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Differential Regulation of the Phosphatidylinositol 3-Kinase/Akt and p70 S6 Kinase Pathways by the α1A-Adrenergic Receptor in Rat-1 Fibroblasts*

Open AccessPublished:February 18, 2000DOI:https://doi.org/10.1074/jbc.275.7.4803
      Phosphatidylinositol (PI) 3-kinase and its downstream effector Akt are thought to be signaling intermediates that link cell surface receptors to p70 S6 kinase. We examined the effect of a Gq-coupled receptor on PI 3-kinase/Akt signaling and p70 S6 kinase activation using Rat-1 fibroblasts stably expressing the human α1A-adrenergic receptor. Treatment of the cells with phenylephrine, a specific α1-adrenergic receptor agonist, activated p70 S6 kinase but did not activate PI 3-kinase or any of the three known isoforms of Akt. Furthermore, phenylephrine blocked the insulin-like growth factor-I (IGF-I)-induced activation of PI 3-kinase and the phosphorylation and activation of Akt-1. The effect of phenylephrine was not confined to signaling pathways that include insulin receptor substrate-1, as the α1-adrenergic receptor agonist also inhibited the platelet-derived growth factor-induced activation of PI 3-kinase and Akt-1. Although increasing the intracellular Ca2+ concentration with the ionophore A23187 inhibited the activation of Akt-1 by IGF-I, Ca2+does not appear to play a role in the phenylephrine-mediated inhibition of the PI 3-kinase/Akt pathway. The differential ability of phenylephrine and IGF-I to activate Akt-1 resulted in a differential ability to protect cells from UV-induced apoptosis. These results demonstrate that activation of p70 S6 kinase by the α1A-adrenergic receptor in Rat-1 fibroblasts occurs in the absence of PI 3-kinase/Akt signaling. Furthermore, this receptor negatively regulates the PI 3-kinase/Akt pathway, resulting in enhanced cell death following apoptotic insult.
      p70 S6 kinase
      M r = 70,000 ribosomal protein S6 kinase
      IGF-I
      insulin-like growth factor I
      PDGF
      platelet-derived growth factor
      PKC
      protein kinase C
      PI
      phosphatidylinositol
      PE
      phenylephrine
      [Ca2+]i
      intracellular Ca2+concentration
      AR
      adrenergic receptor
      PMA
      phorbol 12-myristate 13-acetate
      IRS-1
      insulin receptor substrate-1
      HPLC
      high pressure liquid chromatography
      Cellular growth requires the generation of new translational machinery to accommodate the increased demand for additional proteins. It has been shown that treatment of cells with growth-promoting agents induces the translational up-regulation of ribosomal proteins and protein synthesis elongation factors (
      • Amaldi F.
      • Pierandrei-Amaldi P.
      ,
      • Meyuhas O.
      • Avni D.
      • Silvian S.
      ). This process is controlled in part by phosphorylation of the S6 protein of 40 S ribosomal subunits by the M r = 70,000 S6 kinase (p70 S6 kinase1; Refs.
      • Jefferies H.
      • Thomas G.
      and
      • Jefferies H.B.
      • Fumagalli S.
      • Dennis P.B.
      • Reinhard C.
      • Pearson R.B.
      • Thomas G.
      ). p70 S6 kinase is activated upon treatment of cells with a variety of growth factors, hormones, mitogens, and phosphatase inhibitors, etc. This increase in activity is due to phosphorylation of p70 S6 kinase at multiple sites presumably by multiple kinases (
      • 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.
      ). Due to its importance in the growth response, the signal transduction pathways leading to activation of p70 S6 kinase have received considerable attention. A variety of experimental approaches have led to the identification of phosphatidylinositol (PI) 3-kinase and its downstream effector, the protein kinase Akt, as signaling intermediates that link cell surface receptors to p70 S6 kinase. First, treatment of cells with wortmannin or LY294002, two inhibitors of PI 3-kinase, prevents the activation of Akt (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ) 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.
      ) in response to growth factors or hormones. Second, platelet-derived growth factor (PDGF) receptor mutants that cannot bind PI 3-kinase were unable to induce efficiently the activation of Akt (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ) or p70 S6 kinase (
      • 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.
      ). Third, overexpression of constitutively active forms of PI 3-kinase induces the activation of Akt (
      • Didichenko S.A.
      • Tilton B.
      • Hemmings B.A.
      • Ballmer-Hofer K.
      • Thelen M.
      ,
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Reif K.
      • Burgering B.M.T.
      • Cantrell D.A.
      ) and p70 S6 kinase (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Reif K.
      • Burgering B.M.T.
      • Cantrell D.A.
      ,
      • Weng Q.P.
      • Andrabi K.
      • Klippel A.
      • Kozlowski M.T.
      • Williams L.T.
      • Avruch J.
      ) in the absence of added extracellular ligands. Conversely, expression of dominant-negative mutants of the p85 subunit of PI 3-kinase inhibits the PDGF-induced activation of Akt (
      • Burgering B.M.
      • Coffer P.J.
      ) and p70 S6 kinase (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). Finally, overexpression of a dominant-negative mutant of Akt causes a reduction in insulin-induced activation of 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.
      ), whereas constitutively or conditionally active forms of Akt stimulate p70 S6 kinase (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Reif K.
      • Burgering B.M.T.
      • Cantrell D.A.
      ,
      • 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.
      ,
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.
      • Hemmings B.A.
      • Thomas G.
      ). Together, these results suggest the existence of a linear signaling pathway leading from receptors to PI 3-kinase, Akt and p70 S6 kinase. This pathway is also thought to involve additional components, as Akt does not phosphorylate p70 S6 kinase directly in vitro (
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ).
      Like p70 S6 kinase, Akt is activated by a wide variety of hormones, growth factors, and other stimuli (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ,
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ). Akt is thought to mediate many of the cellular effects of insulin and insulin-like growth factor-I (IGF-I) on glucose metabolism and cell survival (
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ,
      • Alessi D.R.
      • Cohen P.
      ). For example, Akt phosphorylates and inactivates glycogen synthase kinase-3 in cells treated with insulin or IGF-I, thus promoting glycogen synthesis (
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ). Similarly, the anti-apoptotic effect of insulin and IGF-I is partly mediated by Akt phosphorylation of the pro-apoptotic protein BAD (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ). Akt is activated by phosphorylation of Thr-308 in the activation loop of the catalytic domain and Ser-473 in the carboxyl-terminal tail (
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ). It is believed that phosphorylation of both sites requires an interaction between the amino-terminal pleckstrin homology domain of Akt with membrane inositol phospholipids generated by PI 3-kinase (
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ,
      • Alessi D.R.
      • Cohen P.
      ,
      • Andjelkovic M.
      • Alessi D.R.
      • Meier R.
      • Fernandez A.
      • Lamb N.J.C.
      • Frech M.
      • Cron P.
      • Cohen P.
      • Lucocq J.M.
      • Hemmings B.A.
      ). Translocation of Akt to the membrane is thought to induce a conformational change that permits phosphorylation of Thr-308 and Ser-473 by membrane-associated Akt kinases. The kinase that phosphorylates Thr-308 in Akt has been identified as 3-phosphoinositide-dependent protein kinase 1 (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ,
      • Alessi D.R.
      • Deak M.
      • Casamayor A.
      • Caudwell F.B.
      • Morrice N.
      • Norman D.G.
      • Gaffney P.
      • Reese C.B.
      • MacDougall C.N.
      • Harbison D.
      • Ashworth A.
      • Bownes M.
      ). 3-Phosphoinositide-dependent protein kinase 1 also phosphorylates the equivalent site in some other protein kinases, including p70 S6 kinase (
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ,
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ).
      In contrast to insulin and IGF-I, treatment of cells with catecholamines to activate α1-adrenergic receptors (ARs) induces glycogenolysis. Three α1-AR subtypes have been cloned (α1A, α1B, and α1D), and all three receptors activate phospholipase C β, which promotes increased production of inositol 1,4,5-trisphosphate and diacylglycerol, leading to elevation of the intracellular Ca2+ concentration ([Ca2+]i) and activation of protein kinase C (PKC), respectively (
      • 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.
      • Peter B.
      • Bailey D.S.
      ). Recent evidence indicates that α1-ARs also stimulate additional physiologically relevant signaling pathways. For example, treatment of cultured rat neonatal cardiac myocytes with the α1-AR agonist phenylephrine (PE) leads to activation of p70 S6 kinase and an increase in the rate of protein synthesis and cell growth (
      • Boluyt M.O.
      • Zheng J.S.
      • Younes A.
      • Long X.
      • O'Neill L.
      • Silverman H.
      • Lakatta E.G.
      • Crow M.T.
      ).
      Since activation of Akt promotes glycogen synthesis, it seemed inconsistent that α1-ARs, which stimulate glycogen breakdown, would signal to p70 S6 kinase via an Akt-dependent pathway. In this study, we examined this question using Rat-1 fibroblasts expressing the human α1A-AR. We show that treatment of these cells with PE activates p70 S6 kinase but does not activate PI 3-kinase or Akt. Moreover, we show that the α1A-AR inhibits the activation of PI 3-kinase and Akt induced by other growth factors. Finally, we tested whether the differential ability of PE and IGF-I to activate Akt correlates with their ability to protect cells from UV-induced apoptosis.

      DISCUSSION

      The results presented here demonstrate that activation of Akt is not required for activation of p70 S6 kinase. Treatment of Rat-1 cells expressing the α1A-AR with PE did not stimulate any of the three Akt isoforms even though there was a significant increase in p70 S6 kinase activity (Figs. 1 and 2). We further show that p70 S6 kinase can be activated even in cells in which PI 3-kinase is inhibited (Figs. 1 A and 4). This result was unexpected because pharmacological approaches and co-expression studies using highly active or dominant-negative versions of these signaling molecules have indicated that PI 3-kinase and Akt are major upstream regulators of p70 S6 kinase (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • 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.
      ,
      • 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.
      ,
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Reif K.
      • Burgering B.M.T.
      • Cantrell D.A.
      ,
      • Weng Q.P.
      • Andrabi K.
      • Klippel A.
      • Kozlowski M.T.
      • Williams L.T.
      • Avruch 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.
      ). However, not all data are consistent with this hypothesis. First, activation of p70 S6 kinase by some agonists is relatively resistant to wortmannin, suggesting that PI 3-kinase-independent signaling pathways might be involved in activation of the kinase (
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ). Second, a PDGF receptor mutant (Y740F) that failed to activate PI 3-kinase or Akt activated p70 S6 kinase almost as well as the wild-type receptor (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Ming X.F.
      • Burgering B.M.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Heldin C.H.
      • Bos J.L.
      • Kozma S.C.
      • Thomas G.
      ). Third, although expression of a dominant-negative mutant of Akt-1 in CHO cells inhibited insulin-induced activation of p70 S6 kinase by 75%, the same mutant had only a small inhibitory effect on p70 S6 kinase when expressed in 3T3-L1 adipocytes (
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      ). Finally, Thomas and co-workers (
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.
      • Hemmings B.A.
      • Thomas G.
      ) have shown that activated mutants of Akt must be constitutively targeted to the membrane in order to stimulate p70 S6 kinase. They have proposed that these mutants artificially induce p70 S6 kinase activation and that results obtained with these mutants may not reflect wild-type Akt signaling (
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.
      • Hemmings B.A.
      • Thomas G.
      ). Interestingly, membrane-bound versions of active PI 3-kinase were also more efficient than cytosolic mutants at activating p70 S6 kinase (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ). When expressed in COS-7 cells, the two forms of PI 3-kinase generated distinct patterns of phosphoinositides (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ), raising the possibility that novel phospholipids generated in cells expressing these mutants induce p70 S6 kinase activation.
      PI 3,4-bisphosphate and PI 3,4,5-trisphosphate levels in intact cells (Fig. 2 B) and PI 3-kinase activity in phosphotyrosine immunoprecipitates (Fig. 4) do not increase in response to PE treatment, thus leading us to conclude that activation of p70 S6 kinase by the α1A-AR is PI 3-kinase-independent. Why then is the activation of p70 S6 kinase by PE inhibited by LY294002 (Fig.1 B)? One possibility is that LY294002 inhibits a protein distinct from PI 3-kinase that is required for p70 S6 kinase activation. It has been shown that LY294002 and wortmannin inhibit thein vitro autophosphorylation of the mammalian target of rapamycin (mTOR), a kinase that positively regulates p70 S6 kinase (
      • Brunn G.J.
      • Williams J.
      • Sabers C.
      • Wiederrecht G.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      ). Therefore, the inhibitory effect of LY294002 on p70 S6 kinase activation could be exerted through mTOR.
      PKC and Ca2+ have been suggested to participate in PI 3-kinase-independent pathways that lead to p70 S6 kinase activation. Similar to the results obtained here with PE (Figs. 1 and 2), exposure of a T cell leukemia line to PMA resulted in activation of p70 S6 kinase with no increase in Akt-1 activity (
      • Reif K.
      • Burgering B.M.T.
      • Cantrell D.A.
      ). As expected for a PI 3-kinase-independent response, activation of p70 S6 kinase by PMA is relatively resistant to inhibition by wortmannin (
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ,
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). It was reported earlier that a PDGF receptor mutant that couples to the γ subtype of phospholipase C but not to PI 3-kinase induces partial activation of p70 S6 kinase in HEPG2 cells (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). This response was abolished after long term treatment of the cells with PMA, indicating that phospholipase C activates p70 S6 kinase through PKC in this cell type (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ). By contrast, we found that down-regulation of PKC in Rat-1 cells by long term PMA treatment has no effect on the activation of p70 S6 kinase by PE (Fig. 1 C). Therefore, it appears that PI 3-kinase-independent activation of p70 S6 kinase by the α1A-AR in Rat-1 cells is mediated by a pathway that does not involve PMA-sensitive isoforms of PKC.
      It has been known for some time that treatment of cells with A23187 induces the activation of p70 S6 kinase (
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ,
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ), and recent work has demonstrated that Ca2+ ionophores and the Ca2+-mobilizing agent thapsigargin activate p70 S6 kinase independently of Akt-1 (
      • Conus N.M.
      • Hemmings B.A.
      • Pearson R.B.
      ). Consistent with this observation, treatment of cells with these agents causes little or no activation of PI 3-kinase (
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ,
      • Conus N.M.
      • Hemmings B.A.
      • Pearson R.B.
      ). On the other hand, activation of p70 S6 kinase by Ca2+ ionophores and thapsigargin is wortmannin-sensitive (
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.
      • Parker P.J.
      • Ballou L.M.
      ,
      • Conus N.M.
      • Hemmings B.A.
      • Pearson R.B.
      ). These cellular responses are similar to those we observed here using PE (Figs. 1, 2, and 4). Thus, activation of p70 S6 kinase by the α1A-AR in Rat-1 cells may be mediated by a Ca2+-dependent pathway (Fig. 6 C). Recent reports using EGTA-containing medium or BAPTA-AM to deplete cells of free intracellular Ca2+ have indicated that growth factor signaling to p70 S6 kinase is Ca2+-dependent, whereas Akt activation occurs via a Ca2+-independent pathway (
      • Conus N.M.
      • Hemmings B.A.
      • Pearson R.B.
      ,
      • Graves L.M.
      • He Y.
      • Lambert J.
      • Hunter D.
      • Li X.
      • Earp H.S.
      ). Our results here (Fig. 6) and elsewhere (
      • Rybkin I.I.
      • Cross M.E.
      • McReynolds E.M.
      • Lin R.Z.
      • Ballou L.M.
      ) confirm and extend these findings. We have found that the PE-induced activation of p70 S6 kinase is greatly reduced in Rat-1 cells depleted of free intracellular Ca2+(
      • Rybkin I.I.
      • Cross M.E.
      • McReynolds E.M.
      • Lin R.Z.
      • Ballou L.M.
      ). By contrast, the response of Akt to IGF-I is normal in Ca2+-depleted cells (Fig. 6 B). The Ca2+-dependent step in p70 S6 kinase activation has not yet been identified.
      In contrast to its stimulatory effect on p70 S6 kinase (Fig. 1), we found that PE negatively regulates Akt-1 (Fig. 3). The IGF-I-induced activation of Akt was inhibited in cells treated with A23187, suggesting that a high [Ca2+]i inhibits signaling to this kinase (Fig. 6 A). Surprisingly, although PE induces an increase in [Ca2+]i (Fig. 6 C), the inhibitory effect of the α1A-AR on Akt activation does not seem to be exerted through a Ca2+-mediated pathway. In Ca2+-depleted cells, PE was still unable to activate Akt, and its inhibitory effect on the IGF-I response was largely intact (Fig. 6 B). It is not known whether Akt activity might be inhibited in other physiological settings that involve intracellular Ca2+ release.
      Consistent with its effect on Akt-1, we also found that treatment of cells with PE reduced the IGF-I-induced PI 3-kinase activation measured in phosphotyrosine immunoprecipitates (Fig. 4). Prior studies have shown that increased serine phosphorylation of the adaptor protein IRS-1 induced by a variety of factors inhibits the ability of the protein to be tyrosine-phosphorylated by the insulin receptor, thus preventing IRS-1 from binding and activating PI 3-kinase (
      • De Fea K.
      • Roth R.A.
      ,
      • Li J.
      • DeFea K.
      • Roth R.A.
      ,
      • Tanti J.F.
      • Gremeaux T.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ). Two mechanisms have been proposed to mediate the serine phosphorylation of IRS-1. Activators of PKC are thought to promote the phosphorylation of IRS-1 at serine 612 by mitogen-activated protein kinases (
      • De Fea K.
      • Roth R.A.
      ,
      • Li J.
      • DeFea K.
      • Roth R.A.
      ), whereas other factors such as PDGF are thought to regulate IRS-1 function negatively through the phosphorylation of three other serines via an Akt-dependent pathway (
      • Li J.
      • DeFea K.
      • Roth R.A.
      ). Inhibition of PI 3-kinase by the α1A-AR does not appear to involve either of these two mechanisms because (a) PE treatment of Rat-1 cells does not activate Akt (Fig. 2 A) or the mitogen-activated protein kinases Erk1 and Erk2 (
      • Lin R.Z.
      • Chen J.
      • Hu Z.W.
      • Hoffman B.B.
      ) and (b) PE also inhibits the activation of PI 3-kinase induced by the PDGF receptor (Fig. 5 A), which does not utilize IRS-1 for signaling. Alternative mechanisms to explain the inhibitory effect of the α1A-AR on PI 3-kinase activity might be that it inhibits tyrosine phosphorylation of the p85 subunit of PI 3-kinase or prevents association of p85 with the p110 catalytic subunit of PI 3-kinase. In preliminary experiments, we observed no difference on Western blots in the pattern of tyrosine-phosphorylated proteins from cells treated with or without PE.
      M. E. Cross and R. Z. Lin, unpublished data.
      Attempts to examine the p85-p110 interaction in co-immunoprecipitates were unsuccessful due to the low amount of these proteins expressed in Rat-1 cells.3We are continuing to investigate the mechanism by which the α1A-AR negatively regulates PI 3-kinase.
      Our results indicate that the α1A-AR differs from the insulin/IGF-I receptor in its ability to activate PI 3-kinase (Figs.2 B and 4). This result may have significant physiologic implications for cell survival. It is well recognized that Gq-coupled receptors play an important role in the development and ultimate decompensation of cardiac hypertrophy (
      • Dorn G.W., II
      • Brown J.H.
      ). It was recently demonstrated that overexpression of Gq leads to increased apoptosis of cardiac myocytes both in vitro andin vivo (
      • Adams J.W.
      • Sakata Y.
      • Davis M.G.
      • Sah V.P.
      • Wang Y.
      • Liggett S.B.
      • Chien K.R.
      • Brown J.H.
      • Dorn G.W., II
      ). In contrast, treatment with IGF-I stimulates cardiac myocyte hypertrophy but improves cardiac function in experimental models of heart failure (
      • Duerr R.L.
      • Huang S.
      • Miraliakbar H.R.
      • Clark R.
      • Chien K.R.
      • Ross Jr., J.
      ). This disparity may be explained by the differential activation of PI 3-kinase and Akt by the IGF-I receptor versus the α1-AR (or, possibly, Gq-coupled receptors in general). Perhaps exposure of cardiac myocytes to α1-AR agonists or IGF-I activates p70 S6 kinase, leading to increased protein synthesis and cellular hypertrophy, whereas only IGF-I activates Akt, providing a survival signal. Indeed, our results show that IGF-I treatment has a protective effect against UV-induced apoptosis, whereas PE treatment enhanced the apoptotic effect of UV irradiation (Fig. 7). Studies in rat neonatal cardiac myocytes are currently being done to test the validity of this clinically relevant hypothesis.
      To our knowledge, the finding that activation of a Gq-coupled receptor leads to inhibition of Akt and PI 3-kinase has not been previously reported. By using COS-7 cells overexpressing m1 muscarinic acetylcholine receptors and epitope-tagged Akt, Gutkind and co-workers (
      • Murga C.
      • Laguinge L.
      • Wetzker R.
      • Cuadrado A.
      • Gutkind J.S.
      ) found that activation of this Gq-coupled receptor very weakly stimulated Akt. The difference between that finding and the results reported here may be due to cell type differences and alteration in the regulation of Akt when it is overexpressed. Activation of the γ subtype of PI 3-kinase by Gi-coupled receptors via G protein βγ subunits is well described in the literature (
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nurnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ). Not surprisingly, recent reports indicated that Gi-coupled receptors also activate Akt (
      • Takasuga S.
      • Katada T.
      • Ui M.
      • Hazeki O.
      ,
      • Tilton B.
      • Andjelkovic M.
      • Didichenko S.A.
      • Hemmings B.A.
      • Thelen M.
      ). Inhibition of Akt by α1-ARs has important physiological implications for Akt-mediated glucose regulation. Akt is thought to be a critical molecular switch for insulin-mediated regulation of glucose metabolism (
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ). The possibility that Gq-coupled receptors negatively regulate these insulin-induced effects opens new avenues for research examining the role of this large family of receptors in the development of insulin resistance and diabetes mellitus.

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