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α1A Adrenergic Receptor Induces Eukaryotic Initiation Factor 4E-binding Protein 1 Phosphorylation via a Ca2+-dependent Pathway Independent of Phosphatidylinositol 3-kinase/Akt*

Open AccessPublished:February 25, 2000DOI:https://doi.org/10.1074/jbc.275.8.5460
      Phosphorylation of the translation repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is thought to be partly responsible for increased protein synthesis induced by growth factors. This study investigated the effect of a Gq-coupled receptor on protein synthesis and the phosphorylation state and function of 4E-BP1 in Rat-1 fibroblasts expressing the human α1A adrenergic receptor. Treatment of cells with phenylephrine (PE), a specific α1adrenergic receptor agonist, increased protein synthesis and induced the phosphorylation of 4E-BP1 and its release from translation initiation factor 4E. Although the PE-induced phosphorylation of 4E-BP1 was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002, neither phosphatidylinositol 3-kinase nor Akt, its downstream effector, is activated in cells treated with PE (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z., J. Biol. Chem. 275, 4803–4809). The effect of PE on 4E-BP1 phosphorylation was also abolished in cells depleted of intracellular Ca2+ and in cells pretreated with calmodulin antagonists. By contrast, phosphorylation of 4E-BP1 still occurred in cells in which the Ca2+- and diacylglycerol-dependent isoforms of protein kinase C were down-regulated by prolonged exposure to a phorbol ester. We conclude that activation of the α1A adrenergic receptor in Rat-1 fibroblasts leads to phosphorylation of 4E-BP1 via a pathway that is Ca2+- and calmodulin-dependent. Phosphatidylinositol 3-kinase, Akt, and phorbol ester-sensitive protein kinase C isoforms do not appear to be required in this signaling pathway.
      eIF
      eukaryotic initiation factor
      4E-BP1
      eIF4E-binding protein 1
      mTOR
      mammalian target of rapamycin
      PDGF
      platelet-derived growth factor
      AR
      adrenergic receptor
      PE
      phenylephrine
      PI
      phosphatidylinositol
      PKC
      protein kinase C
      TPA
      phorbol 12-myristate 13-acetate
      p70 S6 kinase
      M r = 70,000 ribosomal protein S6 kinase
      MOPS
      4-morpholinepropanesulfonic acid
      BAPTA-AM
      1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester
      Treatment of cells with growth factors induces an increase in the rate of protein synthesis that is required for proliferating cells to undergo DNA synthesis and for nonproliferating cells to undergo hypertrophic growth. In eukaryotes, translational control is exerted mainly at the level of initiation (
      • Pain V.M.
      ,
      • Proud C.G.
      • Denton R.M.
      ). In translation initiation, methionyl-tRNA and several initiation factors bind to the 40 S ribosomal subunit to form the 43 S preinitiation complex, the complex binds to the 5′ end of the mRNA and translocates to the initiation codon, and then the 60 S ribosomal subunit is added to form an active 80 S ribosome. Binding of the 43 S preinitiation complex to mRNA is mediated by eukaryotic initiation factor (eIF)1 4F. eIF4F in mammals contains three subunits; one of them, eIF4E, binds directly to the m7GpppN (where N is any nucleotide) cap on the 5′ end of the mRNA. Together with eIF4B, eIF4F unwinds the secondary structure in the 5′ untranslated region of the mRNA to create a binding site for the 43 S preinitiation complex (
      • Pain V.M.
      ,
      • Proud C.G.
      • Denton R.M.
      ).
      Activation of protein synthesis by growth factors is a complex process that involves phosphorylation of a number of translation initiation factors, regulatory proteins and the 40 S ribosomal subunit (
      • Pain V.M.
      ,
      • Proud C.G.
      • Denton R.M.
      ,
      • Sonenberg N.
      • Gingras A.C.
      ,
      • Jefferies H.
      • Thomas G.
      ). eIF4E-binding protein 1 (4E-BP1) is a 12-kDa translation repressor that is thought to be a key player in the regulation of protein synthesis (
      • Sonenberg N.
      • Gingras A.C.
      ). In resting cells, hypophosphorylated forms of 4E-BP1 bind tightly to eIF4E on the mRNA cap, thus preventing formation of a functional eIF4F complex (
      • Sonenberg N.
      • Gingras A.C.
      ,
      • Pause A.
      • Belsham G.J.
      • Gingras A.C.
      • Donze O.
      • Lin T.A.
      • Lawrence Jr., J.C.
      • Sonenberg N.
      ). Treatment of cells with growth factors leads to phosphorylation of 4E-BP1 on multiple sites and its dissociation from eIF4E, thereby relieving the translational block. Translation of mRNAs with extensive secondary structure at the 5′ end is thought to be particularly sensitive to regulation by 4E-BP1 (
      • Sonenberg N.
      • Gingras A.C.
      ). Phosphorylation of the S6 protein in 40 S ribosomal subunits is another mechanism that mediates growth factor-induced activation of protein synthesis (
      • Jefferies H.
      • Thomas G.
      ). The major kinase that phosphorylates S6 is theM r = 70,000 S6 kinase (p70 S6 kinase) (
      • Jefferies H.
      • Thomas G.
      ,
      • Shima H.
      • Pende M.
      • Chen Y.
      • Fumagalli S.
      • Thomas G.
      • Kozma S.C.
      ). p70 S6 kinase is activated by phosphorylation of the enzyme at multiple sites (
      • Pullen N.
      • Thomas G.
      ,
      • Moser B.A.
      • Dennis P.B.
      • Pullen N.
      • Pearson R.B.
      • Williamson N.A.
      • Wettenhall R.E.
      • Kozma S.C.
      • Thomas G.
      ). Phosphorylation of the 40 S ribosomal subunit by p70 S6 kinase is thought to selectively up-regulate translation of certain mRNAs that contain a polypyrimidine tract adjacent to the mRNA cap (5′-TOP mRNAs; Refs.
      • Amaldi F.
      • Pierandrei-Amaldi P.
      and
      • Jefferies H.B.
      • Fumagalli S.
      • Dennis P.B.
      • Reinhard C.
      • Pearson R.B.
      • Thomas G.
      ).
      Intense study has been aimed at identifying upstream regulators in the signaling pathways that lead to phosphorylation of 4E-BP1 and p70 S6 kinase. These pathways appear to be quite similar. First, it has been demonstrated in many cell systems that growth factor-induced phosphorylation of both proteins is blocked by the immunosuppressant rapamycin (
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ,
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Lin T.-A.
      • Kong X.
      • Saltiel A.R.
      • Blackshear P.J.
      • Lawrence Jr., J.C.
      ,
      • Beretta L.
      • Gingras A.C.
      • Svitkin Y.V.
      • Hall M.N.
      • Sonenberg N.
      ). Rapamycin, when bound to its intracellular receptor FKBP12, inhibits the function of the mammalian target of rapamycin (mTOR), a kinase of which the catalytic domain resembles that of phosphatidylinositol (PI) 3-kinase (
      • Kunz J.
      • Henriquez R.
      • Schneider U.
      • Deuter-Reinhard M.
      • Movva N.R.
      • Hall M.N.
      ,
      • Abraham R.T.
      ). mTOR has been found to undergo autophosphorylation and to phosphorylate exogenous protein substrates in a rapamycin/FKBP12-sensitive manner. Indeed, it was recently reported that mTOR in immunoprecipitates phosphorylates 4E-BP1 and fragments of p70 S6 kinase in vitro (
      • Brunn G.J.
      • Hudson C.C.
      • Sekulic A.
      • Williams J.M.
      • Hosoi H.
      • Houghton P.J.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      ,
      • Burnett P.E.
      • Barrow R.K.
      • Cohen N.A.
      • Snyder S.H.
      • Sabatini D.M.
      ). Phosphorylation of recombinant 4E-BP1 was reported to occur on five Ser/Thr-Pro sites (
      • Brunn G.J.
      • Fadden P.
      • Haystead T.A.J.
      • Lawrence Jr., J.C.
      ) that also become phosphorylated in vivo in response to insulin treatment (
      • Fadden P.
      • Haystead T.A.J.
      • Lawrence Jr., J.C.
      ). However, more recent reports (
      • Burnett P.E.
      • Barrow R.K.
      • Cohen N.A.
      • Snyder S.H.
      • Sabatini D.M.
      ,
      • Gingras A.C.
      • Gygi S.P.
      • Raught B.
      • Polakiewicz R.D.
      • Abraham R.T.
      • Hoekstra M.F.
      • Aebersold R.
      • Sonenberg N.
      ) suggest that mTOR phosphorylates 4E-BP1 only at two sites, which serves as a priming event for subsequent phosphorylation of other Ser/Thr-Pro sites by unknown kinases that co-immunoprecipitate with mTOR (
      • Heesom K.J.
      • Denton R.M.
      ).
      A second similarity between the pathways leading to phosphorylation of 4E-BP1 and p70 S6 kinase is their apparent dependence on PI 3-kinase and its downstream effector, the protein kinase Akt. Treatment of cells with wortmannin or LY294002, two inhibitors of PI 3-kinase, prevents the phosphorylation of both proteins following growth factor treatment (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ,
      • Diggle T.A.
      • Moule S.K.
      • Avison M.B.
      • Flynn A.
      • Foulstone E.J.
      • Proud C.G.
      • Denton R.M.
      ). Likewise, overexpression of a dominant-negative mutant of Akt causes a reduction in insulin-induced phosphorylation of 4E-BP1 (
      • Gingras A.C.
      • Kennedy S.G.
      • O'Leary M.A.
      • Sonenberg N.
      • Hay N.
      ,
      • Takata M.
      • Ogawa W.
      • Kitamura T.
      • Hino Y.
      • Kuroda S.
      • Kotani K.
      • Klip A.
      • Gingras A.-C.
      • Sonenberg N.
      • Kasuga M.
      ) and p70 S6 kinase (
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      ). Conversely, expression of activated forms of PI 3-kinase (
      • Gingras A.C.
      • Kennedy S.G.
      • O'Leary M.A.
      • Sonenberg N.
      • Hay N.
      ,
      • Weng Q.P.
      • Andrabi K.
      • Klippel A.
      • Kozlowski M.T.
      • Williams L.T.
      • Avruch J.
      ) or Akt (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Kohn A.D.
      • Barthel A.
      • Kovacina K.S.
      • Boge A.
      • Wallach B.
      • Summers S.A.
      • Birnbaum M.J.
      • Scott P.H.
      • Lawrence Jr., J.C.
      • Roth R.A.
      ) induces 4E-BP1 phosphorylation and activation of p70 S6 kinase in a rapamycin-sensitive manner. Finally, some mutants of the platelet-derived growth factor (PDGF) receptor that cannot bind PI 3-kinase fail to induce 4E-BP1 phosphorylation (
      • von Manteuffel S.R.
      • Gingras A.C.
      • Ming X.F.
      • Sonenberg N.
      • Thomas G.
      ) and p70 S6 kinase activation (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Ming X.F.
      • Burgering B.M.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Heldin C.H.
      • Bos J.L.
      • Kozma S.C.
      • Thomas G.
      ) upon PDGF treatment. These results have led to the proposal of a signaling pathway leading from growth factor receptors to PI 3-kinase, Akt, mTOR, and phosphorylation of 4E-BP1 and p70 S6 kinase (
      • Gingras A.C.
      • Kennedy S.G.
      • O'Leary M.A.
      • Sonenberg N.
      • Hay N.
      ).
      In contrast to this proposed signaling pathway, we recently found that stimulation of the α1A adrenergic receptor (AR) leads to an increase in p70 S6 kinase activity without activation of PI 3-kinase or Akt (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ). α1 ARs have been implicated in the pathogenesis of cardiac hypertrophy, but little is known about the signaling pathways utilized by these receptors to regulate translation (
      • Schluter K.D.
      • Piper H.M.
      ). Treatment of rat neonatal cardiac myocytes in vitrowith the α1 AR agonist phenylephrine (PE) was reported to activate p70 S6 kinase and stimulate protein synthesis and hypertrophic cell growth (
      • Boluyt M.O.
      • Zheng J.S.
      • Younes A.
      • Long X.
      • O'Neill L.
      • Silverman H.
      • Lakatta E.G.
      • Crow M.T.
      ). However, the study of these events in cardiac myocytes is complicated by the fact that they express all three of the known α1 AR subtypes (α1A, α1B, and α1D; Refs.
      • Schwinn D.A.
      • Johnston G.I.
      • Page S.O.
      • Mosley M.J.
      • Wilson K.H.
      • Worman N.P.
      • Campbell S.
      • Fidock M.D.
      • Furness L.M.
      • Parry-Smith D.J.
      • et al.
      and
      • Rokosh D.G.
      • Stewart A.F.R.
      • Chang K.C.
      • Bailey B.A.
      • Karliner J.S.
      • Camacho S.A.
      • Long C.S.
      • Simpson P.C.
      ). In this and our recent (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ) study, we used Rat-1 fibroblasts that stably express the human α1A AR as a simplified model system to study signaling pathways that regulate translation. We show here that stimulation of the α1A AR leads to an increase in protein synthesis accompanied by phosphorylation of 4E-BP1 and its dissociation from eIF4E. This response is not mediated by PI 3-kinase/Akt signaling, but rather by a Ca2+- and calmodulin-dependent pathway.

      DISCUSSION

      The results presented here demonstrate that stimulation of the α1A AR in Rat-1 cells promotes increased phosphorylation of the translation repressor 4E-BP1. Even though phosphorylation of 4E-BP1 as judged by gel mobility shift assays was modest in cells treated with PE as compared with PDGF (Fig. 2), PE-induced phosphorylation caused almost all of the protein to be released from eIF4E (Fig. 3 B). Furthermore, the results in this and our previous study (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ) indicate that functional phosphorylation of 4E-BP1 in response to α1A AR stimulation is independent of PI 3-kinase/Akt signaling. We base this conclusion on the observations that PI 3-kinase activity in phosphotyrosine immunoprecipitates did not increase, the three known isoforms of Akt were not activated, and the levels of PI 3,4-bisphosphate and PI 3,4,5-trisphosphate were not elevated in cells treated with PE (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ). One possible explanation for why 4E-BP1 phosphorylation is blocked by LY294002 (Fig. 3) is that the compound inhibits a protein distinct from PI 3-kinase, such as mTOR (
      • Brunn G.J.
      • Hudson C.C.
      • Sekulic A.
      • Williams J.M.
      • Hosoi H.
      • Houghton P.J.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      ,
      • Brunn G.J.
      • Fadden P.
      • Haystead T.A.J.
      • Lawrence Jr., J.C.
      ,
      • Brunn G.J.
      • Williams J.
      • Sabers C.
      • Wiederrecht G.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      ), that is required for 4E-BP1 phosphorylation.
      To our knowledge, this is the first report of growth factor receptor-mediated stimulation of 4E-BP1 phosphorylation in the absence of PI 3-kinase/Akt activation. The functional consequences of growth factor-induced phosphorylation of 4E-BP1 have been most intensively studied in cells treated with insulin, which acts through a tyrosine kinase receptor to activate the PI 3-kinase/Akt pathway. Although other G protein-coupled receptors, including the μ-opioid (
      • Polakiewicz R.D.
      • Schieferl S.M.
      • Gingras A.-C.
      • Sonenberg N.
      • Comb M.J.
      ), gastrin/cholecystokinin type B (
      • Pyronnet S.
      • Gingras A.C.
      • Bouisson M.
      • Kowalski-Chauvel A.
      • Seva C.
      • Vaysse N.
      • Sonenberg N.
      • Pradayrol L.
      ), prostaglandin F(
      • Rao G.N.
      • Madamanchi N.R.
      • Lele M.
      • Gadiparthi L.
      • Gingras A.-C.
      • Eling T.E.
      • Sonenberg N.
      ), and angiotensin II type 1 (
      • Fleurent M.
      • Gingras A.-C.
      • Sonenberg N.
      • Meloche S.
      ) receptors, can also signal to 4E-BP1, stimulation of these four receptors has been reported to activate PI 3-kinase and/or Akt in addition to inducing 4E-BP1 phosphorylation in the same cellular context (
      • Polakiewicz R.D.
      • Schieferl S.M.
      • Gingras A.-C.
      • Sonenberg N.
      • Comb M.J.
      ,
      • Pyronnet S.
      • Gingras A.C.
      • Bouisson M.
      • Kowalski-Chauvel A.
      • Seva C.
      • Vaysse N.
      • Sonenberg N.
      • Pradayrol L.
      ,
      • Rao G.N.
      • Madamanchi N.R.
      • Lele M.
      • Gadiparthi L.
      • Gingras A.-C.
      • Eling T.E.
      • Sonenberg N.
      ,
      • Fleurent M.
      • Gingras A.-C.
      • Sonenberg N.
      • Meloche S.
      ,
      • Kowalski-Chauvel A.
      • Pradayrol L.
      • Vaysse N.
      • Seva C.
      ,
      • Takahashi T.
      • Taniguchi T.
      • Konishi H.
      • Kikkawa U.
      • Ishikawa Y.
      • Yokoyama M.
      ). Use of PI 3-kinase inhibitors and co-expression studies using highly active or dominant-negative mutants of PI 3-kinase and Akt have suggested that these signaling molecules act upstream of 4E-BP1 and p70 S6 kinase (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ,
      • Diggle T.A.
      • Moule S.K.
      • Avison M.B.
      • Flynn A.
      • Foulstone E.J.
      • Proud C.G.
      • Denton R.M.
      ,
      • Gingras A.C.
      • Kennedy S.G.
      • O'Leary M.A.
      • Sonenberg N.
      • Hay N.
      ,
      • Takata M.
      • Ogawa W.
      • Kitamura T.
      • Hino Y.
      • Kuroda S.
      • Kotani K.
      • Klip A.
      • Gingras A.-C.
      • Sonenberg N.
      • Kasuga M.
      ,
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      ,
      • Weng Q.P.
      • Andrabi K.
      • Klippel A.
      • Kozlowski M.T.
      • Williams L.T.
      • Avruch J.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Kohn A.D.
      • Barthel A.
      • Kovacina K.S.
      • Boge A.
      • Wallach B.
      • Summers S.A.
      • Birnbaum M.J.
      • Scott P.H.
      • Lawrence Jr., J.C.
      • Roth R.A.
      ,
      • von Manteuffel S.R.
      • Gingras A.C.
      • Ming X.F.
      • Sonenberg N.
      • Thomas G.
      ,
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Ming X.F.
      • Burgering B.M.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Heldin C.H.
      • Bos J.L.
      • Kozma S.C.
      • Thomas G.
      ). However, recent work by Dufner et al. (
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.
      • Hemmings B.A.
      • Thomas G.
      ) has shown that although expression of membrane-bound and cytosolic active mutants of Akt induces phosphorylation of 4E-BP1, only those Akt mutants that are constitutively targeted to the membrane can activate p70 S6 kinase. In addition, a membrane-targeted kinase-dead mutant of Akt blocked insulin-induced phosphorylation of 4E-BP1 but had no effect on insulin-induced p70 S6 kinase activation. These workers concluded that Akt plays a dominant role in signaling to 4E-BP1 but is not necessary for p70 S6 kinase activation (
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.
      • Hemmings B.A.
      • Thomas G.
      ). Our data indicate that activation of Akt is not necessary for either the functional phosphorylation of 4E-BP1 (this study) or the activation of p70 S6 kinase (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ).
      Similar to the results shown here, amino acids have also been reported to elicit 4E-BP1 phosphorylation independently of PI 3-kinase/Akt signaling. Cells incubated in medium lacking amino acids exhibit reduced 4E-BP1 phosphorylation and p70 S6 kinase activity, and readdition of amino acids to these cells induces the functional phosphorylation of 4E-BP1 and activation of p70 S6 kinase (
      • Patti M.E.
      • Brambilla E.
      • Luzi L.
      • Landaker E.J.
      • Kahn C.R.
      ,
      • Hara K.
      • Yonezawa K.
      • Weng Q.P.
      • Kozlowski M.T.
      • Belham C.
      • Avruch J.
      ,
      • Shigemitsu K.
      • Tsujishita Y.
      • Hara K.
      • Nanahoshi M.
      • Avruch J.
      • Yonezawa K.
      ). Even though these events are inhibited by wortmannin treatment in some cell types, amino acids do not promote an increase in PI 3-kinase or Akt activity. It would be of interest to determine whether amino acids and the α1A AR use a similar PI 3-kinase-independent signal transduction pathway to promote phosphorylation of 4E-BP1 and p70 S6 kinase.
      It was shown earlier that incubation of a variety of cell types in medium containing EGTA to deplete intracellular Ca2+ stores leads to a sharp and rapid decrease in the rate of protein synthesis (reviewed in Ref.
      • Palfrey H.C.
      • Nairn A.C.
      ). Polysomes were converted to monosomes in cells treated with EGTA, and analysis of ribosome transit times indicated that average elongation rates were relatively unaffected in Ca2+-depleted cells. These observations led to the conclusion that one or more steps in translation initiation require Ca2+. It has been proposed that Ca2+ depletion might inhibit protein synthesis initiation by increasing the phosphorylation of eIF2α, but not all data support this hypothesis (
      • Palfrey H.C.
      • Nairn A.C.
      ). Our results suggest that 4E-BP1 and p70 S6 kinase are two proteins that confer Ca2+ dependence on translation initiation. Treatment of Rat-1 fibroblasts with EGTA leads to the loss of PE-induced 4E-BP1 phosphorylation and p70 S6 kinase activation (Fig.4). In addition, use of BAPTA-AM to chelate intracellular Ca2+ reduces both the basal and hormone-activated levels of phosphorylation of the two proteins (Fig. 4). Thus, our expectation is that cap-dependent translation (regulated by 4E-BP1) and translation of 5′-TOP mRNAs (regulated by p70 S6 kinase) are inhibited in Ca2+-depleted cells. This idea could be tested by analyzing translation of specific mRNAs on polysome gradients.
      The Ca2+ dependence of 4E-BP1 phosphorylation and p70 S6 kinase activation does not appear to be mediated by PKCs. Down-regulation of Ca2+-dependent PKCs by long term TPA treatment had little effect on the PE-induced phosphorylation of 4E-BP1 (Fig. 5 A) or the activation of p70 S6 kinase (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ). Instead, it appears that these Ca2+-dependent events require calmodulin or a closely related protein. We show here that treatment of Rat-1 cells with two structurally unrelated calmodulin antagonists inhibits the functional phosphorylation of 4E-BP1 induced by the α1A AR (Fig. 5 B). It was reported earlier that calmidazolium and other calmodulin antagonists inhibit protein synthesis when added to Ehrlich ascites tumor cells (
      • Kumar R.V.
      • Panniers R.
      • Wolfman A.
      • Henshaw E.C.
      ). Treatment of cells with calmidazolium induced the disaggregation of polysomes, indicating that a step in translation initiation was inhibited. Interestingly, the concentration of calmidazolium that gave half-maximal inhibition of protein synthesis in intact cells (10 μm) was much lower than that required to inhibit cell-free translation (125 μm; Ref.
      • Kumar R.V.
      • Panniers R.
      • Wolfman A.
      • Henshaw E.C.
      ). One interpretation of this result is that the major target of calmidazolium is a calmodulin-dependent signaling pathway that mediates 4E-BP1 phosphorylation in intact cells.
      Functional phosphorylation of 4E-BP1 and activation of p70 S6 kinase in response to stimulation of the α1A AR were sensitive to rapamycin (Fig. 3). This suggests that mTOR is a positive regulator for both of these events, as mTOR is the only known intracellular target of the rapamycin-FKBP12 complex (
      • Abraham R.T.
      ). Thomas and co-workers (
      • von Manteuffel S.R.
      • Dennis P.B.
      • Pullen N.
      • Gingras A.C.
      • Sonenberg N.
      • Thomas G.
      ) showed that overexpression of catalytically active or inactive versions of p70 S6 kinase blocked the insulin-induced phosphorylation of 4E-BP1. They suggested that a signaling pathway leading to phosphorylation of 4E-BP1 and p70 S6 kinase bifurcates immediately upstream of the two proteins and that overexpressed p70 S6 kinase proteins inhibit 4E-BP1 phosphorylation by sequestering a common rapamycin-sensitive upstream activator that might be mTOR. Furthermore, other investigators have proposed that insulin positively regulates 4E-BP1 phosphorylation by increasing the kinase activity of mTOR through a PI 3-kinase/Akt-dependent pathway (
      • Scott P.H.
      • Brunn G.J.
      • Kohn A.D.
      • Roth R.A.
      • Lawrence Jr., J.C.
      ). 4E-BP1 and p70 S6 kinase responded similarly to all the cell treatments examined herein, supporting the idea that phosphorylation of the two proteins is controlled by overlapping signal transduction pathways. However, activation of mTOR by PI 3-kinase/Akt cannot account for the functional phosphorylation of 4E-BP1 induced by the α1AAR (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ). We are currently testing whether mTOR activity might be controlled by a Ca2+/calmodulin-dependent pathway.
      Up-regulation of protein synthesis plays a critical role in physiologic and pathologic cell growth and proliferation. Recent advances usingin vitro models to delineate the signal transduction pathways that regulate translation have furthered our understanding of this important cellular process. Additional studies using in vivo models are needed to determine whether observations made in cell culture systems are relevant at the organism level.

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