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Constitutively Active Phosphatidylinositol 3-Kinase and AKT Are Sufficient to Stimulate the Epithelial Na+/H+ Exchanger 3*

Open AccessPublished:August 01, 2001DOI:https://doi.org/10.1074/jbc.M103900200
      Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na+/H+ exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.
      NHE3
      Na+/H+ exchanger 3
      BCECF
      2′7′-bis(carboxyethyl) 5–6-carboxyfluorescein
      EGF
      epidermal growth factor
      eGFP
      enhanced green fluorescent protein
      FGF
      fibroblast growth factor
      GLUT4
      glucose transporter 4
      HA
      hemagglutinin
      moi
      multiplicity of infection
      PI 3-kinase
      phosphatidylinositol 3-kinase
      RXR
      retinoid X receptor
      In the mammalian intestine, the brush border Na+/H+ exchanger 3 (NHE3)1 is a component of neutral NaCl absorption. This process explains basal ileal NaCl absorption and the increase in ileal sodium absorption that occurs after meals, and it is also the sodium absorptive process in ileal sodium absorbing cells that is inhibited in most diarrheal diseases. NHE3 is the component of neutral NaCl absorption that has been shown to be acutely stimulated and inhibited under these conditions. NHE3 activity is regulated by multiple growth factors and protein kinases, which mimic the changes associated with the digestive process (
      • Donowitz M.
      • Tse M.
      ). The mechanisms of inhibition and stimulation are only partially defined. For instance, EGF and clonidine stimulate ileal sodium absorbing cells by increasing the percentage of total NHE3 in the brush border (
      • Donowitz M.
      • Montgomery J.L.
      • Walker M.S.
      • Cohen M.E.
      ). In PS120 fibroblasts stably expressing NHE3, FGF exposure increases NHE3 transport activity and the percentage of total NHE3 in the plasma membrane (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ,
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ). The percent increase in Na+/H+ exchange is quantitatively similar to the increase in percentage of NHE3 on the plasma membrane. In contrast, protein kinase C inhibition of NHE3 in Caco-2 cells is associated with a decrease in percentage of NHE3 on the plasma membrane, but in this case, the change in transport exceeds the change in percentage of surface NHE3, indicating change in turnover number in addition to change in NHE3 trafficking (
      • Janecki A.J.
      • Montrose M.H.
      • Zimniak P.
      • Zweibaum A.
      • Tse C.M.
      • Khurana S.
      • Donowitz M.
      ).
      Insights are just beginning to be achieved in identifying the signal transduction processes involved in short-term regulation of NHE3. For instance, phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to be necessary for 1) the basal level of plasma membrane NHE3 amount and NHE3 transport activity (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ,
      • Kurashima K.
      • Szabo E.Z.
      • Lukacs G.
      • Orlowski J.
      • Grinstein S.
      ,
      • Akhter S.
      • Cavet M.E.
      • Tse C.M.
      • Donowitz M.
      ) and 2) EGF/FGF stimulation of NHE3 activity in PS120 fibroblasts and Caco-2 cells (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ). PI 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors (
      • Krasilnikov M.A.
      ,
      • Toker A.
      • Cantley L.C.
      ,
      • Rameh L.E.
      • Cantley L.C.
      ). Activation of PI 3-kinase results in increased intracellular levels of 3′-phosphorylated inositol phospholipids and induction of signaling responses, including the activation of the protein kinase Akt, which is also known as protein kinase B (
      • Chan T.O.
      • Rittenhouse S.E.
      • Tsichlis P.N.
      ,
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ). Activation of PI 3-kinase plays a role in growth factor signaling cascades, leading to metabolic and mitogenic cellular responses. In addition, PI 3-kinase activity has been implicated in regulated exocytosis and endocytosis. For instance, its activation is sufficient to stimulate glucose transporter 4 (GLUT4) translocation to the plasma membrane in 3T3-L1 adipocytes in the absence of insulin (
      • Frevert E.U.
      • Bjorbaek C.
      • Venable C.L.
      • Keller S.R.
      • Kahn B.B.
      ).
      The conclusions of the above previous studies of PI 3-kinase and NHE3 were largely based on pharmacological approaches; inhibition of PI 3-kinase by wortmannin and LY294002 were utilized, although they were supported by biochemical evidence of activation of PI 3-kinase by growth factors. No previous studies have attempted to determine whether Akt has any role in regulation of NHE3. This is largely due to lack of Akt inhibitors.
      In the current study, we tested the hypothesis that activation of PI 3-kinase or Akt alone is sufficient for the stimulation of NHE3 activity. This hypothesis was tested in PS120/NHE3 cells after somatic gene transfer using recombinant, replication-deficient adenovirus containing constitutively active PI 3-kinase or Akt. These studies demonstrate that an inducible adenoviral expression system can induce significant increases in PI 3-kinase and Akt activity and amount in PS120 cells, and activated PI 3-kinase and Akt were sufficient to stimulate NHE3. In addition, activating PI 3-kinase with a peptide that stimulates PI 3-kinase activity similarly stimulated NHE3 (
      • Hubner S.
      • Couvillon A.D.
      • Kas J.A.
      • Bankaitis V.A.
      • Vegners R.
      • Carpenter C.L.
      • Janmey P.A.
      ,
      • Janmey P.A.
      • Xian W.
      • Flanagan L.A.
      ). This study demonstrates that activation of PI 3-kinase and Akt is sufficient to stimulate NHE3 in a fibroblast cell model.

      MATERIALS AND METHODS

      The synthetic rhodamine-linked 10-mer peptide, to be referred to as PI 3-kinase peptide, that is based on the phosphoinositide binding sequence of gelsolin was a gift from P. Janmey (
      • Hubner S.
      • Couvillon A.D.
      • Kas J.A.
      • Bankaitis V.A.
      • Vegners R.
      • Carpenter C.L.
      • Janmey P.A.
      ,
      • Janmey P.A.
      • Xian W.
      • Flanagan L.A.
      ). Nigericin, Hoechst dye 33342, and 2′,7′-bis(2-carboxy-ethyl)-5(6)-carboxyfluorescein acetoxymethyl ester were obtained from Molecular Probes, Inc. (Eugene, OR). Enhanced chemiluminescence reagents were from PerkinElmer Life Sciences. Thin-layer chromatography (TLC) plates were from Merck.l-α-Phosphatidylinositol was from Sigma. [γ-32P]ATP (3000 Ci/mmol) was from PerkinElmer Life Sciences. XAR-5 film was obtained from Amersham Pharmacia Biotech. Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Life Technologies, Inc. Monoclonal mouse antibodies to the influenza virus HA (HA1) and Myc epitopes (9E10) were from Babco (Berkeley, CA). Phosphospecific-Akt antibody was from New England BioLabs, Inc. (Beverly, MA). A polyclonal anti-p110 PI 3-kinase antibody and protein A-Sepharose were from Upstate Biotechnology Inc. (Lake Placid, NY). Peroxidase-conjugated donkey anti-mouse IgG was from Jackson ImmunoResearch Laboratories, Inc. The general anti-Akt antibody was previously reported by Tsichlis (
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ). The NHS-SS-biotin was from Pierce. Other agents were from Sigma.

      Cell Culture

      Stably transfected full-length rabbit NHE3V (NHE3 with epitope of vesicular stomatitis versus G protein on the C terminus, as previously described (
      • Hoogerwerf W.A.
      • Tsao S.C.
      • Devuyst O.
      • Levine S.A.
      • Yun C.H.
      • Yip J.W.
      • Cohen M.E.
      • Wilson P.D.
      • Lazenby A.J.
      • Tse C.M.
      • Donowitz M.
      )) in Chinese hamster lung-derived fibroblasts, the PS120/E3V cell line, were grown in Dulbecco's modified Eagle's medium supplemented with 25 mm NaHCO3, 10 mm HEPES, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2/95% 02 humidified incubator at 32 °C, as described (
      • Levine S.A.
      • Montrose M.H.
      • Tse C.M.
      • Donowitz M.
      ). OK/E3V cells (generously provided by J. Noel, University of Montreal) were maintained in Dulbecco's modified Eagle's medium supplemented with 25 mm NaHCO3, 10 mm HEPES, penicillin (50 IU/ml), streptomycin (50 µg/ml), and 10% fetal bovine serum in a 5% CO2/95% O2 humidified incubator at 37 °C. For all experiments, cells were grown on glass coverslips and studied after being serum-starved for 3 days after reaching confluence. Both the OK/E3V and PS120/E3V cell lines were selected for Na+/H+exchange activity (every other passage) by exposing cells to an acid load consisting of 50 mm NH4Cl/94 mm NaCl solution for 1 h, followed by an isotonic 2 mm Na+ solution as described (
      • Levine S.A.
      • Nath S.K.
      • Yun C.H.
      • Yip J.W.
      • Montrose M.
      • Donowitz M.
      • Tse C.M.
      ). CRE8 cells were grown in Dulbecco's modified Eagle's medium supplemented with 2 mm GlutaMAX (Invitrogen), 15 mm HEPES (pH 7.4), 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2/95% 02humidified incubator at 37 °C.

      Plasmid Vector Construction

      Mammalian expression vectors directing the expression of Myc-tagged p110* (PI 3-kinase) and HA-tagged myr-Akt constructs, as well as these inserted constructs, were described (
      • Kohn A.D.
      • Barthel A.
      • Kovacina K.S.
      • Boge A.
      • Wallach B.
      • Summers S.A.
      • Birnbaum M.J.
      • Scott P.H.
      • Lawrence J.C.
      • Roth R.A.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Hirano M.
      • Williams L.T.
      ,
      • Bellacosa A.
      • Chan T.O.
      • Ahmed N.N.
      • Datta K.
      • Malstrom S.
      • Stokoe D.
      • McCormick F.
      • Feng J.
      • Tsichlis P.
      ). The adenovirus shuttle vector used which contains an ecdysone-inducible promoter (pAdEcd), and pAdVgRXR encoding the ecdysone and RXR receptors were as described (Fig.1) (
      • Johns D.C.
      • Marx R.
      • Mains R.E.
      • O'Rourke B.
      • Marban E.
      ). The p110* encodes for a constitutively active form of PI 3-kinase in which the inter-SH2 domain of the p85 regulatory subunit was ligated to the NH2terminus of the p110 catalytic subunit of PI 3-kinase. The p110* protein was tagged at the COOH terminus with the Myc epitope (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ). In order to subclone the epitope-tagged p110*-Myc from a mammalian expression vector to pAdEcd, BamHI-digested p110*-Myc DNA was ligated into the multiple cloning site of pAdEcd. The plasmid containing the BamHI fragment in pAdEcd, the pAdEcd-BamHI plasmid, was digested with XbaI andNheI, and then the XbaI fragment from the p110*-Myc construct was subcloned into pAdEcd-BamHI. The Akt construct contains the HA epitope-tagged to its C terminus and is catalytically active due to its myristylation-related membrane location. The myr-Akt-HA plasmid was digested with HindIII and EcoRI, and the DNA fragment was subcloned into the multiple cloning site of pAdEcd for construction of pAdEmyr-Akt-HA (Fig. 1). The expression cassette of eGFP from pEGFP was subcloned into the multiple cloning site of pAdEcd, making vector pAdEGI (Fig. 1), which expressed enhanced green fluorescence protein and was used to assess adenovirus infection efficiency.
      Figure thumbnail gr1
      Figure 1Linear representation of the ecdysone-inducible adenovirus constructs described in this study.Schematic representations of the adenovirus vector AdVgRXR (receptor virus) and the viral constructs AdEp110*-Myc, AdE myr-Akt-HA, and AdEGI are shown. ψ, packing signal; Ecd promoter,ecdysone-inducible promoter; ITR, inverted terminal repeat;RSV, Rous sarcoma virus; VgEcR, modified ecdysone receptor; pA, SV40 polyadenylation signal; MCS,multiple cloning site.

      Recombinant Adenoviral Vectors

      The recombinant adenoviruses containing the cDNA encoding either the C-terminal Myc epitope-tagged constitutively active form of PI 3-kinase (AdEp110*-Myc) or myristoylated Akt-HA (AdEmyr-Akt-HA) were generated by Cre lox recombination (
      • Johns D.C.
      • Marx R.
      • Mains R.E.
      • O'Rourke B.
      • Marban E.
      ,
      • Hardy S.
      • Kitamura M.
      • Harris-Stansil T.
      • Dai Y.
      • Phipps M.L.
      ) using CRE8 cells that stably express Cre recombinase. Construction of recombinant adenovirus AdEp110*-Myc and AdEmyr-Akt-HA was as described (
      • Johns D.C.
      • Marx R.
      • Mains R.E.
      • O'Rourke B.
      • Marban E.
      ) as was determination of multiplicity of infection (moi) (
      • Mittereder N.
      • March K.L.
      • Trapnell B.C.
      ).

      Adenoviral Infections

      PS120/E3V cells were co-infected with AdVgRXR plus (i) AdEp110*-Myc, (ii) AdEmyr-Akt-HA, (iii) AdEGI, or (iv) empty virus (Ψ5) (Fig. 1) in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum and the appropriate amount of virus for 24 h. Then, expression was induced by addition of the ecdysone promoter ligand ponasterone A (3 µm) for 24 h before further assays were performed. The dose of ponasterone A was selected to activate the ecdysone receptor maximally on the basis of previously reported dose-response curves (
      • No D.
      • Yao T.P.
      • Evans R.M.
      ).

      Confocal Microscopy

      Images were taken after cells were fixed with 3% paraformaldehyde at 4 °C for 20 min. Nuclei were stained with Hoechst 33342, washed with phosphate-buffered saline, and examined using a confocal fluorescent microscope (Zeiss LSC410). The excitation/emission wavelengths were set at 488/510 nm and 351/364 nm for eGFP and Hoechst 33342, respectively.

      Detection of Recombinant AdEp110*-Myc and AdEmyr-Akt-HA Expression Using Western Blot Analysis

      PS120/E3V cells were co-infected with AdEp110*-Myc or AdEmyr-Akt-HA plus AdVgRXR. Expression of AdEp110*-Myc and AdEmyr-Akt-HA was induced by the addition of ponasterone A (3 µm). Uninfected or infected cells were lysed in a solubilizing buffer containing 50 mm Tris (pH 7.4), 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 1 mm vanadate, 50 mm NaF, 5 mm NaPPi, 10 mm 2-β-glycerophosphate, and protease inhibitors (1:1000 dilution of Sigma mixture p8340). The cell lysates were centrifuged (10,000 × g for 10 min) to remove insoluble materials. For the Western blot analyses, whole cell lysates (30–40 µg of protein/lane) were denatured by boiling with Laemmli sample buffer containing 100 mm dithiothreitol and resolved by SDS-polyacrylamide gel electrophoresis. Gels were transferred to nitrocellulose membrane by electroblotting in transfer buffer containing 25 mm Tris base, 192 mm glycine, 20% MeOH, and 0.05% SDS and immunoblotted with indicated antibodies. AdEp110*-Myc was detected with murine Myc monoclonal antibody 9E10, and AdEmyr-Akt-HA was detected with murine HA-monoclonal antibody HA1. Blots were then incubated with horseradish peroxidase-linked secondary antibody followed by chemiluminescence detection.

      Phosphatidylinositol 3-Kinase Activity Assay

      PI 3-kinase activity was measured as described previously using TLC with phosphatidylinositol 4-phosphate, which co-migrates with PI-3P (Sigma mixture p9638), as a standard (
      • Lee-Kwon W.
      • Park D.
      • Bernier M.
      ). Uninfected cells or adenovirus-infected PS120/NHE3V cells were lysed, and lysates were immunoprecipitated with a polyclonal anti-p110 antibody (Upstate Biotechnology Inc.). Immune complexes were precipitated from the supernatant with protein A-Sepharose (Upstate Biotechnology Inc.) and washed as described (
      • Lee-Kwon W.
      • Park D.
      • Bernier M.
      ,
      • Lee-Kwon W.
      • Park D.
      • Baskar P.V.
      • Kole S.
      • Bernier M.
      ). The washed immune complexes were incubated with l-α-phosphatidylinositol (Sigma) as substrate and [γ-32P]ATP (3000 Ci/mmol) for 10 min at room temperature. Reaction was stopped with 20 µl of 6 n HCl (final volume, 90 µl), extracted with 160 µl of chloroform:methanol (1:1), and centrifuged. The lower organic phase was removed and applied to a silica gel thin-layer chromatography plate (Merck) coated with 1% potassium oxalate. The TLC plates were developed in methanol:chloroform:water:ammonium hydroxide (60:47:11.3:2), dried, visualized by autoradiography with products identified as co-migrating with standard, and quantitated by scanning densitometry/Imagequant software.

      Measurement of Na+/H+Exchange

      Cellular Na+/H+ exchange activity in PS120/E3V cells was determined fluorometrically using the intracellular pH-sensitive dye BCECF with cells grown to 60–70% confluency on glass coverslips, as described previously (
      • Levine S.A.
      • Montrose M.H.
      • Tse C.M.
      • Donowitz M.
      ,
      • Tse C.M.
      • Levine S.A.
      • Yun C.H.
      • Brant S.R.
      • Pouyssegur J.
      • Montrose M.H.
      • Donowitz M.
      ). The effects of expressing p110* and Akt-myr in the absence or presence of ponasterone A on NHE3 were studied. The cells were loaded with the acetoxymethyl ester of 2′7′-bis(carboxyethyl) 5–6-carboxyfluorescein (BCECF-AM), 5 µm) in Na+ medium (130 mm NaCl, 5 mm KCl, 2 mmCaCl2, 1 mm MgSO4, 1 mmNaH2PO4, 25 mm glucose, 20 mm HEPES, pH 7.4) for 20 min at 22 °C and then washed with TMA+ medium (130 mm tetramethylammonium chloride, 5 mm KCl, 2 mm CaCl2, 1 mm MgSO4, 1 mmNaH2PO4, 25 mm glucose, 20 mm HEPES, pH 7.4) to remove the extracellular dye; the coverslip was then mounted at an angle of 60o in a 100-µl fluorometer cuvette designed for perfusion and thermostated at 37 °C. The cells were pulsed with 40 mmNH4Cl in TMA+ medium for 3 min, followed by TMA+ medium, which resulted in the acidification of the cells. Na+ medium was then added, which induced alkalinization of cells. Na+/H+ exchange (H+ efflux) was calculated as described previously (
      • Levine S.A.
      • Montrose M.H.
      • Tse C.M.
      • Donowitz M.
      ,
      • Levine S.A.
      • Nath S.K.
      • Yun C.H.
      • Yip J.W.
      • Montrose M.
      • Donowitz M.
      • Tse C.M.
      ,
      • Tse C.M.
      • Levine S.A.
      • Yun C.H.
      • Brant S.R.
      • Pouyssegur J.
      • Montrose M.H.
      • Donowitz M.
      ), as the product of sodium-dependent change in pHi times the buffering capacity at each pHi, and it was analyzed using a nonlinear regression data analysis program (Origin) that allowed fitting of data to a general allosteric model described by the Hill equation, with estimates forV max andK′[H+]i and their respective errors (S.E.).

      Biotinylation and Immunoblotting

      Plasma membrane NHE3 was measured by surface biotinylation, as described (
      • Akhter S.
      • Cavet M.E.
      • Tse C.M.
      • Donowitz M.
      ). Confluent PS120/E3V cells were serum-starved for 5 h, and then 10 µm PI 3-kinase peptide was incubated for 15 min. All subsequent manipulations were performed at 4 °C. Cells were rinsed twice with ice-cold phosphate-buffered saline (150 mm NaCl and 20 mm Na2HPO4, pH 7.4) and once in borate buffer (154 mm NaCl, 1.0 mm boric acid, 7.2 mm KCl, and 1.8 mm CaCl2, pH 9.0). Plasma membrane surface was then exposed to 0.5 mg/ml sulfo-NHS-SS-biotin in borate buffer for 40 min with horizontal shaking. After labeling, cells were washed with the quenching buffer (20 mm Tris and 120 mm NaCl, pH 7.4) to scavenge the unreacted biotin. Cells were washed three times with ice-cold phosphate-buffered saline and lysed in 1 ml of N+buffer (60 mm HEPES, pH 7.4, 150 mm NaCl, 3 mm KCl, 5 mm Na3EDTA, 3 mm EGTA, and 1% Triton X-100). Cells were sonicated for 20 s and agitated on a rotating rocker for 30 min at 4 °C. Insoluble cell debris was removed by centrifugation for 30 min at 12,000 × g. Supernatant representing the total fraction was incubated with avidin-agarose for 2 h. After avidin precipitation, the supernatant was retained as the intracellular fraction. The avidin-agarose beads were washed five times in N+ buffer to remove the all of the nonspecifically bound proteins. The avidin-agarose bead bound proteins, representing plasma membrane NHE3, were solubilized in equivalent volumes of loading buffer (5 mm Tris-HCl, pH 6.8, 1% SDS, 10% glycerol, 1% 2-mercaptoethanol), boiled for 5 min, size-fractionated by SDS-polyacrylamide gel electrophoresis on 9% gels, and then electrophoretically transferred to nitrocellulose. After blocking with 5% nonfat milk, the blots were probed with a monoclonal anti-VSV-G antibody (P5D4 hybridoma supernatant) as the primary antibody and horseradish peroxidase-conjugated anti-mouse as the secondary antibody. Bands were visualized by enhanced chemiluminescence.

      DISCUSSION

      In this study, the question was asked whether the activation of PI 3-kinase or Akt was sufficient to stimulate NHE3 in PS120 cells. Previous studies demonstrated a role for PI 3-kinase in rapid stimulation of NHE3 (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ,
      • Kurashima K.
      • Szabo E.Z.
      • Lukacs G.
      • Orlowski J.
      • Grinstein S.
      ,
      • Akhter S.
      • Cavet M.E.
      • Tse C.M.
      • Donowitz M.
      ,
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.M.
      • Tse C.M.
      • Cohen M.E.
      • Donowitz M.
      ), but there have been no previous studies implicating Akt in regulation of NHE3. PI 3-kinase activity has previously been shown to be necessary for basal NHE3 activity in the polarized epithelial cell lines Caco-2 and OK, as well as in the fibroblast cell line PS120 and in AP-1 cells, and also in EGF/FGF stimulation of NHE3 in fibroblasts and EGF stimulation of NHE3 in ileal brush border (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ,
      • Kurashima K.
      • Szabo E.Z.
      • Lukacs G.
      • Orlowski J.
      • Grinstein S.
      ,
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.M.
      • Tse C.M.
      • Cohen M.E.
      • Donowitz M.
      ). Based on inhibitor studies in PS120, AP-1, OK, and Caco-2 cells, basal NHE3 activity is lowered by the PI 3-kinase inhibitor wortmannin (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ,
      • Kurashima K.
      • Szabo E.Z.
      • Lukacs G.
      • Orlowski J.
      • Grinstein S.
      ,
      • Akhter S.
      • Cavet M.E.
      • Tse C.M.
      • Donowitz M.
      ). EGF/FGF stimulation of NHE3 is blocked in PS120 cells by the same inhibitors (
      • Donowitz M.
      • Tse M.
      ,
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ,
      • Akhter S.
      • Cavet M.E.
      • Tse C.M.
      • Donowitz M.
      ). FGF stimulated NHE3 in PS120 cells by 50%. 50% of this stimulation appears to be due to PI 3-kinase because wortmannin caused a 50% reduction of this stimulation (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ). Thus, FGF stimulates NHE3 by both PI 3-kinase-dependent and PI 3-kinase-independent mechanisms (
      • Donowitz M.
      • Tse M.
      ,
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ,
      • Akhter S.
      • Cavet M.E.
      • Tse C.M.
      • Donowitz M.
      ).
      Whether increased PI 3-kinase or AKT activity were sufficient to stimulate NHE3 had not been addressed before this study. However, this issue has begun to be studied in regards to the glucose transporter GLUT4 in adipocytes and smooth muscle cells. Insulin and growth factor stimulate glucose uptake and GLUT4 translocation to the plasma membrane by a process associated with activation and movement to the plasma membrane of PI 3-kinase (
      • Wang L.
      • Hayashi H.
      • Ebina Y.
      ,
      • Valverde A.M.
      • Navarro P.
      • Teruel T.
      • Conejo R.
      • Benito M.
      • Lorenzo M.
      ,
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ). Both glucose uptake and GLUT4 translocation are inhibited by the PI 3-kinase inhibitors wortmannin and LY290042 (
      • Klippel A.
      • Escobedo J.A.
      • Hirano M.
      • Williams L.T.
      ,
      • No D.
      • Yao T.P.
      • Evans R.M.
      ). Thus, PI 3-kinase activation is necessary for the insulin stimulation of glucose uptake and GLUT4 translocation. Initial studies demonstrated that constitutively active PI 3-kinase expression (p110) increased both glucose uptake and GLUT4 translocation in the absence of insulin, and dominant negative PI 3-kinase (p85) inhibited insulin-stimulated glucose uptake and GLUT4 translocation (
      • Sharma P.M.
      • Egawa K.
      • Huang Y.
      • Martin J.L.
      • Huvar I.
      • Boss G.R.
      • Olefsky J.M.
      ,
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Marchand-Brustel Y.
      ,
      • Frevert E.U.
      • Kahn B.B.
      ). These data were contradictory, however, concerning whether the magnitude of the insulin stimulation of glucose uptake was duplicated by overexpression of constitutively active PI 3-kinase. Recent interpretation is that increasing PI 3-kinase activity alone is quantitatively less than the insulin stimulation of glucose uptake (
      • Sharma P.M.
      • Egawa K.
      • Huang Y.
      • Martin J.L.
      • Huvar I.
      • Boss G.R.
      • Olefsky J.M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). In addition, increasing PI 3-kinase by separate mechanisms, including activation with platelet-derived growth factor and integrin receptor stimulation, did not increase glucose uptake at all (
      • Guilherme A.
      • Czech M.P.
      ,
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ). Moreover, exposure to membrane-permeant PIP3, which is converted intracellularly to end products of activated PI 3-kinase, failed to alter glucose transport in the absence of insulin (
      • Jiang T.
      • Sweeney G.
      • Rudolf M.T.
      • Klip A.
      • Traynor-Kaplan A.
      • Tsien R.Y.
      ). These results are currently interpreted as indicating that whereas activated PI 3-kinase is sufficient to induce a partial stimulation of GLUT4-related glucose uptake and membrane translocation, a second (or more) insulin-stimulated non-PI 3-kinase-dependent pathway(s) is necessary to reproduce the full insulin stimulation of glucose uptake (
      • Guilherme A.
      • Czech M.P.
      ,
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ,
      • Lazar D.F.
      • Wiese R.J.
      • Brady M.J.
      • Mastick C.C.
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      • Cuatrecasas P.
      • Saltiel A.R.
      ). A candidate second pathway has been described with the recognition that insulin brings the adapter protein CAP to the insulin receptor, where it recruits Cbl and interacts with flotillin resulting in phosphorylation of Cbl and direction of the Cbl-CAP complex to lipid rafts (or caveolae) in the plasma membrane (
      • Baumann C.A.
      • Ribon V.
      • Kanzaki M.
      • Thurmond D.C.
      • Mora S.
      • Shigematsu S.
      • Bickel P.E.
      • Pessin J.E.
      • Saltiel A.R.
      ).
      Our results demonstrate that stimulation of PI 3-kinase activity either by use of a peptide stimulator of PI 3-kinase activity (
      • Hubner S.
      • Couvillon A.D.
      • Kas J.A.
      • Bankaitis V.A.
      • Vegners R.
      • Carpenter C.L.
      • Janmey P.A.
      ) or transient infection with adenovirus containing constitutively active PI 3-kinase increases NHE3 activity. This stimulation is similar in magnitude to the PI 3-kinase-dependent component of rapid growth factor stimulation of NHE3 (
      • Janecki A.J.
      • Janecki M.
      • Akhter S.
      • Donowitz M.
      ). The quantitative similarity suggests that an increase in PI 3-kinase activity is not only sufficient to increase NHE3 activity but that this component of the growth factor stimulation is entirely due to the increase in PI 3-kinase activity. The growth factor-stimulated messenger that initiates the PI 3-kinase-dependent growth factor stimulation of NHE3 remains unidentified.
      Thus, similarities and differences between insulin stimulation of GLUT4 and growth factor stimulation of NHE3 have been identified. Both involve stimulation by trafficking and increases in exocytosis, with PI 3-kinase being necessary and sufficient for the stimulation. In addition, there is a second component of stimulation in addition to a pathway mediated through PI 3-kinase. The recently recognized pathway of insulin stimulation of GLUT4 that may include CAP-flotillin-Cbl has not been studied in regulation of NHE3, and no additional specific factor has been identified for the growth factor stimulation of NHE3. Concerning differences between growth factor stimulation of NHE3 and insulin stimulation of GLUT4, NHE3 trafficking under basal conditions is more than for GLUT4, which is nearly entirely intracellular. This basal stimulation of NHE3 is also PI 3-kinase-dependent. This difference is not surprising in that NHE3 is rapidly regulated by both stimulation and inhibition in the intestine and kidney, whereas regulation of glucose uptake is stimulated by insulin, and no inhibitory mechanism from basal rate has yet been identified. Importantly all GLUT4 mobilization is PI 3-kinase-dependent, while only part of NHE3 depends on PI 3-kinase. Another difference of the stimulatory mechanisms of GLUT4 and NHE3 is the suggestion that insulin mobilizes glucose from both the recycling endosomes and a special storage compartment, whereas growth factor mobilization of NHE3 has only been suggested as coming from the recycling compartment (
      • Foster L.J.
      • Klip A.
      ), with no specific storage compartment for NHE3 yet identified.
      The current studies also represent the initial demonstration of involvement of Akt in growth factor stimulation of NHE3. Akt represents a major downstream signaling molecule in the PI 3-kinase cascade. In unpublished studies, we have shown that EGF stimulation of ileal NaCl absorption and brush border NHE3 activity is associated with a rapid increase in brush border Akt activity.
      Li, X.-H., Shih, C., and Donowitz, M., unpublished observations.
      Due to lack of available Akt inhibitors, the functional significance of this stimulation could not be determined. These studies show that increasing Akt activity and amount stimulates NHE3. In addition, the similarity in magnitude of stimulation of NHE3 with transient transfection with constitutively active PI 3-kinase and Akt mutants and the fact that increasing PI 3-kinase activity stimulates Akt activity without altering Akt amount suggests that most, if not all, of the PI 3-kinase stimulation of NHE3 may be due to activation of Akt in PS120 cells.
      Results assessing the contribution of AKT to insulin/PI 3-kinase stimulation of glucose uptake in adipocytes and muscle cells indicates that AKT activation is necessary and appears sufficient to stimulate glucose uptake. Constitutively active AKT (using several different constitutively active constructs different than that used in our studies consisting of MyrΔ-4–129 Akt (in which the Akt plextin homology domain is deleted) and a v-Akt analogue (called Gag-protein kinase B) stimulated glucose uptake and GLUT4 plasma membrane translocation. The magnitude of the effects was between 69 and 100% of the insulin stimulation (
      • Ueki K.
      • Yamamoto-Honda R.
      • Kaburagi Y.
      • Yamauchi T.
      • Tobe K.
      • Burgering B.M.
      • Coffer P.J.
      • Komuro I.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Tanti J.F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Van Obberghen E.
      • Marchand-Brustel Y.
      ). However, disagreement exists in that studies performed with several dominant negative AKT constructs either inhibited most of the insulin-stimulated glucose uptake or had inhibitory effects of only 0–20% (
      • Wang Q.
      • Somwar R.
      • Bilan P.J.
      • Liu Z.
      • Jin J.
      • Woodgett J.R.
      • Klip A.
      ,
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      ,
      • Cong L.N.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.A.
      • Taylor S.I.
      • Quon M.J.
      ).
      Other systems, in which the downstream signaling of PI 3-kinase involves Akt, have had additional downstream signals identified. In addition to Akt, atypical forms of protein kinase C (especially ζ and λ), but also the conventional isoforms β-2 and the novel isoform δ have been shown to represent downstream signaling molecules following PI 3-kinase activation in some cells (
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • Le Good J.A.
      • Gigg R.
      • Parker P.J.
      ). The current studies have not considered involvement of protein kinase C in the PI 3-kinase stimulation of NHE3. However, the similarity in magnitude of FGF stimulation of NHE3 and effects of constitutively active PI 3-kinase and Akt, as well as similarity in the stimulation of NHE3 with the increase in Akt activation when constitutively active PI 3-kinase is transfected, make it likely that the contribution of atypical forms of protein kinase C to PI 3-kinase stimulation of NHE3 is small, if it is present at all, in these cells.
      Attempts were made to examine the contribution of PI 3-kinase and Akt in regulation of NHE3 by expressing dominant negative forms using the same adenoviral expression system used for these activation studies. The constructs used were described previously and consisted of p110* mutated in the kinase domain and Akt-AA (T308A/S473A) (
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ,
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      ). In both cases, there was significant loss of cell viability, which we presumed, but did not demonstrate, was due to induction of apoptosis.
      The adenovirus infection system was used for transient infection in order to develop a method of obtaining transient expression in a high percentage of cells to allow correlation of biochemistry and functional data, in this case, NHE3 transport. The motivation for developing an inducible system was that in studying molecules that alter cell division and state of differentiation, we wanted to be able to select conditions in which changes in the differentiation status of cells studied could be minimized by controlling the time of expression of selected signaling molecules. Thus, after use of eGFP as a marker to determine conditions to infect nearly all cells, we selected a time window of expression of the signaling molecule of interest. The level of expression of PI 3-kinase due to the inducible expression was not compared with the endogenous level; however, the increase in activity due to ponasterone A was similar to that which occurred with growth factor exposure (
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.M.
      • Tse C.M.
      • Cohen M.E.
      • Donowitz M.
      ). In addition, our studies documented the low level of leakiness of this expression system in the absence of activation of the ecdysone receptor with ponasterone A (Fig. 3). Thus, we suggest the usefulness of this inducible adenovirus system for expressing signaling molecules that are to be activated at a relatively fixed time and studied over a limited period. In addition, the relatively small percentage of increase in amount of expressed protein may prove to be an advantage compared with other methods that lead to problems from large amounts of protein expression.

      Acknowledgments

      We acknowledge the expert editorial assistance of H. McCann. We thank Paul Janmey, University of Pennsylvania, for supplying the PI 3-kinase10-mer peptide. We thank L. T. Williams for the constitutively active PI 3-kinase and useful discussion concerning its use.

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