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Wortmannin and Its Structural Analogue Demethoxyviridin Inhibit Stimulated Phospholipase A2 Activity in Swiss 3T3 Cells

WORTMANNIN IS NOT A SPECIFIC INHIBITOR OF PHOSPHATIDYLINOSITOL 3-KINASE (∗)
  • Michael J. Cross
    Affiliations
    Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, United Kingdom
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  • Allison Stewart
    Affiliations
    Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, United Kingdom
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  • Matthew N. Hodgkin
    Affiliations
    Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, United Kingdom
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  • David J. Kerr
    Affiliations
    Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, United Kingdom
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  • Michael J.O. Wakelam
    Correspondence
    To whom correspondence should be addressed: Institute for Cancer Studies, University of Birmingham, P. O. Box 363, Birmingham B15 2TT, UK
    Affiliations
    Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TT, United Kingdom
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  • Author Footnotes
    ∗ This work was supported by grants from the Wellcome Trust, the Medical Research Council UK and the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:October 27, 1995DOI:https://doi.org/10.1074/jbc.270.43.25352
      Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that these compounds are not as selective as assumed and demonstrate inhibition of bombesin-stimulated phospholipase A2 activity by both wortmannin and DMV with an IC40 (2 nM) which is slightly more potent than the inhibition of insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate generation in these cells (⌼10 nM). While it has not been possible to fully block in vitro phospholipase A2 activity with wortmannin, inhibition cannot be a consequence of inhibition of PI 3-kinase activity since bombesin fails to generate 3-phosphorylated lipids in the intact cell. Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is not as specific as previously reported, and experimental conclusions based solely on its use should be treated with caution.

      INTRODUCTION

      The fungal metabolite wortmannin and its structural analogue demethoxyviridin (DMV)
      The abbreviations used are: DMV
      demethoxyviridin
      PLA2
      phospholipase A2
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PLC
      phospholipase C
      PLD
      phospholipase D
      PIP2
      phosphatidylinositol 4,5-bisphosphate
      PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      HPLC
      high performance liquid chromatography.
      have been demonstrated to have an inhibitory effect upon phosphatidylinositol 3-kinase (PI 3-kinase) activity at nanomolar concentrations(
      • Arcaro A.
      • Wymann M.P.
      ). Inhibition has been demonstrated upon both PI 3-kinase activity in anti-p85 immunoprecipitates and the stimulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) generation in N-formylmethionylleucylphenylalanine (fMLP)-stimulated neutrophils(
      • Arcaro A.
      • Wymann M.P.
      ). The binding of 17-[3H]hydroxywortmannin to a number of neutrophil proteins has been demonstrated, and one of these has been identified as the 110-kDa subunit of PI 3-kinase(
      • Thelen M.
      • Wymann M.P.
      • Langen H.
      ). On the basis of these findings, it has been proposed that wortmannin and DMV are specific inhibitors of PI 3-kinase, and, thus, addition of these compounds to cells will result in the specific inhibition of the PI 3-kinase pathway. Consequently, an increasing number of papers have described the use of wortmannin and, on the sole basis of such experiments, assigned a role for PI 3-kinase in a number of physiological responses (e.g. (
      • Nakamura I.
      • Takahashi N.
      • Sasaki T.
      • Tanaka S.
      • Udagawa N.
      • Murakami H.
      • Kimura K.
      • Kabuyama Y.
      • Kurokawa T.
      • Suda T.
      • Fukui Y.
      ,
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ,
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      )).
      Wortmannin has also been reported to inhibit phospholipase D (PLD)(
      • Bonser R.W.
      • Thompson N.T.
      • Randall R.W.
      • Tateson J.E.
      • Spacey G.D.
      • Hodson H.F.
      • Garland L.G.
      ), myosin light chain kinase, and plekstrin phosphorylation (see (
      • Arcaro A.
      • Wymann M.P.
      )). Although these results could cast doubt upon the specificity of wortmannin, these effects have been shown to occur at concentrations greater than those reported to inhibit PI 3-kinase. However, the specificity of wortmannin for other lipid-metabolizing enzymes has not been examined. In this paper, we have examined the specificity of wortmannin and DMV and show that they inhibit stimulated PIP2-phospholipase C (PI-PLC), PLD, phospholipase A2 (PLA2) as well as PI 3-kinase in Swiss 3T3 cells and, in addition, in vitro PI 3-kinase and PLA2 activities. We also demonstrate that both compounds are more potent inhibitors of PLA2 than PI 3-kinase.

      EXPERIMENTAL PROCEDURES

      Materials

      Radiochemicals were from Amersham International plc, except for the inositol phosphate standards which were from DuPont NEN, all tissue culture media and sera were from Life Technologies, Inc., thin layer chromatography plates and HPLC columns were from Whatman, and lipids were from Sigma, Avanti Polar Lipids, or Lipid Products, Nutley, Surrey, UK. Wortmannin was from Sigma, and DMV was a generous gift from Dr G. MacAully, Dept. of Chemistry, Glasgow University, UK. Anti-p85, clone U13 was from Serotec. SF9 cellexpressed human cytosolic PLA2 (85-kDa enzyme) was a generous gift from Dr. C. Jackson, Fisons Pharmaceuticals, Loughborough, UK.

      Culture and Labeling of Cells

      Swiss 3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% newborn calf serum at 37°C in a humidified atmosphere of 5% (v/v) CO2 in air, all cells were quiesced in 2% (v/v) serum-containing medium for 24 h, with radiolabel as appropriate, prior to experiment. The cells were labeled in medium containing 2% (v/v) serum with 1 μCi/ml [3H]inositol for PI-PLC experiments, 4 μCi/ml [3H]palmitate for PLD experiments, and 1 μCi/ml [3H]arachidonate for the PLA2 experiments; in each case, labeling was for 24 h and the cells were grown in 24-well plates. For the measurement of 3-phosphorylated lipids, confluent cells were washed twice with phosphate-free Dulbecco's modified Eagle's medium containing 0.1% (w/v) fatty acid-free bovine serum albumin and 20 mM Hepes, pH 7.4, and subsequently labeled for 90 min in 0.25 mCi/ml [32P]phosphate in the same medium.

      Phospholipase Assays in Intact Cells

      The measurement of PI-PLC activity as the stimulated accumulation of [3H]inositol phosphates in the presence of 10 mM LiCl, PLD activity as the accumulation of [3H]phosphatidylbutanol in the presence of 30 mM butanol, and PLA2 activity by the stimulated increase in [3H]arachidonate generation were as described previously(
      • Cook S.J.
      • Briscoe C.P.
      • Wakelam M.J.O.
      ,
      • Currie S.
      • Smith G.L.
      • Crichton C.A.
      • Jackson C.G.
      • Hallam C.
      • Wakelam M.J.O.
      ,
      • Plevin R.
      • Palmer S.
      • Gardner S.D.
      • Wakelam M.J.O.
      ).

      PI 3-Kinase Activity

      Confluent quiesced cells were washed in Hanks' buffered saline containing 10 mM glucose and 0.1% bovine serum albumin, stimulated for the required period of time, washed with ice-cold phosphate-buffered saline, and then lysed in 1% (w/v) Nonidet P-40, 10% (v/v) glycerol, 20 mM Tris-HCl (pH 8), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.5 mM Na3VO4, containing 10 μg/ml leupeptin and 0.2 mM phenylmethanesulfonyl fluoride. The lysates were cleared by centrifugation, and their protein concentrations were determined by the BCA method (Sigma). A solution containing 100 μg of protein was incubated with 15 μl of anti-p85 PI 3-kinase subunit antibody (U13 hybridoma supernatant) for 2 h at 4°C, and 20 μl of 50% (v/v) Protein G-Sepharose was then added for 2 h at 4°C. The immunoprecipitates were washed successively at 4°C as follows: 2 × 1 ml of lysis buffer, 2 × 1 ml of 0.5 M LiCl, 0.1 M Tris-HCl, pH 8.0, 1 × 1 ml of 0.1 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.6, and finally 1 × 1 ml of 5 mM MgCl2, 20 mM Hepes, pH 7.4. 20 μl of substrate (3 mg/ml phosphatidylinositol in 1% cholate) was added to each tube and allowed to stand for 10 min at room temperature; PI 3-kinase activity was then determined by the method of Jackson et al.(
      • Jackson T.R.
      • Stephens L.R.
      • Hawkins P.T.
      ). For studies using DMV, the immunoprecipitate and phosphatidylinositol were incubated with inhibitor for 5 min at room temperature prior to addition of [γ-32P]ATP to start the reaction.

      3-Phosphorylated Lipids

      Cells labeled with [32P]phosphate were washed twice with phosphate-free Dulbecco's modified Eagle's medium and then stimulated. Incubations were terminated by the addition of 4 ml of ice-cold methanol, 1 M HCl (3:1, v/v). The cells were scraped from the flask, and total lipids were extracted. They were then deacylated with methylamine and deglycerated using mild sodium periodate treatment (10 mM for 20 min in the dark)(
      • Wong N.S.
      • Barker C.J.
      • Morris A.J.
      • Craxton A.
      • Kirk C.J.
      • Michell R.H.
      ). The lipid-derived head groups were reconstituted in 1 ml of water, dried in vacuo, and reconstituted in an additional 1 ml of water. The phosphate-containing head groups were then separated by anion exchange HPLC on a 25-cm Partisphere 5 SAX column eluted with a linear gradient of ammonium dihydrogen phosphate (0-0.5 M, pH 3.8 with phosphoric acid) in water at 1 ml/min over 110 min. Fractions were collected every 0.5 min, and 32P radioactivity was determined by static liquid scintillation spectrometry. The retention times of radioactive peaks were compared to authentic external 3H standards of Ins-1-P, Ins(1,4)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, and 32Pi. Standards, run every 5th injection, were detected by on-line liquid scintillation spectrometry (Radiomatic A500, Canberra Packard). The retention times of peaks and external standards throughout all the runs varied by less than 1 min.

      Phospholipase A2Activity

      In vitro phospholipase A2 assays utilized a bilayer assay with 7.4 μM 1-stearoyl,2-[14C]arachidonyl phosphatidylcholine (3.45 Ci/mol), 0.24 μM phosphatidylinositol 4,5-bisphosphate, 1.7 μM diacylglycerol, in a 50 mM Tris-HCl pH 7.4 buffer containing 10 μM CaCl2 and 100 mM KCl. SF9 cell-expressed human cytosolic phospholipase A2 or Swiss 3T3 cell lysates (prepared in 20 mM Hepes, pH 7.4, 2.5 mM EGTA, 150 nM CaCl2) were added, and reactions were performed at 37°C for 10 min. The released arachidonate was extracted into hexane, 0.4% acetic acid, and radioactivity was determined by liquid scintillation spectrometry.

      Preparation of Demethoxyviridin (DMV) and Wortmannin

      DMV and wortmannin were dissolved in dimethyl sulfoxide. For the phospholipase studies using whole cells, the inhibitors were diluted in buffer immediately prior to addition in the assay. A 5-min preincubation with the inhibitors was used prior to the addition of agonist. The level of dimethyl sulfoxide was below 0.1% (v/v) which had no observable effect in any assay.

      RESULTS AND DISCUSSION

      In the Swiss 3T3 fibroblast cell line, bombesin stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (PI-PLC) and phosphatidylcholine by phospholipase D (PLD) and phospholipase A2 (PLA2) (
      • Wakelam M.J.O.
      • Briscoe C.P.
      • Stewart A.
      • Pettitt T.R.
      • Cross M.J.
      • Paul A.
      • Yule J.M.
      • Gardner S.D.
      • Hodgkin M.
      ). Bombesin has been reported to have no effect upon the activation of PI 3-kinase in Swiss 3T3 cells(
      • Jackson T.R.
      • Stephens L.R.
      • Hawkins P.T.
      ); therefore, insulin was used to stimulate PI 3-kinase activity.
      DMV inhibited bombesin-stimulated PI-PLC activity, as determined by inositol phosphate accumulation, in Swiss 3T3 cells with an IC40 value of approximately 30 nM (Table 1). The wortmannin analogue also inhibited phosphatidylcholine-PLD activity, as measured by phosphatidylalcohol generation, with an IC40 of approximately 50 nM, although maximum inhibition was never greater than 60% (Table 1). Similar potency values were reported for fMLP-stimulated neutrophils (
      • Bonser R.W.
      • Thompson N.T.
      • Randall R.W.
      • Tateson J.E.
      • Spacey G.D.
      • Hodson H.F.
      • Garland L.G.
      ) except that the sensitivity of the PLC response to DMV was greater in the 3T3 cells, and, in the neutrophil, the PLD response was completely inhibited. These differences may reflect different phospholipase isoforms in the different cell types.
      DMV was an extremely potent inhibitor of bombesin-stimulated phosphatidylcholine-PLA2 activity in Swiss 3T3 cells. Fig. 1 shows that inhibition was observable at concentrations as low as 0.1 nM, and that the IC40 was approximately 2 nM. This inhibition of PLA2 was also observed with wortmannin (Fig. 1), demonstrating the potency of this class of compounds as PLA2 inhibitors. Inhibition of PLA2 was also observed in an in vitro assay. However, inhibition was only partial with approximately 30% inhibition being detected at 0.1 nM (Table 2). This inhibition was dependent upon the inclusion of PIP2 in the assay, which has been reported to enhance phospholipase A2 activity(
      • Leslie C.C.
      • Channon J.Y.
      ). Incubation of a Swiss 3T3 cell lysate or an anti-PLA2 immunoprecipitate with wortmannin for 10 min prior to addition of the lipid substrate failed to induce any PLA2 inhibition in vitro.
      Figure thumbnail gr1
      Figure 1Inhibition of bombesin-stimulated phospholipase A2 in Swiss 3T3 cells by increasing concentrations of DMV (A) and wortmannin (B). Phospholipase A2 was determined as described under “Experimental Procedures,” the results are means ± S.D. where n = 3 and from single experiments typical of three. For the experiments shown, basal and maximum stimulated disintegrations/min in arachidonate values were 190 ± 60 and 600 ± 40 (A) and 860 ± 93 and 2354 ± 252 (B).
      Bombesin has been reported not to stimulate PI 3-kinase activity in Swiss 3T3 cells(
      • Jackson T.R.
      • Stephens L.R.
      • Hawkins P.T.
      ), and we have confirmed this observation both by assaying the activity of immunoprecipitated kinase (Fig. 2) and by determining changes in PIP3 levels (see Fig. 3). Therefore, we examined the effects of wortmannin and DMV in insulin-stimulated cells. Fig. 2 shows that insulin and platelet-derived growth factor, but not bombesin, increase the PI 3-kinase activity in anti-p85 immunoprecipitates. In the in vitro PI 3-kinase assay, where the insulin-stimulated kinase was immunoprecipitated using an anti-p85 subunit antibody, DMV inhibited enzyme activity with an IC40 value between 1 and 2 nM (Fig. 2). This is similar to the value of 3.4 nM previously reported (
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ) and compares with reported IC40 values between 1 and 10 nM for PI 3-kinase inhibition by wortmannin(
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ).
      Figure thumbnail gr2
      Figure 2Inhibition of PI 3-kinase activity by DMV. PI 3-kinase activity was determined in anti-p85 immunoprecipitates prepared from insulin-stimulated cells. Increasing concentrations of DMV were added prior to the addition of the [32P]ATP. Results are presented as percent inhibition ± S.D. of the activity in the absence of DMV which was 0.0129 ± 0.0005 pmol of phosphate incorporated into PI 3-P/min/mg of cellular protein. The inset shows the generation of PI 3-P in immunoprecipitates prepared from cells stimulated with insulin, plateletderived growth factor (PDGF) or bombesin.
      Figure thumbnail gr3
      Figure 3The generation of PIP3 in Swiss 3T3 cells. 32P-Labeled Swiss 3T3 cells were stimulated with insulin or bombesin ± DMV or ± wortmannin, the lipids were isolated, deacylated, and deglycerated, and the Ins(1,3,4,5)P4 fraction which is generated from PIP3 was isolated by HPLC. A, generation of PIP3 in response to insulin (□) and bombesin (○). B, generation of PIP3 in response to insulin - DMV (□) or + 1 μM DMV (○). C, generation of PIP3 in response to insulin - wortmannin (□) or + 1 μM wortmannin (○).
      Inhibition of PI 3-kinase activity in anti-p85 immunoprecipitates where phosphatidylinositol rather than phosphatidylinositol 4,5-bisphosphate is presented as the substrate can only give an indication of the effects of a compound in the whole cell. The only definitive measure of effects upon in vivo PI 3-kinase activity is to examine changes in the cellular levels of the 3-phosphorylated lipids. Therefore, lipids were isolated from control and stimulated [32P]Pi-labeled cells, deacylated, and deglycerated, and the generated water-soluble phosphates were separated by ion exchange HPLC. Fig. 3 shows that insulin stimulates PIP3 generation in Swiss 3T3 cells, the figure also shows that bombesin did not stimulate PIP3 generation in agreement with Jackson et al.(
      • Jackson T.R.
      • Stephens L.R.
      • Hawkins P.T.
      ). Insulin-stimulated PIP3 generation was inhibited by both DMV and wortmannin (Fig. 3) with DMV being slightly more potent with an approximate IC40 of 10 nM compared to a value greater than 10 nM for wortmannin (Fig. 4). The more potent effect of DMV compared to wortmannin upon 3-phosphorylated lipid generation is similar to that reported for effects upon PI 3-kinase activity in immunoprecipitates(
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ).
      Figure thumbnail gr4
      Figure 4Inhibition of PIP3 generation by DMV and wortmannin. Cells were treated as in but with increasing concentrations of DMV or wortmannin. Results are expressed as percent insulin-stimulated PIP3 generation.
      The data presented in Fig. 1 clearly define wortmannin and DMV as potent and complete inhibitors of stimulated phospholipase A2 activity in Swiss 3T3 cells. However, in vitro inhibition was incomplete when the enzyme was activated by PIP2, diacylglycerol, and calcium (Table 2). The contrast between the in vitro and in vivo inhibition of phospholipase A2 suggests a complex mode of action for wortmannin and DMV. It is possible that they interact directly with the phospholipase and that the incomplete effect was due to a nonphysiological conformation taken up by the protein in the test tube. An alternative possibility is that inhibition may be due to an interaction with a protein which regulates phospholipase A2 activity in the stimulated cell. Should this be the case, it is unlikely that the protein involved is PI 3-kinase since in Swiss 3T3 cells bombesin activates phospholipase A2 without stimulating PI 3-kinase. Additionally, when stimulated phospholipase A2 and PI 3-kinase activities are inhibited in vivo by wortmannin and DMV, both compounds are more potent against the release of arachidonate than the generation of 3-phosphorylated lipids.
      The potency of DMV was greater than that of wortmannin for each of the enzymes examined. However, this difference is offset by the reduced stability of DMV when compared to wortmannin. Indeed, when DMV was incubated in a physiological buffer at 37°C for 10 min prior to addition to cells, it was rendered inactive (results not shown). In view of the instability of these compounds in aqueous solution, their experimental and therapeutic use will remain limited until the development of more stable analogues.
      The results presented in this paper demonstrate that wortmannin and its structural analogue DMV are not specific or selective inhibitors of PI 3-kinase in intact cells. Inhibition of other enzymes by wortmannin, e.g. phospholipase D, myosin light chain kinase, has been reported previously; however, the concentrations required were much greater than those reported to inhibit PI 3-kinase. The inhibition of bombesin-stimulated phospholipase A2 activity in Swiss 3T3 cells is apparent at subnanomolar concentrations and has an IC40 (approximately 5 nM) which is similar to that previously reported for the inhibition of PI 3-kinase in vitro and lower than that determined for the inhibition of insulin-stimulated PIP3 generation (Fig. 4). This value is also similar to that measured for the inhibition of PI 3-kinase activity in anti-p85 immunoprecipitates from Swiss 3T3 cells (Fig. 2). The proposed specificity of action of wortmannin upon PI 3-kinase has been supported by the demonstration of wortmannin binding to a 110-kDa protein in an SDS-polyacrylamide gel and to the 110-kDa subunit of PI 3-kinase(
      • Thelen M.
      • Wymann M.P.
      • Langen H.
      ). However, the 85-kDa phospholipase A2 migrates at 110 kDa on SDS-polyacrylamide gel electrophoresis, highlighting the possibility that one of the bands shown to bind [3H]wortmannin could be phospholipase A2.
      Accordingly, where a role for PI 3-kinase in a particular pathway or cellular function has been assigned on the basis purely of its inhibition by wortmannin, the conclusions may be incorrect and a role for other signaling pathways such as phospholipase A2 must be considered. A clear example of this is in the regulation of neutrophil responses where a central role for PI 3-kinase has been proposed on the basis of wortmannin sensitivity(
      • Arcaro A.
      • Wymann M.P.
      ). Other studies have suggested a role for the phospholipase A2 product arachidonate in the activation of neutrophils(
      • Dana R.
      • Malech H.L.
      • Levy R.
      ), and, thus, the nonselective action of wortmannin would suggest a need for a reassessment. Many papers reporting the use of wortmannin have utilized concentrations of 100 nM and above; however, at these levels, as the results presented here demonstrate, there will be inhibition of PLC, PLD, PLA2, and PI 3-kinase. Despite these caveats, it is clear that in many cases where wortmannin has been used to identify a role for PI 3-kinase the conclusions are correct, but these studies have utilized further experimental evidence, e.g. dominant negative p85 subunit transfections(
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ), to confirm their findings. In other cases, it could be phospholipase A2 that is the enzyme of importance.

      Acknowledgments

      We thank Dr. C. G. Jackson, Fisons plc, Loughborough, UK, for his assistance with the in vitro phospholipase A2 assays.

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