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Signal Transduction Pathways That Regulate Eukaryotic Protein Synthesis

1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês

10.1074/jbc.274.43.30337

ISSN

1083-351X

Autores

Robert E. Rhoads,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

eukaryotic initiation factor amino acids adenylate cyclase calmodulin diacylglycerol eukaryotic elongation factor eEF2 kinase epidermal growth factor extracellular signal-regulated kinase guanine nucleotide exchange factor G-protein-coupled receptors glycogen synthase kinase-3 inositol insulin receptor substrate mitogen-activated protein kinase mitogen-activated protein/ERK kinase MAPK-interacting kinase mammalian target of rapamycin 70-kDa ribosomal S6 kinase 90-kDa ribosomal S6 kinase PtdIns-dependent kinase pleckstrin homology phosphorylated, heat- and acid-stable protein phosphatidylinositol 3-kinase protein kinase B protein kinase C phospholipase C phosphatidylinositol receptor kinase substrates receptor tyrosine kinase Src homology domain 2 SH2-containing phosphotyrosine phosphatase-2 terminal oligopyrimidine untranslated region The last several years have witnessed an explosion in the published literature on two topics, the pathways that transduce extracellular signals to their intracellular targets and modification of the core translational apparatus in response to these signals. Most of these pathways result in cell growth and cell division. Synthesis of the entire complement of proteins is necessary to double the cell size, but synthesis of the so-called “growth-regulated” proteins (1Baserga R. Cancer Res. 1990; 50: 6769-6771PubMed Google Scholar) is needed for cell division. This article summarizes recent advances in our understanding of how a single mitogenic stimulus can simultaneously lead to an increase in both global and growth-regulated protein synthesis. The three stages of protein synthesis are catalyzed by initiation, elongation, and release factors (Ref. 2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 31-69Google Scholar; a guide to current and previous nomenclature can be found in Ref. 3Clark B.F.C. Grunberg-Manago M. Gupta N.K. Hershey J.W.B. Hinnebusch A.G. Jackson R.J. Maitra U. Mathews M.B. Merrick W.C. Rhoads R.E. Sonenberg N. Spremulli L. Trachsel H. Voorma H.O. Biochimie ( Paris ). 1996; 78: 11-12Crossref Scopus (41) Google Scholar). A ternary complex of eIF2·GTP·Met-tRNAi1 binds to the 40 S ribosomal subunit to form the 43 S initiation complex (Fig.1). The eIF4 factors plus poly(A)-binding protein recognize the 5′-terminal cap or 3′-terminal poly(A) tract of mRNA, unwind mRNA secondary structure, and transfer it to the 43 S initiation complex, resulting in the 48 S initiation complex. Scanning for the first initiation codon in good sequence context requires eIF4A and the presence of eIF1 and eIF1A (4Pestova T.V. Borukhov S.I. Hellen C.U.T. Nature. 1998; 394: 854-859Crossref PubMed Scopus (320) Google Scholar). Then eIF5 stimulates GTP hydrolysis by eIF2, after which the initiation factors are replaced by the 60 S subunit to form the 80 S initiation complex. The released eIF2·GDP is recycled to eIF2·GTP by the GEF eIF2B. The first elongator aminoacyl-tRNA is brought to the A-site by eEF1, followed by a cycle of GTP hydrolysis and exchange analogous to that of eIF2. Translocation is catalyzed by eEF2, again with a GTP hydrolysis cycle. The binding of growth factors to the extracellular domain of RTKs causes a conformational change that induces oligomerization and activation of the intracellular protein Tyr kinase domain (Ref. 5Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar; Fig. 2). Substrates for the kinase can be either the RTK itself or a separate RKS. The SH2 domains of several different signaling molecules dock to the resulting Tyr(P)s in a sequence-specific manner, thereby activating separate downstream signaling cascades. These receptors are coupled to heterotrimeric G-proteins (6Vaughan M. J. Biol. Chem. 1998; 273: 17297Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Dissociation of the G-protein subunits activates AC, PLC, and other downstream effectors. PLC hydrolyzes PtdIns(4,5)P2 to DAG and Ins(1,4,5)P3. These are docking proteins for downstream effectors of RTKs (7White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (625) Google Scholar). The best studied RKS are the insulin receptor substrates, which include IRS-1, IRS-2, IRS-3, Gab-1, and p62DOK. Members of the IRS family bind to insulin receptor via an NH2-terminal PH domain and a Tyr(P)-binding domain. The COOH-terminal portions of the proteins contain numerous Tyr phosphorylation sites. IRS-1 alone provides docking sites for PI3-K, SH-PTP2, Grb-2, Fyn, Nck, and Crk. This phosphatase contains two SH2 domains, and enzyme activity is maximally activated when both are occupied by Tyr(P)-containing peptides (8Pluskey S. Wandless T.J. Walsh C.T. Shoelson S.E. J. Biol. Chem. 1995; 270: 2897-2900Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). SH-PTP2 is activated by docking to EGF receptor, platelet-derived growth factor receptor, c-kit, insulin receptor, IRS-1, IRS-2, and IRS-3 and may serve to attenuate the Tyr(P) signal in these molecules (7White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (625) Google Scholar). This G-protein is bound to the plasma membrane by COOH-terminal prenylation and myristoylation (9Vojtek A.B. Der C.J. J. Biol. Chem. 1998; 273: 19925-19928Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). GEF activity is provided by SOS, which associates constitutively with the SH2- and SH3-containing protein Grb-2. The Grb-2·SOS complex is recruited to the plasma membrane by binding to specific Tyr(P)s in IRS-1, IRS-2, Shc, or SH-PTP2 (7White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (625) Google Scholar). Another GEF, Ras-GEF, is stimulated by Ca2+/calmodulin (CaM) downstream of GPCR (10Downward J. Nature. 1998; 396: 416-417Crossref PubMed Scopus (36) Google Scholar). The hydrolysis of GTP by Ras is stimulated by GTPase-activating proteins such as p120GAP and NF1 (9Vojtek A.B. Der C.J. J. Biol. Chem. 1998; 273: 19925-19928Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Ras·GTP activates the Ser/Thr kinase Raf-1 by recruiting it to the plasma membrane. Raf-1, in turn, phosphorylates and activates MEK1 and MEK2. The MEKs are dual specificity kinases, phosphorylating both Thr and Tyr residues in ERK1 and ERK2 (p42 and p44 MAPKs). This kinase is composed of a catalytic subunit and a SH2-containing regulatory subunit that binds to Tyr(P)s in RTKs and RKS (7White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (625) Google Scholar). PI3-K is also activated synergistically by direct binding to Ras·GTP (11Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (501) Google Scholar). PI3-K is a dual specificity kinase that phosphorylates PtdIns at the 3-position and proteins on Ser/Thr residues (12Divecha N. Irvine R.F. Cell. 1995; 80: 269-278Abstract Full Text PDF PubMed Scopus (590) Google Scholar). The lipid phosphorylation signal activates PDK and PKB, and the protein phosphorylation signal activates MAPK (13Bondeva T. Pirola L. Bulgarelli-Leva G. Rubio I. Wetzker R. Wymann M.P. Science. 1998; 282: 293-296Crossref PubMed Scopus (302) Google Scholar). Both activities are inhibited by wortmannin and LY294002 (14Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). These recently discovered kinases, with at least four isoforms, bind to and are activated by PtdIns(3,4,5)P3 by their COOH-terminal PH domains (15Stokoe D. Stephens L. Copeland T. Gaffney R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1054) Google Scholar, 16Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (916) Google Scholar). PKB exists in at least four isoforms (α, β1, β2, γ) and is activated by both RTKs and GPCR. In the former case, PI3-K is involved (17Wijkander J. Holst L.S. Rahn T. Resjo S. Castan I. Manganiello V. Belfrage P. Degerman E. J. Biol. Chem. 1997; 272: 21520-21526Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), but in the latter, there are both PI3-K-dependent (18Murga C. Laguinge L. Wetzker R. Cuadrado A. Gutkind J.S. J. Biol. Chem. 1998; 273: 19080-19085Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) and -independent (19Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) pathways. PKB is targeted to the plasma membrane by direct binding to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 through its PH domain (20Sable C.L. Filippa N. Filloux C. Hemmings B.A. Obberghen E.V. J. Biol. Chem. 1998; 273: 29600-29606Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), where it is activated by phosphorylation at Thr-308 by PDK (15Stokoe D. Stephens L. Copeland T. Gaffney R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1054) Google Scholar). There are at least 10 isoforms of PKC (α–ζ) that differ in responsiveness to phospholipids and Ca2+ (21Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar). Classical PKCs (α, β, and γ) are activated and eventually down-regulated by phorbol esters, which are structural analogs of the physiological signal DAG, but atypical isoforms (λ and ζ) are not. Insulin activates both classical and atypical isoforms (22Bandyopadhyay G. Standaer M.L. Zhao L., Yu, B. Avignon A. Galloway L. Karnam P. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 2551-2558Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 23Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1997; 17: 5184-5192Crossref PubMed Google Scholar). PKCζ is activated downstream of PI3-K (24Herrera-Velit P. Knutson K.L. Reiner N.E. J. Biol. Chem. 1997; 272: 16445-16452Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) via direct phosphorylation at Thr-410 by PDK1 (25Le Good J.A. Ziegler W.H. Parekh D.B. Allessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar). Changes in cytosolic Ca2+levels can occur by at least two mechanisms (26Nairn A. Palfrey H.C. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 295-318Google Scholar). First, GPCR operate Ca2+ channels that allow influx from the extracellular space. Second, Ins(1,4,5)P3, released in response to both RTKs and GPCR, binds to receptors in the endoplasmic reticulum and releases Ca2+ into the cytosol. This kinase regulates numerous cellular processes besides phosphorylation of glycogen synthase, including insulin-stimulated protein synthesis (27Cross D.A.E. Allessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4397) Google Scholar). Phosphorylation of GSK-3 at Ser-9 inactivates the enzyme and is correlated with the activation of protein synthesis. GSK-3 is phosphorylated directly by PKB in vivo (28Hajduch E. Alessi D.R. Hemmings B.A. Hundal H.S. Diabetes. 1998; 47: 1006-1013Crossref PubMed Scopus (296) Google Scholar, 29van Weeren P.C. Bruyn M.T. de Vries-Smits M.M. van Lint J. Burgering B.M. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). mTOR is a 290-kDa protein kinase that is activated by PKB (30Scott P.H. Brunn G.J. Kohn R.A. Lawrence J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (414) Google Scholar) and phosphorylates Ser/Thr-Pro motifs (31Abraham R.T. Wiederrecht G.J. Annu. Rev. Immunol. 1996; 14: 483-510Crossref PubMed Scopus (575) Google Scholar). As the name implies, its kinase activity is inhibited by the immunosuppressant rapamycin (31Abraham R.T. Wiederrecht G.J. Annu. Rev. Immunol. 1996; 14: 483-510Crossref PubMed Scopus (575) Google Scholar). The ability of insulin to cause phosphorylation and activation of mTOR is attenuated by cAMP (32Scott P.H. Lawrence J.C. J. Biol. Chem. 1998; 273: 34496-34501Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The major activity responsible for phosphorylation of ribosomal protein S6 is p70S6K (33Proud C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar), which is activated by hierarchical phosphorylation of seven Ser/Thr sites. Phosphorylation of COOH-terminal sites occurs first, making Thr-389 available for phosphorylation by a rapamycin-sensitive pathway (34Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (947) Google Scholar) and culminating in phosphorylation of Thr-229 by constitutively active PDK1 (35Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar). Multipotential S6 kinase is activated and becomes membrane-associated in response to insulin (36Chang Y.W. Traugh J.A. J. Biol. Chem. 1997; 272: 28252-28257Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). p90S6K is activated in response to growth factors by forming complexes with ERK1 and -2 and undergoing phosphorylation (37Smith J.A. Poteet-Smith C.E. Malarkey K. Sturgill T.W. J. Biol. Chem. 1999; 274: 2893-2898Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Additional S6 kinases continue to be discovered (38Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar, 39New L. Zhao M. Li Y. Bassett W.W. Feng Y. Ludwig S. Padova F.D. Gram H. Han J. J. Biol. Chem. 1999; 274: 1026-1032Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). PHAS-I was originally observed as a phosphorylated protein that increased upon insulin treatment of cells but was later found to bind eIF4E specifically (40Lawrence J.C. Abraham R.T. Trends Biochem. Sci. 1997; 22: 345-349Abstract Full Text PDF PubMed Scopus (186) Google Scholar). Two additional isoforms have since been found, PHAS-II and PHAS-III (41Poulin F. Gingras A.C. Olsen H. Chevalier S. Sonenberg N. J. Biol. Chem. 1998; 273: 14002-14007Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Phosphorylation of PHAS occurs at six or more sites, two to five of which appear to result from direct phosphorylation by mTOR (34Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (947) Google Scholar,42Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence J.C. Abraham R.T. Science. 1997; 277: 99-101Crossref PubMed Scopus (814) Google Scholar). These kinases, homologous to p90S6K, were identified as binding partners of ERK1 and -2 (43Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (793) Google Scholar) and in a screen for ERK1 substrates (44Fukunaga R. Hunter T. EMBO J. 1997; 16: 1921-1933Crossref PubMed Scopus (559) Google Scholar). Mnk is activated by phosphorylation at Thr-197 and Thr-202 both by a mitogen-activated pathway via ERK1 or -2 and also by a stress-activated pathway via p38 (45Waskiewicz A.J. Johnson J.C. Penn B. Mahalingam M. Kimball S.R. Cooper J.A. Mol. Cell. Biol. 1999; 19: 1871-1880Crossref PubMed Scopus (404) Google Scholar). Interestingly, Mnk1 also binds to the COOH terminus of eIF4G (45Waskiewicz A.J. Johnson J.C. Penn B. Mahalingam M. Kimball S.R. Cooper J.A. Mol. Cell. Biol. 1999; 19: 1871-1880Crossref PubMed Scopus (404) Google Scholar,46Pyronnet S. Imataka H. Gingras A.-C. Fukunaga R. Hunter T. Sonenberg N. EMBO J. 1999; 18: 270-279Crossref PubMed Scopus (537) Google Scholar). This kinase is activated by Ca2+/CaM and contains a putative CaM-binding domain COOH-terminal to the catalytic domain (47Redpath N.T. Price N.T. Proud C.G. J. Biol. Chem. 1996; 271: 17547-17554Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). eEF2 kinase is also activated by cAMP, independently of Ca2+ (48Diggle T.A. Redpath N.T. Heesom K.J. Denton R.M. Biochem. J. 1998; 336: 525-529Crossref PubMed Scopus (52) Google Scholar). Two types of signal-induced modifications of translation factors have been described to date: changing the intrinsic activity or binding properties of the factor by phosphorylation and sequestration of the factor in an inactive complex, the formation or dissociation of which may be controlled by phosphorylation. Most situations that lead to phosphorylation of eIF2α represent cellular stress, but eIF2α phosphorylation also changes as a result of normal signaling pathways, e.g. those regulated by amino acids (49Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) and interleukin-3 (50Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar). Phosphorylation of eIF2α on Ser-51 by several different kinases causes formation of a stable complex with eIF2B (Fig. 1), thereby reducing the concentration of active eIF2B (51Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar). This GEF is phosphorylated on the ε subunit and inactivated by GSK-3α and -β (52Welsh G.I. Miyamoto S. Price N.T. Safer B. Proud C.G. J. Biol. Chem. 1996; 271: 11410-11413Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Phosphorylation occurs at Ser-540, although evidence suggests that a priming phosphorylation at Ser-544 by some other kinase is needed (53Welsh G.I. Loughlin A.J. Foulstone E.J. Price N.T. Proud C.G. Biochem. Soc. Trans. 1997; 25 (suppl.): 191Crossref Scopus (7) Google Scholar). The mRNA cap-binding protein is phosphorylated at Ser-209 in vivo (54Joshi B. Cai A.-L. Keiper B.D. Minich W.B. Mendez R. Beach C.M. Stepinski J. Stolarski R. Darzynkiewicz E. Rhoads R.E. J. Biol. Chem. 1995; 270: 14597-14603Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 55Flynn A. Proud C.G. J. Biol. Chem. 1995; 270: 21684-21688Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) by Mnk1 and -2 (43Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (793) Google Scholar, 45Waskiewicz A.J. Johnson J.C. Penn B. Mahalingam M. Kimball S.R. Cooper J.A. Mol. Cell. Biol. 1999; 19: 1871-1880Crossref PubMed Scopus (404) Google Scholar), resulting in an increase of its affinity for caps (56Minich W.B. Balasta M.L. Goss D.J. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7668-7672Crossref PubMed Scopus (261) Google Scholar). eIF4E variants unable to bind eIF4G are poorly phosphorylated (46Pyronnet S. Imataka H. Gingras A.-C. Fukunaga R. Hunter T. Sonenberg N. EMBO J. 1999; 18: 270-279Crossref PubMed Scopus (537) Google Scholar). The availability of eIF4E is also regulated by formation of an inactive complex with PHAS, and phosphorylation of PHAS causes dissociation of the complex (57Lin T. Kong X. Haystead T.A.J. Pause A. Belsham G. Sonenberg N. Lawrence J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (602) Google Scholar, 58Pause A. Belsham G.J. Gingras A. Donze O. Lin T. Lawrence J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1063) Google Scholar). The relationship between these two eIF4E regulatory mechanisms is unclear. In vitro, PHAS inhibits eIF4E phosphorylation by Mnk1 (59Wang X. Flynn A. Waskiewicz A.J. Webb B.L.J. Vries R.G. Baines I.A. Cooper J.A. Proud C.G. J. Biol. Chem. 1998; 273: 9373-9377Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), but in vivo, eIF4E is extensively phosphorylated in cells overexpressing PHAS, suggesting that eIF4E phosphorylation is independent of PHAS (45Waskiewicz A.J. Johnson J.C. Penn B. Mahalingam M. Kimball S.R. Cooper J.A. Mol. Cell. Biol. 1999; 19: 1871-1880Crossref PubMed Scopus (404) Google Scholar). This linking protein, as part of the eIF4F complex, is phosphorylated at unknown sites by multipotential S6 kinase (60Morley S.J. Dever T.E. Etchison D. Traugh J.A. J. Biol. Chem. 1991; 266: 4669-4672Abstract Full Text PDF PubMed Google Scholar). The phosphorylated complex is more stimulatory for in vitroprotein synthesis and binding of mRNA to the 43 S initiation complex. This ribosomal protein is phosphorylated at five Ser residues, located at 235, 236, 240, 244, and 247 (33Proud C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). However, the effect of S6 phosphorylation on protein synthesis is unclear. There is a correlation between activation of p70S6K and translation of mRNAs containing a 5′-TOP tract (61Jefferies B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (813) Google Scholar), and treatment of 80 S ribosomes with multipotential S6 kinase increases the rate of elongation 2-fold (36Chang Y.W. Traugh J.A. J. Biol. Chem. 1997; 272: 28252-28257Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). A recent study implicates S6 phosphorylation in the interaction between 40 S subunits and mRNA (62Chiaberge S. Cassarino E. Mangiarotti G. J. Biol. Chem. 1998; 273: 27070-27075Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The first of the elongation factors is composed of α, β, γ, and δ subunits. Various subunits are phosphorylatedin vitro by several kinases, including casein kinase II, multipotential S6 kinase, and PKC (36Chang Y.W. Traugh J.A. J. Biol. Chem. 1997; 272: 28252-28257Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In the latter two cases, these phosphorylations result in a stimulation of elongation and GDP/GTP exchange, respectively. This elongation factor is inhibited by phosphorylation via eEF2 kinase on Thr-56 and Thr-58, the phosphorylation of the latter site requiring prior phosphorylation of the former (26Nairn A. Palfrey H.C. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 295-318Google Scholar, 63Redpath N.T. Price N.T. Severinov K.V. Proud C.G. Eur. J. Biochem. 1993; 213: 689-699Crossref PubMed Scopus (157) Google Scholar). Insulin, by far the best characterized effector of protein synthesis, stimulates both initiation and elongation. eIF2α phosphorylation does not change, but eIF2B activity nonetheless increases by the pathway IRS-1 (Tyr-608, -628, and -658; Ref. 64Mendez R. Myers M.G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1996; 16: 2857-2864Crossref PubMed Scopus (208) Google Scholar) → PI3-K (64Mendez R. Myers M.G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1996; 16: 2857-2864Crossref PubMed Scopus (208) Google Scholar) → PDK → PKB (65Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Masuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar) → GSK-3 (28Hajduch E. Alessi D.R. Hemmings B.A. Hundal H.S. Diabetes. 1998; 47: 1006-1013Crossref PubMed Scopus (296) Google Scholar) → eIF2Bε (53Welsh G.I. Loughlin A.J. Foulstone E.J. Price N.T. Proud C.G. Biochem. Soc. Trans. 1997; 25 (suppl.): 191Crossref Scopus (7) Google Scholar) (see Fig. 2). However, insulin may activate eIF2B through additional routes, because constitutively active PKCζ stimulates general protein synthesis in an insulin-dependent manner without activating p70S6K, suggesting that PKB is not involved (23Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1997; 17: 5184-5192Crossref PubMed Google Scholar). Binding of SH-PTP2 to IRS-1(Tyr-1172 and -1222) attenuates insulin-stimulated protein synthesis (66Myers M.G. Mendez R. Shi P. Pierce J.H. Rhoads R. White M.W. J. Biol. Chem. 1998; 273: 26908-26914Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). PHAS-I is phosphorylated in response to insulin (58Pause A. Belsham G.J. Gingras A. Donze O. Lin T. Lawrence J.C. Sonenberg N. 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Phosphorylation of eIF4E is by the pathway IRS-1 (Tyr-895) → Grb-2·SOS → Ras → Raf → MEK → ERK → Mnk. (There are numerous other RKS that contribute to insulin stimulation of MAPK, especially Shc.) eIF4E association with eIF4G is stimulated by insulin (70Sinaud S. Balage M. Bayle G. Dardevet D. Vary T.C. Kimball S.R. Jefferson L.S. Grizard J. Am. J. Physiol. 1999; 39: E50-E61Google Scholar) as is eIF4G phosphorylation (60Morley S.J. Dever T.E. Etchison D. Traugh J.A. J. Biol. Chem. 1991; 266: 4669-4672Abstract Full Text PDF PubMed Google Scholar). Insulin also stimulates elongation by two mechanisms: phosphorylation of eEF1 and S6, the latter occurring by both multipotential S6 kinase (36Chang Y.W. Traugh J.A. J. Biol. Chem. 1997; 272: 28252-28257Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and p70S6K (71Myers M.G. Grammer T.C. Wang L. Sun X.J. Pierce J.H. Blenis J. White M.F. J. Biol. 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The activity of eIF2B is increased, partly through dephosphorylation of eIF2α and partly through phosphorylation of eIF2Bε, but GSK-3 activity is not altered (75Kimball S.R. Horetsky R.L. Jefferson L.S. J. Biol. Chem. 1998; 273: 30945-30953Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Like insulin, amino acids cause phosphorylation of p70S6Kand PHAS through a mTOR-dependent pathway (76Xu G. Kwon G. Marshall C.A. Lin T.-A. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 77Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (295) Google Scholar, 78Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 79Shigemitsu K. Tsujishita Y. Hara K. Nanahoshi M. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 1058-1065Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Unlike insulin, however, the pathway does not involve PKB (78Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 77Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (295) Google Scholar) but does involve tRNA aminoacylation (80Iiboshi Y. Pabst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The insulin- or IGF-1-mediated phosphorylation of p70S6K and PHAS-I requires amino acids (76Xu G. Kwon G. Marshall C.A. Lin T.-A. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 79Shigemitsu K. Tsujishita Y. Hara K. Nanahoshi M. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 1058-1065Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). In the presence of insulin, amino acids activate eIF2B but not eIF4E (75Kimball S.R. Horetsky R.L. Jefferson L.S. J. Biol. Chem. 1998; 273: 30945-30953Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Less is known about the regulation of protein synthesis by GPCR per se. Angiotensin causes phosphorylation of eIF4E (81Rao G.N. Griendling K.K. Frederickson R.M. Sonenberg N. Alexander R.W. J. Biol. Chem. 1994; 269: 7180-7184Abstract Full Text PDF PubMed Google Scholar) and PHAS-I (82Fleurent M. Gingras A.-C. Sonenberg N. Meloche S. J. Biol. Chem. 1997; 272: 4006-4012Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), the μ-opioid receptor activates p70S6K (83Polakiewicz R.D. Schiefer S.M. Gingras A.C. Sonenberg N. Comb M.J. J. Biol. 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Factors contributing to low efficiency of translation include a highly structured 5′-UTR, the presence of upstream AUGs, and poor sequence context for the initiating AUG (89De Benedetti A. Harris A.L. Int. J. Biochem. Cell Biol. 1999; 31: 59-72Crossref PubMed Scopus (303) Google Scholar), all of which are found in the 5′-UTRs of mRNAs for scarce proteins (90Kochetov A.V. Ischenko I.V. Vorobiev D.G. Kel A.E. Babenko V.N. Kisselev L.L. Kolchanov N.A. FEBS Lett. 1998; 440: 351-355Crossref PubMed Scopus (88) Google Scholar). mRNAs with these properties encode a disproportionate share of proteins involved in cell growth and cell cycle progression (89De Benedetti A. Harris A.L. Int. J. Biochem. Cell Biol. 1999; 31: 59-72Crossref PubMed Scopus (303) Google Scholar). These mRNAs are poorly translated in quiescent cells but preferentially recruited to ribosomes after a mitogenic signal (91Darveau A. Pelletier J. Sonenberg N. Proc. Natl. Acad. Sci. U. S. 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Chem. 1998; 273: 29864-29872Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar) requires mTOR, and inhibition of mTOR with rapamycin prolongs the G1 phase in both T-cells (95Terada N. Takase K. Pabst P. Nairn A.C. Gelfand E.W. J. Immunol. 1995; 155: 3418-3426PubMed Google Scholar) and yeast (96Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. Mol. Biol. Cell. 1996; 7: 25-42Crossref PubMed Scopus (603) Google Scholar). These results suggest that pathways which activate the unwinding machinery, i.e. by phosphorylation of eIF4E, eIF4G, or PHAS, disproportionately stimulate translation of growth-regulated mRNAs with high 5′-UTR secondary structure. 5′-TOP mRNAs are also differentially regulated in response to extracellular signals (97Hornstein E. Git A. Braunstein I. Avnil D. Meyuhas O. J. Biol. Chem. 1999; 274: 1708-1714Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These encode many translational components, including ribosomal proteins, eEF1α, eEF2, and poly(A)-binding protein. 5′-TOP mRNAs are recruited to polysomes in a growth-dependent fashion that is selectively inhibited by rapamycin (72Redpath N.T. Foulstone E.J. Proud C.G. EMBO J. 1996; 15: 2291-2297Crossref PubMed Scopus (231) Google Scholar, 98Jefferies H.B.J. Reinhard C. Kozma S.C. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4441-4445Crossref PubMed Scopus (560) Google Scholar). This finding alone does not distinguish between signaling through PHAS and signaling through p70S6K, because the pathway bifurcates downstream of mTOR (99von Manteuffel S. Dennis P.B. Pullen N. Gingras A. Sonenberg N. Thomas G. Mol. Cell. Biol. 1997; 17: 5426-5436Crossref PubMed Scopus (212) Google Scholar) (see Fig. 2). However, ectopic expression of a dominant interfering p70S6K blocks both activation of p70S6K and 5′-TOP mRNA translation, indicating a direct role for p70S6K (61Jefferies B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (813) Google Scholar). The insulin-stimulated pathways to general protein synthesis and to growth-regulated protein synthesis in 32D cells can be dissected with rapamycin (64Mendez R. Myers M.G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1996; 16: 2857-2864Crossref PubMed Scopus (208) Google Scholar). Insulin-stimulated total protein synthesis is inhibited only ∼10%, and actin synthesis is not affected at all, but insulin-stimulated c-Myc synthesis is completely inhibited. Also, expression of constitutively active PKCζ in the absence of IRS-1, which bypasses the mTOR pathway, permits insulin-stimulated general protein synthesis but not c-Myc synthesis (23Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1997; 17: 5184-5192Crossref PubMed Google Scholar). This suggests that the insulin signal bifurcates at some point after PI3-K, one pathway stimulating general protein synthesis through eIF2B and one stimulating growth-regulated protein synthesis, accounting for ∼10% of the total, through eIF4E and S6. Similarly, T-cell activation causes 13% of the pre-existing mRNAs to be recruited to ribosomes (100Garcia-Sanz J.A. Mikulits W. Livingstone A. Lefkovits I. Mullner E.W. FASEB J. 1998; 12: 299-306Crossref PubMed Scopus (49) Google Scholar), and rapamycin causes a 15% decrease in protein synthesis in activated T-cells (95Terada N. Takase K. Pabst P. Nairn A.C. Gelfand E.W. J. Immunol. 1995; 155: 3418-3426PubMed Google Scholar). Protein synthesis is one of the most complicated biochemical processes undertaken by the cell, requiring roughly 150 different polypeptides and 70 different RNAs. Yet only seven polypeptides (eIF2α, eIF2Bε, eIF4E, eIF4G, S6, eEF1, and eEF2) have been identified as targets for regulatory pathways to date. Early observations that multiple initiation and elongation factors were phosphorylated in response to a single extracellular signal (101Duncan R. Hershey J.W.B. J. Biol. Chem. 1985; 260: 5493-5497Abstract Full Text PDF PubMed Google Scholar, 102Morley S.J. Traugh J.A. J. Biol. Chem. 1989; 264: 2401-2404Abstract Full Text PDF PubMed Google Scholar) may have suggested unnecessary redundancy. This now seems more comprehensible when it is realized that modification of some factors affects the overall rate of translation whereas modification of others affects the spectrum of mRNAs translated. Understanding the pathways for regulation of protein synthesis holds promise for novel approaches for cancer intervention (e.g. Ref. 103Aktas H. Fluckiger R. Acosta J.A. Savage J.M. Palakurthi S.S. Halperin J.A. Proc. Natl. Acad. Sci. U. S. 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