs.193 This observation, together with the additive

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nature of the repression of TOP mRNAs by both serum and amino acids, suggest that although these signals converge through the TSC1–TSC2 complex, the latter conveys the amino acid signal to TOP mRNA translation through mTOR, as well as through an additional pathway. Unexpectedly, several lines of evidence contradict the possibility that mTOR operates within mTORC1 to control the translation efficiency of TOP mRNAs. (a) Rapamycin ubiquitously and completely inhibits mTORC1 activity, yet fails to repress the translation of TOP mRNAs or has just a minor inhibitory effect in most experiments conducted thus far (Ref. 188 and references therein). (b) The translation of TOP mRNAs is refractory to raptor knockdown in human cells or only slightly repressed in knockout mouse cells, even though mTORC1 activity toward S6K1 activity is markedly reduced in both cases.188 (c) TOP mRNA translation is rendered rapamycin hypersensitive in raptordeficient cells.188 (d) Inhibition of TOP mRNA translation upon amino acid deprivation of HEK293 displays faster kinetics and is more effective than that exhibited by rapamycin.42 Having shown that mTORC1 has a minor, if any, role in translational control of TOP mRNAs, has suggested that TOP mRNAs are controlled primarily by mTORC2. Conceptually, this possibility seemed consistent with the sporadic sensitivity to rapamycin of both TOP mRNA translation and TORC2 activity, as well as the delayed response of these two readouts to rapamycin, relative to the rapamycin sensitivity of mTORC1 activity.26,188 Nevertheless, the ability of insulin to activate TOP mRNA translation in rictor knockout cells has refuted the requirement for mTORC2 in this mode of regulation.188 Several explanations can be proposed in an attempt to reconcile the relative independence of TOP mRNA translation on raptor or rictor with the canonical two mTOR complex model. (a) mTOR controls TOP mRNA translation through a third, as yet unidentified complex (mTORC3?); (b) mTOR can control TOP mRNAs in a complex-independent fashion. According to either of these explanations, neither raptor nor rictor is critical for TOP mRNA regulation; and (c) we cannot exclude formally the possibility that, in the absence of mTORC1, the translational regulation of TOP mRNAs is mediated by mTORC2, and vice versa. Examining the latter explanation would have to wait till the establishment of conditional rictor and raptor double knockout MEFs.

D. Candidate Trans-Acting Factors The discrete translational behavior of TOP mRNAs suggests that the 50 TOP motif is recognized by a specific translational trans-acting factor. This contention is supported by circumstantial evidence that in vitro translational repression of TOP mRNAs is caused by the accumulation of a titratable repressor in cell-free translation systems.199,200

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A search for proteins that interact with the 50 -TOP motif has yielded throughout the years a number of candidates: (a) a cytoplasmic protein of about 56 kDa from mouse T-lymphocytes, p56L32;201 (b) La autoantigen and cellular nucleic acid-binding protein (CNBP) that bind the 50 -TOP and the CG-rich sequence immediately downstream, respectively.202,203 Moreover, it has been claimed that it is the unphosphorylated form of La that selectively binds TOP mRNAs,204 yet TOP mRNAs are nearly completely excluded from La-associated mRNAs, when examined by genome-wide analysis;205 and (c) La-related protein 7 (LARP 7) was identified as a 50 -TOP motif-binding protein.206 However, no experimental data are currently available to unequivocally show that any of these proteins can affect the translation of TOP mRNA in vitro or in vivo.207 miRNAs are short (about 22 nt) oligonucleotides, which are major regulators of gene expression and function at the posttranscriptional level (see chapter 5 by Cara T. Pager, Karen A. Wehner, Gabriele Fuchs, P. Sarnow in this volume). A recent affinity purification of miR-10a-associated mRNAs has disclosed 55 TOP mRNAs out of 100 most enriched mRNAs.208 However, unlike most miRs characterized so far that bind their target mRNAs through interaction with the 30 -UTR, miR-10a appears to bind the CG-rich sequence immediately downstream of the 50 -TOP motif. Furthermore, overexpression of miR-10a selectively enhances the synthesis of rps in untreated cells and increases the polysomal association of the respective mRNAs in amino acid-starved cells. Likewise, functional analyses, using chimeric mRNAs, have demonstrated that ectopic expression of miR-10a alleviates the translational repression of TOP, but not of non-TOP mRNAs in anisomycin (translational inhibitor)-treated cells.208 These results imply, therefore, that overexpressed miR-10a exerts its positive role in the translational control of TOP mRNAs in a 50 -TOP motif-dependent manner. However, based on multiple examples of erroneous conclusions regarding the function of an overexpressed protein (discussed in Ref. 178), additional evidence is required for an unequivocal establishment of miR-10a as a physiological relevant regulator of TOP mRNA translation. These include (a) loss-of-function experiments, using downregulation of endogenous miR expression and (b) demonstration that miR-10a binding to TOP mRNAs correlates with their translation efficiency.

E. S6K and TOP mRNAs: The Rise and Fall of a Dogma The apparent correlation between the pathways that transduce external signals to S6K activity and TOP mRNA translation, as well as the partial similarity in their rapamycin sensitivity, led to a model assuming a causal relationship between these two variables.209 This model, however, has later been refuted by biochemical and genetic studies with cultured cells42,178,185,210 and subsequently, by genetically manipulated animals that have provided the

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‘‘last nail in the coffin’’ of the model. Thus, the translation of TOP mRNAs is normally regulated in hepatocytes from S6K1/;S6K2/ mice79 or in regenerating liver- and serum-stimulated MEFs from rpS6 phosphorylationdeficient mice.82 Collectively, all these observations indicate that TOP mRNAs are translationally controlled in an S6K- and rpS6 phosphorylationindependent fashion.

IV. Physiological Roles of S6K A. Cell Size Genetic and pharmacological manipulations in Drosophila, as well as in mammalian cells, have established all mediators along the pathway from growth factor receptors to mTOR that are involved in regulation of cell size (reviewed in Ref. 211). Accordingly, in parallel to the refutation of the role of S6K in the translational control of TOP mRNAs, it has emerged as a critical determinant of cell size. Thus, most Drosophila lacking their single S6K gene, dS6K, exhibit embryonic lethality, with the few surviving adults having a severely reduced body size, due to a decrease in cell size rather than a decrease in cell number. In addition, the surviving flies have a shorter life span, and females are sterile.212 S6K1/ mice are significantly smaller at birth, due to a proportional decrease in the size of all organs.213 A smaller cell size in these mice was reported for pancreatic b-cells214 and myoblasts.215 In contrast, the birth weight of S6K2/ mice, as well as the size of their myoblasts, is similar to those recorded for wild-type mice.214,215 The embryonic and postnatal growth, as well as the size of myoblasts and binucleated hepatocytes of the double knockout mice, S6K1/;S6K2/, are similar to those of S6K1/ mice.79,86,215 Nonetheless, the deficiency of both S6Ks, unlike the deficiency of each of them alone, is associated with a profound decrease in viability.79 rpS6 is the only protein, of the known S6K1 substrates, that has been shown, so far, to be directly involved in the control of cell size. Thus, a wide variety of cell types derived from rpS6P/ mice are significantly smaller than their wild-type counterparts. These include pancreatic b-cells, interleukin-7dependent cells derived from fetal livers, MEFs,82 muscle myotubes,216 and hepatocytes (Y. Bolkier, A. Binder, and O. Meyuhas, unpublished results). It appears, however, that the small cell phenotype is not ubiquitous, as acinar cells in the pancreas display a similar size regardless of the absence of S6K1214 or phosphorylatable serine residues in rpS6.82

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Previous studies have pointed out the fact that signals from PI3K to cell growth are not exclusively transduced through S6K. Thus, PTEN/ human cancer cells undergo a rapid increase in size after irradiation as compared with their PTENþ/þ counterparts. However, while inhibition of PI3K by wortmannin treatment was able to reduce cell size to the wild-type levels, rapamycin only led to a partial recovery in cell size.217 This may imply that execution of cell size control is only partially mediated through rapamycin-sensitive mTOR targets. Several lines of evidence support this hypothesis. (a) Conditional knockout of PTEN in the cerebellum and dentate gyrus causes cell autonomous increase in neuronal soma size, progressive macrocephaly, seizures, and premature death.218,219 Interestingly, these manifestations resemble phenotypic abnormalities associated with Lhermitte–Duclos disease, which occurs in humans with germ line mutations in PTEN. However, despite being downstream of PTEN, S6K1 deficiency fails to block the growth of these neuronal cells.220 (b) The expression of constitutively active Akt1 (myrAkt1) results in increased b-cell size and improved glucose tolerance,221,222 which is quite the opposite of the phenotype observed in S6K1/ or rpS6P/ b-cells.82,214 Nonetheless, the increase in b-cell size in myrAkt1 transgenic mice neither was affected by the deficiency of S6K1223 nor of rpS6 phosphorylation (A. Dreazen and O. Meyuhas, unpublished results). Collectively, these results imply that Akt1 signals cell size predominantly in an mTORC1-independent fashion. Interestingly, pancreata in about 30% of mice expressing myrAkt1 undergo hyperplastic transformation leading to insulinoma formation. However, deficiency of S6K1, but not of S6K2, fully protects the animals from myrAkt1-mediated pancreatic tumorigenesis.223 The readily detectable phosphorylated rpS6 in myrAkt1 transgenic islets, despite S6K1 deficiency, might argue against a tumorigenic role of rpS6 phosphorylation. Strikingly, however, the myrAkt1 oncogene also failed to trigger insulinoma formation in rpS6P/ b-cells lacking rpS6 phosphorylation (A. Dreazen and O. Meyuhas, unpublished results). It appears, therefore, that both S6K1 and rpS6 phosphorylation can promote malignant transformation, yet in an independent manner. The small size phenotype of S6K1–/–;S6K2–/– or rpS6P/ cells can result from a defect in cell growth, or alternatively, from accelerated cell cycle in the face of an unchanged rate of cell growth. Several lines of evidence support the notion that the small cell size phenotype in both mutants reflects impaired growth, rather than being a by-product of enhanced cell division. (a) S6K1–/–; S6K2–/– MEFs or primary myoblasts display similar cell doubling time as do their wild-type counterparts.79,215 (b) rpS6P/ MEFs remain smaller than their wild-type counterparts, even when progression through the cell cycle is arrested by aphidicolin, an inhibitor of DNA polymerase-a.82 (c) The size of immortalized rpS6P/ MEFs is increased to the extent that it equals that of

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rpS6Pþ/þ MEFs. Nevertheless, this increase is not accompanied by lengthening of the doubling time, as would be expected if the size was inversely proportional to the division rate.82 (d) rpS6Pþ/ primary MEFs are still smaller than rpS6Pþ/þ MEFs, even though they have a similar doubling time (M. Katz and O. Meyuhas, unpublished results).

B. Myoblast Size and Energy Charge S6K1 deficiency results in reduced size of myoblasts and cross-sectional area of muscle fibers due to a reduction of cytosolic volume with no defect in cell fusion. Moreover, genetic and pharmacological manipulations of myotubes have established that muscle cell number and size are regulated by distinct branches downstream of mTORC1, and have identified S6K1 as an essential effector for muscle cell growth.215 The small size of S6K-deficient muscle and myoblasts seems to be mediated by upregulation of AMPK in response to an increased AMP:ATP ratio in the mutant muscle.224 Moreover, the increased content of mitochondria and the reduced level of triacylglycerol in this muscle are consistent with the apparent elevated AMPK activity.225,226 Accordingly, downregulation of AMPK protects S6K-deficient myotubes or myofibers from size decrease, suggesting that AMPK activity negatively contributes to the growth control of muscle cells.224 rpS6 phosphorylation-deficient mice suffer from muscle weakness, as assessed by a variety of physical performance tests. This physical inferiority appears to result from two defects: (a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins and (b) a reduced content of ATP and phosphocreatine, two readily available energy sources.216 Notably, despite partial similarity in the phenotypic manifestations between the rpS6P/ and S6K/ muscles, the mechanism underlying the growth defect in rpS6P/ muscle, unlike that of the S6K/ mouse, does not seem to involve AMPK activation.216 The distinction between the mechanisms operated by these two related deficiencies suggests that S6K1 regulates cell size predominantly through one or more of its other substrates, rather than rpS6. One such candidate is SKAR, also involved in cell size regulation. It should be mentioned, however, that knockdown of SKAR leads to a reduction in cell size that is not as dramatic as knockdown of S6K1.103

C. Global Protein Synthesis The multiplicity of S6K substrates, which are bona fide components of the translational machinery, has raised the possibility that it plays a critical regulatory role in global protein synthesis. However, analysis of the proportion of ribosomes engaged in polysomes versus monosomes, which reflects the global

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translational rate, has revealed comparable profiles in myoblasts, hepatocytes, and fibroblasts from both wild-type and S6K1/-/-;S6K2/ mice. Similarly, measuring protein synthesis by methionine incorporation, showed no difference between wild-type and S6K-deficient cells.86 Numerous early studies showing temporal correlation between rpS6 phosphorylation and initiation of protein synthesis following mitogenic or nutritional stimuli227 provide a basis for the claim that rpS6 phosphorylation is involved in regulation of protein synthesis. This model has been further supported by UV cross-linking experiments that have localized rpS6 to the interface between the two ribosomal subunits and demonstrated its interaction with tRNA, initiation factors, and mRNA (reviewed in Ref. 228). However, monitoring the relative proportion of ribosomes engaged in translation (associated with polysomes) has demonstrated a similar proportion in the liver of both rpS6P/ and wild-type mice. Furthermore, this similarity was apparent even in regenerating liver, in which rpS6 undergoes extensive phosphorylation only in the wild type.82 Interestingly, MEFs derived from the rpS6P/ mouse, showed a significant increase in the rate of global protein synthesis (incorporation of radiolabeled amino acids), relative to those measured in wild-type MEFs.82 This superior protein synthesis rate is indeed reflected in a higher proliferation rate of rpS6P/ MEFs. It appears, therefore, that protein synthesis, at least in this cell type, is downregulated by rpS6 phosphorylation. Though a slightly faster elongation rate was determined in rpS6P/ MEFs, the augmentation in overall protein synthesis in these cells is mainly attributed to enhanced translation initiation by an as yet unknown mechanism. Taken together, these results indicate that the small cell phenotype of S6K- or rpS6 phosphorylation-deficient cells cannot be ascribed to compromised global protein synthesis.

D. Glucose Homeostasis and Insulin Resistance Insulin secretion closely correlates with the size of pancreatic b-cells.229 Indeed, mice deficient in S6K1 or rpS6 phosphorylation exhibit impaired glucose homeostasis, due to insufficient insulin secretion in response to glucose load. Furthermore, this defect is associated with the small size of b-cells in these mice, as well as a twofold reduction in both circulating levels and pancreatic content of insulin.82,214 Notably, the similarity in these phenotypic manifestations strongly suggests that it is the failure to phosphorylate rpS6 that can account for the common defects in both types of mutants. Nevertheless, verifying this hypothesis should await the availability of data on the phosphorylation status of rpS6 in S6K1/ b-cells.

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Despite the fact that S6K1/ and rpS6P/ mice are mildly glucose intolerant and display hypoinsulinemia, both maintain normal fasting glucose levels, which is consistent with the higher insulin sensitivity displayed by their peripheral tissues relative to that of wild-type tissues.82,214 This observation has led to the hypothesis that S6K is involved in insulin resistance that plays an important role in the pathogenesis of type 2 diabetes. Indeed, the mTOR/ S6K pathway signals not only downstream, but also upstream as a negative regulator, because S6K phosphorylates several serine residues of IRS-1 (see Section II.D) to inhibit insulin signaling at the level of IRS-1.107–110 It should be noted, however, that if increased insulin sensitivity in S6K1/ mice indeed results from the elimination of serine phosphorylation, it is quite puzzling how the lack of phosphorylatable serine residues in rpS6 is able to phenocopy the effect of S6K1 deficiency. One plausible explanation is that unphosphorylatable rpS6 is associated with an increased activity of an IRS‐1 serine phosphatase. It is well established that excess nutrient intake and chronic hyperinsulinemia cause insulin resistance, therefore, the negative regulation of insulin signaling exerted by S6K might have evolved to suppress this signaling under conditions of nutrient overload. Consistent with this model, S6K1/ mice maintained on a high-fat diet (HFD), normally promoting insulin resistance, remain insulin sensitive. Furthermore, knockdown of S6K1 in cells has no effect on insulin-induced activation of its receptor, but promotes insulininduced PKB phosphorylation.109 When wild-type mice were placed on an HFD they rapidly accumulated fat concomitantly with marked elevation of their S6K1 activity. Contrarily, when S6K1/ mice were similarly treated, the rate of weight accumulation was dramatically reduced as compared to wildtype mice, due to a dramatic increase in lipolysis and metabolic rate, which is linked to enhanced oxidative phosphorylation.109 The S6K1/IRS‐1 homeostatic negative feedback loop is not confined to HFD, as amino acid treatment of mouse muscle cells or human muscle biopsies leads to the concomitant activation of S6K1, downregulation of IRS-1 through its phosphorylation of S1097 or S1101, respectively, and insulin resistance.110 Taken together these observations have posed S6K1 as a critical component of insulin or nutrient in the development of insulin resistance through phosphorylation and inhibition of IRS-1 function. HFD feeding is sufficient to induce insulin resistance of hepatic gluconeogenesis within 3 days, and this response involves activation of hypothalamic S6K. Thus, overexpression of constitutively active S6K in the mediobasal hypothalamus (MBH) mimics the HFD effect in normal chow-fed animals, and blunts the ability of insulin to suppress glucose production. In contrast, suppression of S6K by overexpression of dominant-negative S6K or dominantnegative raptor in the MBH restored the ability of MBH insulin to suppress

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hepatic glucose production after HFD feeding. These results suggest that inhibition of S6K within the MBH may prevent the earliest stage of dietinduced insulin resistance.230

E. S6K and LTP and Memory A recent study has demonstrated that S6K1 and S6K2 differentially contribute toward the normal expression of behavioral learning and hippocampal synaptic plasticity. This is exemplified by the observation that S6K1-deficient mice display impaired early LTP and are deficient in multiple forms of learning and memory, whereas S6K2-deficient mice also display several memory phenotypes.231

V. Concluding Remarks The preceding sections have shown that S6K is a critical determinant of cell size and affects whole animal physiology. S6K was initially implicated in translational control of TOP mRNAs, yet later studies have unequivocally shown that it exerts its effects in a TOP mRNAs-independent fashion. Nonetheless, despite extensive experimental work, many questions regarding the mode of action of S6K and the translational control of TOP mRNAs remain unresolved, including the following major issues: (a) Which of the S6K1 substrate(s) mediates its regulatory role on processes as diverse as cell growth, glucose homeostasis, peripheral insulin resistance, and muscle energy balance, and why does S6K2 activity fail to compensate for S6K1 deficiency? Does it reflect partial selectivity of each of these kinases toward some substrates, and which of the unique substrates do(es) indeed play a major role in these distinct manifestations? (b) Why S6K deficiency has marginal, if any, effect on global protein synthesis, even though several of its substrates are bona fide translational factors? Is it possible that S6K accounts for the translation efficiency of a subgroup of mRNAs, via these factors, rather than general translational activity? (c) Many of the phenotypic manifestations of rpS6 phosphorylation deficiency recapitulate those observed in S6K1 knockout mice. However, among the common defects, some cannot be simply attributed to the lack of rpS6 phosphorylation. Thus, the smaller cross-sectional area of myofibers and the diminished energy content in S6K/ muscle are reminiscent of that of rpS6P/ muscle, even though rpS6 is still phosphorylated in the former. Similarly, the deficiency of either rpS6

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phosphorylation or S6K can protect b-cells from hyperplastic transformation, despite apparent rpS6 phosphorylation in the absence of S6K. Hence, it would be of great importance to figure out how S6K1 exerts these effects in a rpS6 phosphorylation-independent fashion. (d) The evolutionary conservation of TOP mRNAs, their ubiquitous distribution, and their translational control by multiple signals, imply that their translation efficiency plays a critical role in cellular physiology. However, examining this hypothesis will have to await the identification of the respective trans-acting factor(s). Thus, loss-of-function or gain-of function experiments in cells and whole animals will enable: (a) defining its mode of action as activator or repressor and (b) establishing the role of the translational control of TOP mRNAs during normal development, cell-cycle progression, and adaptation to various stress conditions.

Acknowledgments This work was supported by grants to O.M. from United States-Israel Binational Science Foundation (BSF 2005034), the Israel Science Foundation (Grant No. 296/05), the German-Israeli Foundation (Grant No. 819/05f), and the Otto Stieber Foundation. The authors thank Robert P. Perry for his critical comments, Wayne Sossin for his comments on the response of TOP mRNAs to LTP, and Steve Marygold, Philip East, and Riu Yamashita for the provision of unpublished sequences of Drosophila and human 50 -TOP motifs.

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