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Distinct Phosphatidylinositol 3-Kinase Lipid Products Accumulate upon Oxidative and Osmotic Stress and Lead to Different Cellular Responses*

Open AccessPublished:December 10, 1999DOI:https://doi.org/10.1074/jbc.274.50.35963
      Signaling by phosphatidylinositol (PI) 3-kinases is mediated by 3-phosphoinositides, which bind to Pleckstrin homology (PH) domains that are present in a wide spectrum of proteins. PH domains can be classified into three groups based on their different lipid binding specificities. Distinct 3-phosphoinositides can accumulate upon PI 3-kinase activation in cells in response to different stimuli and mediate specific cellular responses. In Swiss 3T3 mouse fibroblasts, oxidative stress induced by 1 mmH2O2 caused almost exclusive accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), whereas osmotic stress increased both phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PtdIns(3,4)P2 levels. The increase in PtdIns(3,4)P2 levels, caused by oxidative stress, correlated with the activation of protein kinase B, which has a promiscuous PH domain that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2. p70 S6 kinase, another signaling component downstream of PI 3-kinase, however, was not activated by this oxidative stress-induced increase in PtdIns(3,4)P2 levels. Increased PtdIns(3,4,5)P3 and PtdIns(3,4)P2levels in response to osmotic stress did not correlate with protein kinase B activation, because of concomitant activation of an inhibitory pathway, but p70 S6 kinase was activated by osmotic stress. These results demonstrate that PtdIns(3,4)P2 can accumulate independently of PtdIns(3,4,5)P3 and exerts a pattern of cellular responses that is distinct from that induced by accumulation of PtdIns(3,4,5)P3.
      PI
      phosphatidylinositol
      PtdIns
      phosphatidylinositol
      PtdIns4P
      phosphatidylinositol 4-phosphate
      PtdIns(4
      5)P2, phosphatidylinositol 4,5-bisphosphate
      PtdIns3P
      phosphatidylinositol 3-phosphate
      PtdIns(3
      4)P2, phosphatidylinositol 3,4-bisphosphate
      PtdIns(3
      4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
      PH
      Pleckstrin homology
      MAP
      mitogen-activated protein
      MAPKAP-K2
      mitogen-activated protein kinase-activated protein kinase 2
      SAPK
      stress-activated protein kinase
      IGF-1
      insulin-like growth factor I
      HPLC
      high performance liquid chromatography
      PKB
      protein kinase B
      The significance of PI13-kinases in the regulation of a wide spectrum of cellular signaling events has been well established (
      • Rameh L.E.
      • Cantley L.C.
      ,
      • Van Haesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). Based on structural features and substrate specificity, three classes of catalytic subunits of PI 3-kinases have so far been recognized (
      • Van Haesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ). The Type I class, members of which phosphorylate PtdIns, PtdIns4P, and PtdIns(4,5)P2 in vitro but utilize PtdIns(4,5)P2 as the likely substrate in vivo, is subdivided into Type Ia and Type 1b. Type Ia PI 3-kinases interact with adaptor proteins containing SH2 domains that bind phosphotyrosine residues, linking this class to tyrosine kinase signaling cascades. Type 1b PI 3-kinases are stimulated by G-protein βγ subunits and do not interact with SH2 domain containing adaptor proteins but with p101, a novel adaptor protein that has no homology with known proteins. Type II PI 3-kinases phosphorylate PtdIns and PtdIns4P in vitro (and not PtdIns(4,5)P2) and contain a C2 domain, implicated in lipid binding. Type III PI 3-kinases only phosphorylate PtdIns and are thought to be constitutively active. They can be recruited and regulated by their adaptor, a dual specificity protein kinase, with which they form heterodimers.
      The principle enzymatic activity of PI 3-kinases involves the phosphorylation of phosphoinositides on the 3-position of the inositol ring, resulting in the formation of 3-phosphoinositides, which can either directly interact with appropriate modules of specific target proteins or first undergo further phosphorylation. Examples of the latter mechanism include the observation that in platelets, PtdIns3P can be phosphorylated by a PtdIns3P 4-kinase activity to give PtdIns(3,4)P2, which activates PKB (
      • Banfic H.
      • Tang X.
      • Batty I.H.
      • Downes C.P.
      • Chen C.
      • Rittenhouse S.E.
      ), and in yeast, a PtdIns3P 5-kinase activity encoded by the FAB1p gene has been shown to be essential for vacuolar functioning (
      • Cooke F.T.
      • Dove S.K.
      • McEwen R.K.
      • Painter G.
      • Holmes A.B.
      • Hall M.N.
      • Michell R.H.
      • Parker P.J.
      ). FYVE domains are the structural modules that specifically bind PtdIns3P (
      • Gaullier J.
      • Simonson A.
      • D'Arrigo A.
      • Bremnes B.
      • Stenmark H.
      • Aasland R.
      ,
      • Patki V.
      • Lawe D.C.
      • Corvera S.
      • Virbasius F.V.
      • Chawla A.
      ), whereas PH domains confer specificity for polyphosphoinositides (PtdIns(4,5)P2, PtdIns(3,4)P2, and PtdIns(3,4,5)P3) (
      • Kavran J.M.
      • Klein D.E.
      • Lee A.
      • Falasaca M.
      • Isakoff S.J.
      • Skolnik E.Y.
      • Lemmon M.A.
      ,
      • Lemmon M.A.
      • Falasca M.
      • Ferguson K.M.
      • Schlessinger J.
      ). More than 100 different proteins containing PH domains have been identified, and the majority require membrane association for their function, which is mediated via interaction with phosphoinositides. PH domains can be assigned to one of three groups based on their affinities for PtdIns(4,5)P2, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (or their polar headgroup), although many details involved in binding specificity still have to be elucidated (
      • Kavran J.M.
      • Klein D.E.
      • Lee A.
      • Falasaca M.
      • Isakoff S.J.
      • Skolnik E.Y.
      • Lemmon M.A.
      ). Modelling of PH domains, based on sequence alignments, resolved crystal structures, and binding studies, are beginning to allow predictions of binding specifities and hence likely physiological ligands.
      Cells respond to different extracellular inputs from their environment by activation of protein kinase cascades. These complex networks allow amplification of the signal and can mediate the required diversity of cellular responses. The classical MAP kinase pathway is activated by agonists that include growth factors and tumor-promoting agents and the SAPK/p38 pathway is activated by cellular stresses and inflammatory cytokines. PI 3-kinases can mediate cellular responses to stress, but their direct role and the mechanism of activation is not well defined.
      There are relatively few reports on direct activation of PI 3-kinase upon exposure to cellular stresses. Heat shock and oxidative stress have both been reported to activate PI 3-kinase activity directly in NIH 3T3 cells (
      • Lin R.Z.
      • Hu Z-W.
      • Chin J.H.
      • Hoffman B.B.
      ,
      • Tanaka K.
      • Horiguchi K.
      • Yoshida T.
      • Takeda M.
      • Fujisawa H.
      • Takeuchi K.
      • Umeda M.
      • Kato S.
      • Ihara S.
      • Nagata N.
      • Fukui Y.
      ) or indirectly by manipulation of downstream signaling components (
      • Guyton K.Z.
      • Liu Y.
      • Gorospe M.
      • Xu Q.
      • Holbrook N.J.
      ). Both heat shock and oxidative stress effects were suramin-sensitive (
      • Lin R.Z.
      • Hu Z-W.
      • Chin J.H.
      • Hoffman B.B.
      ,
      • Guyton K.Z.
      • Liu Y.
      • Gorospe M.
      • Xu Q.
      • Holbrook N.J.
      ), suggesting the involvement of growth factor receptors. Konishi et al. (
      • Konishi H.
      • Matsuzaki H.
      • Tanaka M.
      • Ono Y.
      • Tokunaga C.
      • Kuroda S.
      • Kikkawa U.
      ) showed that osmotic shock activated wild type but not mutated PKB in transfected COS-7 cells, whereas others failed to detect this in Swiss3T3 cells (
      • Shaw M.
      • Cohen P.
      • Alessi D.R.
      ,
      • Meier R.
      • Thelen M.
      • Hemmings B.A.
      ). The involvement of PI 3-kinase in stress responses inferred by activation of PKB or other downstream signaling components, however, can be misleading as demonstrated by the following two observations in this report. Firstly, wortmannin-sensitive PKB activation was strongly enhanced upon H2O2 treatment in the absence of a detectable increase of PtdIns(3,4,5)P3. Secondly, osmotic shock greatly increased PtdIns(3,4,5)P3 concentrations, which did not result in the expected strong activation of PKB. Because oxidative stress induced by direct exposure of cells to H2O2 results in a complex array of signaling events and osmotic shock activates many different components of several kinase cascades (
      • Cohen P.
      ,
      • Han S.J.
      • Choi K.Y.
      • Brey P.T
      • Lee W.J.
      ,
      • Qin S.
      • Minami Y.
      • Hibi M.
      • Kurosaki T.
      • Yamamura H.
      ), the role of PI 3-kinase in these events has been analyzed in more detail.
      This report shows the accumulation of distinct PI 3-kinase lipid products, in response to oxidative stress and osmotic shock upon treatment of Swiss3T3 mouse fibroblasts with 1 mmH2O2 and 0.5 m sucrose, respectively. These products result in the activation of different metabolic pathways and lead to distinct patterns of cellular response. Whereas oxidative stress caused a sustained accumulation of PtdIns(3,4)P2 and to subsequent activation of PKB but not of p70 S6 kinase, osmotic shock increased PtdIns(3,4,5)P3concentration and subsequent activation of p70 S6 kinase but did not activate PKB.

      EXPERIMENTAL PROCEDURES

      Materials

      [γ-32P]ATP (3000 Ci/mmol) and Enhanced Chemoluminescence kit was from Amersham Pharmacia Biotech. [3H]Inositol was from NEN Life Science Products. Partisphere SAX column was from Whatman. Insulin-like growth factor I (IGF-1) and protein G-agarose were from Sigma. Anti-phosphotyrosine monoclonal antibody G410 was from Upstate Biotechnology, Inc. The horseradish peroxidase-conjugated anti-sheep/goat antibody was from Scottish Antibody Production Unit, whereas the PKBα C-terminal antibody and the anti-PKB Ser(P)473 antibody, both raised in sheep, were a gift from the Medical Research Council protein phosphorylation unit in Dundee.

      Methods

      Cell Culture and PKB Activity Assay

      Swiss 3T3 mouse fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 5% CO2atmosphere at 37 °C. Cells were grown to confluence in 6-well plates (sometimes in inositol-free Dulbecco's modified Eagle's medium with 10% dialyzed fetal bovine serum for parallel comparison with cell labeling experiments; see below). Prior to treatment, the medium was aspirated, and the cells were washed twice with 2 ml of modified Krebs Henseleit (KRH) buffer (0.1 m NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.3 mmCaCl2, 1.2 mm KH2PO4, 25 mm HEPES/NaOH, pH 7.4, and 2% D(+)-glucose) and then conditioned for 60 min in 3 ml of KRH buffer in a 37 °C waterbath.
      Cells were stimulated with 100 ng/ml IGF-1, 1 mmH2O2, and 0.5 m sucrose for the indicated times, after which the medium was aspirated and 1 ml of ice-cold lysis-buffer (50 mm Tris/HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 0.1% Triton X-100, 0.1% β-mercaptoethanol, 1 mm sodium ortho-vanadate, 50 mm NaF, 5 mm sodium pyrophosphate, 10 mm di-sodium β-glycerophosphate, 1 mmbenzamidin, and 1 mm phenylmethylsulfonyl fluoride) was added. Cells were scraped on ice, and the lysates were transferred into Eppendorf tubes and spun for 1 min at 20,800 × g. The supernatant was transferred to a new tube, snap frozen in liquid nitrogen, and stored at −80 °C.
      1 μg of PKB antibody coupled to 5 μl of protein G-agarose beads was added to each sample. After 90 min on a shaking platform at 4 °C, the beads were spun down, the supernatant was aspirated, and the beads were washed twice with ice-cold lysis-buffer containing 0.5m NaCl and twice with ice-cold wash-buffer (50 mm Tris/HCl, pH 7.5, 0.1 mm EDTA, 0.03% Brij-35, and 0.1% β-mercaptoethanol). After the last wash, the buffer was removed carefully to a volume of ∼10 μl, and the reaction was started by adding 50 μl of assay-buffer (50 mm Tris/HCl, pH 7.5, 0.1 mm EDTA, 0.1% β-mercaptoethanol, 20 μm cAMP-dependent protein kinase inhibitor peptide, 50 μm Crosstide (PKB substrate peptide), 10 mm Mg(OAc)2, 100 μm ATP, and 1 μCi of [γ-32P]ATP. After 30 min at 30 °C on a shaking platform, the reactions were terminated by pipetting the suspension onto 2.5 × 2.5-cm phosphocellulose papers that were washed four times for 15 min in 0.5%ortho-phosphoric acid and then washed once with acetone. The dried filters were counted for radioactivity in 4 ml of scintillant (FloScint IV).

      p70 S6 Kinase Activity Assay

      Measurement of p70 S6 kinase was done exactly as described for the PKB assay except that the antibody was raised against p70 S6 kinase, and the substrate peptide used was KKRNRTLTV at 100 μm.

      PI 3-Kinase Activity Assay

      PI 3-kinase was immunoprecipitated from Swiss 3T3 mouse fibroblast cell lysates, andin vitro activity was measured as follows. After treatment (described above) the cell medium was aspirated, ice-cold lysis-buffer was added, cells were scraped, and the lysates were spun for 2 min at 20,800 × g at 4 °C. The supernatants were snap frozen in liquid nitrogen and stored at −80 °C.
      Anti-phosphotyrosine antibody (1 μg/sample) was precoupled to protein G-agarose beads (5 μl/sample) and added to each sample. After 90 min on a shaking platform at 4 °C, the beads were spun down, the supernatant was aspirated, and the beads were washed twice with 1 ml of ice-cold lysis-buffer containing 0.5 m NaCl and twice with 1 ml of assay buffer (0.1 m NaCl, 2.5 mmMgCl2, 1 mm EGTA, 0.2 mm EDTA, and 25 mm HEPES, pH 7.5). 40 μl of 2× assay buffer, 10 μl of lipid vesicles, and 20 μl of H2O was then added to each 20 μl of beads. Lipid vesicles were prepared by drying PtdIns and phosphatidyl serine down, resuspending them in 0.5 ml of lipid buffer (10 mm HEPES, pH 7.5, 1 mm EGTA, 0.1m NaCl) to a final concentration of 1 mm each. Vesicles were made by four blasts of 15 s with a probe sonicator at a power setting of 50%, with 1 min cooling on ice in between. Reactions were started by adding 10 μl of 100 μm[γ-32P]ATP (10 μCi/assay) and incubated for 20 min on a platform shaker at 37 °C. The reactions were terminated with 0.75 ml of CHCl3:MeOH:concentrated HCl (40:80:1), and two phases were obtained by adding 0.25 ml of CHCl3 and 0.35 ml of 0.1m HCl. After centrifugation the upper phase was aspirated, and the organic phase was washed twice with 0.5 ml of synthetic upper phase and dried down. The lipids were analyzed by TLC on silica 60 plates using CHCl3:MeOH:ammonia:H2O (75:100:15:25) as the mobile phase. The bands were visualized by autoradiography and scraped, and the radioactivity was measured in a scintillation counter.

      Cell Labeling and Phosphoinositide Analysis

      Swiss 3T3 mouse fibroblasts were seeded in 6-well plates at 104cells/cm2 in Dulbecco's modified Eagle's medium without inositol and supplemented with 10% dialyzed fetal bovine serum. After 48 h, the medium was replaced with 4 ml of fresh medium containing 10 μCi/ml [3H]inositol and left for 48 h. The medium was aspirated, and the cells were washed twice with 2 ml of KRH and then conditioned for 60 min in 3 ml of KRH buffer in a 37 °C waterbath. Cells were treated as described above but quenched with 1 ml of ice-cold 10% trichloroacetic acid (w/v), scraped on ice and transferred to Eppendorf tubes. The wells were rinsed once with 0.5 ml of ice-cold 10% trichloroacetic acid, and the particulate material was pelleted by centrifugation (5 min at 20,800 × g). The supernatant was removed, and the pellet was washed once with 1 ml of ice-cold 5% trichloroacetic acid/1 mm EDTA. The lipids were extracted on ice for 20 min in 0.75 ml of CHCl3:MeOH:concentrated HCl (40:80:1). Two phases were obtained by adding 0.25 ml of CHCl3 and 0.45 ml of 0.1 m HCl to the samples, which were thoroughly vortexed and spun for 2 min at 17,500 × g. The lower, organic phases were transferred to screw cap tubes containing 50 μl of 1m ammonia in methanol. The upper phases and interphases were re-extracted once with 0.5 ml of synthetic lower phase, and the organic phases were pooled, dried down in a vacuum concentrator, and stored at −80 °C.
      The deacylation was carried out as described in Ref.
      • Clark N.G.
      • Dawson R.M.
      , and the glycerophosphoinositolphosphates were analyzed by HPLC using a Partisphere SAX column (5-μm mesh size; 4.6 × 250 mm). The column was eluted at a flow rate of 1 ml/min, with 5 min H2O followed by a gradient made from H2O to 1m NH4H2PO4 (pH 3.8 withortho-phosphoric acid) in 100 min with an increment of 1%/min. Three fractions/min were collected, to each of which 3 ml of scintillant was added, and the radioactivity was measured in a scintillation counter.

      PtdIns(3,4,5)P3 Mass Determination

      The PtdIns(3,4,5)P3 mass assay was done as decribed in Ref.
      • Van der Kaay J.
      • Cullen P.J.
      • Downes C.P.
      , which is modified from Ref.
      • Van der Kaay J.
      • Batty I.H.
      • Cross D.A.E.
      • Watt P.W.
      • Downes C.P.
      , by the use of a different inositol 1,3,4,5-tetraphosphate-binding protein that had essentially the same characteristics. Recombinant GST-GAP1IP4BP (expressed inEscherichia coli and purified on glutathione-agarose beads) replaced crude cerebellar membrane from sheep. Total cellular lipids were isolated from confluent 6-well plates as described above.

      Western Blot

      Cell lysates were obtained exactly as described for the PKB activity assay. Approximately 20 μg of cytosolic protein from each sample was subjected to SDS-polyacrylamide gel electrophoresis on 2 gels and transferred to polyvinylidene difluoride membranes. One membrane was incubated with the anti-PKBα C-terminal antibody, and the other was incubated with anti-PKB Ser(P)473 antibody each at 1 μg/ml in Tris-buffered saline, 0.1% Tween-20, 3% dried milk. The secondary horseradish peroxidase-conjugated anti-sheep/goat antibody allowed visualization by enhanced chemoluminescence according to the manufacturer's protocol.

      ACKNOWLEDGEMENT

      P. J. Cullen provided the expression vector for the inositol 1,3,4,5-tetraphosphate-binding protein.

      REFERENCES

        • Rameh L.E.
        • Cantley L.C.
        J. Biol. Chem. 1999; 274: 8347-8350
        • Van Haesebroeck B.
        • Leevers S.J.
        • Panayotou G.
        • Waterfield M.D.
        Trends Biochem. Sci. 1997; 22: 267-272
        • Stephens L.R.
        • Jackson T.R.
        • Hawkins P.T.
        Biochem. Biophys. Acta. 1993; 1179: 27-75
        • Banfic H.
        • Tang X.
        • Batty I.H.
        • Downes C.P.
        • Chen C.
        • Rittenhouse S.E.
        J. Biol. Chem. 1998; 273: 13-16
        • Cooke F.T.
        • Dove S.K.
        • McEwen R.K.
        • Painter G.
        • Holmes A.B.
        • Hall M.N.
        • Michell R.H.
        • Parker P.J.
        Curr. Biol. 1998; 8: 1219-1222
        • Gaullier J.
        • Simonson A.
        • D'Arrigo A.
        • Bremnes B.
        • Stenmark H.
        • Aasland R.
        Nature. 1998; 394: 432-433
        • Patki V.
        • Lawe D.C.
        • Corvera S.
        • Virbasius F.V.
        • Chawla A.
        Nature. 1998; 394: 433-443
        • Kavran J.M.
        • Klein D.E.
        • Lee A.
        • Falasaca M.
        • Isakoff S.J.
        • Skolnik E.Y.
        • Lemmon M.A.
        J. Biol. Chem. 1998; 273: 30497-30508
        • Lemmon M.A.
        • Falasca M.
        • Ferguson K.M.
        • Schlessinger J.
        Trends Cell Biol. 1997; 7: 237-242
        • Lin R.Z.
        • Hu Z-W.
        • Chin J.H.
        • Hoffman B.B.
        J. Biol. Chem. 1997; 272: 31196-31202
        • Tanaka K.
        • Horiguchi K.
        • Yoshida T.
        • Takeda M.
        • Fujisawa H.
        • Takeuchi K.
        • Umeda M.
        • Kato S.
        • Ihara S.
        • Nagata N.
        • Fukui Y.
        J. Biol. Chem. 1999; 274: 3919-3922
        • Guyton K.Z.
        • Liu Y.
        • Gorospe M.
        • Xu Q.
        • Holbrook N.J.
        J. Biol. Chem. 1996; 271: 4138-4142
        • Konishi H.
        • Matsuzaki H.
        • Tanaka M.
        • Ono Y.
        • Tokunaga C.
        • Kuroda S.
        • Kikkawa U.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7639-7643
        • Shaw M.
        • Cohen P.
        • Alessi D.R.
        Biochem. J. 1998; 336: 241-246
        • Meier R.
        • Thelen M.
        • Hemmings B.A.
        EMBO J. 1998; 17: 7294-7303
        • Cohen P.
        Trends Cell Biol. 1997; 7: 353-361
        • Han S.J.
        • Choi K.Y.
        • Brey P.T
        • Lee W.J.
        J. Biol. Chem. 1998; 273: 369-374
        • Qin S.
        • Minami Y.
        • Hibi M.
        • Kurosaki T.
        • Yamamura H.
        J. Biol. Chem. 1997; 272: 2098-2103
        • Clark N.G.
        • Dawson R.M.
        Biochem. J. 1981; 195: 301-306
        • Van der Kaay J.
        • Cullen P.J.
        • Downes C.P.
        Methods in Molecular Biology: Phospholipid Signalling Protocols. 105. Humana Press, Inc., Totowa, NJ1998: 109-125
        • Van der Kaay J.
        • Batty I.H.
        • Cross D.A.E.
        • Watt P.W.
        • Downes C.P.
        J. Biol. Chem. 1997; 272: 5477-5481
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        • Toker A.
        Science. 1997; 275: 665-668
        • Stokoe D.
        • Stephens L.R.
        • Copeland T.
        • Gaffney P.R.
        • Reese C.B.
        • Painter G.F.
        • Holmes A.B.
        • McCormick F.
        • Hawkins P.T.
        Science. 1998; 279: 673-674
        • Alessi D.R.
        • Andjelkovic M.
        • Caudwell F.B.
        • Cron P.
        • Morrice N.
        • Cohen P.
        • Hemmings B.A.
        EMBO J. 1996; 15: 6541-6551
        • Balendran A.
        • Deak M.
        • Paterson A.
        • Gaffney P.
        • Currie R.A.
        • Downes C.P.
        • Alessi D.R.
        Curr. Biol. 1999; 9: 393-404
        • Currie R.A.
        • Walker K.S.
        • Gray A.
        • Deak M.
        • Casamayor A.
        • Downes CP.
        • Cohen P.
        • Alessi D.R.
        • Lucocq J.
        Biochem. J. 1999; 337: 575-583
        • Klarlund J.K.
        • Rameh L.E.
        • Cantley L.C.
        • Buxton J.M.
        • Holik J.L.
        • Sakelis C.
        • Patki V.
        • Corvera S.
        • Czech M.P.
        J. Biol. Chem. 1998; 273: 1859-1862
        • Alessi D.R.
        • Kozlowski M.T.
        • Weng Q.-P.
        • Morrice N.
        • Avruch J.
        Curr. Biol. 1997; 8: 69-81
        • Pullen N.
        • Dennis P.B.
        • Andjelkovic M.
        • Dufner A.
        • Kozma S.C.
        • Hemmings B.A.
        • Thomas G.
        Science. 1998; 279: 707-710
        • Chou M.M.
        • Blenis J.
        Cell. 1996; 85: 573-583
        • Burgering B.M.T.
        • Coffer P.J.
        Nature. 1995; 376: 599-602
        • Czech M.P.
        • Lawrence J.C.
        • Lynn A.C.
        Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4173-4177
        • Heldin C.
        Cell. 1995; 80: 213-223
        • 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.
        J. Biol. Chem. 1998; 273: 11937-11943
        • Peterson R.T.
        • Desai B.N.
        • Hardwick J.S.
        • Schreiber S.L.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442
        • Joyal J.L.
        • Burks D.J.
        • Pons S.
        • Matter W.F.
        • Vlahos C.J.
        • White M.F.
        • Sacks D.B.
        J. Biol. Chem. 1997; 272: 28183-28186