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Structural and Biochemical Evaluation of the Interaction of the Phosphatidylinositol 3-Kinase p85α Src Homology 2 Domains with Phosphoinositides and Inositol Polyphosphates*

Open AccessPublished:May 28, 1999DOI:https://doi.org/10.1074/jbc.274.22.15678
      Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85α subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85α SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241–6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85α SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85α SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85α. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85α SH2 domains.
      Src homology 2 (SH2)
      The abbreviations used are: SH2, Src homology 2; di-C6-PIP3, rac-dihexanoylphosphatidyl-d/l-myo-inositol 3,4,5-trisphosphate; HSQC, heteronuclear single quantum coherence; GST, glutathione S-transferase; Ins, d-myo-inositol; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI, phosphoinositide; Ptd, phosphatidyl; Tyr(P), phosphotyrosine; PBS, phosphate-buffered saline
      1The abbreviations used are: SH2, Src homology 2; di-C6-PIP3, rac-dihexanoylphosphatidyl-d/l-myo-inositol 3,4,5-trisphosphate; HSQC, heteronuclear single quantum coherence; GST, glutathione S-transferase; Ins, d-myo-inositol; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI, phosphoinositide; Ptd, phosphatidyl; Tyr(P), phosphotyrosine; PBS, phosphate-buffered saline
      domains are conserved, noncatalytic sequences of about 100 amino acids that adopt a common three-dimensional fold. These domains are commonly found in signal transduction proteins that regulate a variety of cellular processes, such as phospholipid metabolism, protein phosphorylation, and dephosphorylation, protein trafficking, and gene expression (
      • Pawson T.
      • Schlessinger J.
      ). SH2 domains mediate high affinity binding to phosphotyrosine (Tyr(P)) residues in proteins such as activated membrane receptors and cytosolic adaptor proteins. Three to five amino acids C-terminal to the target Tyr(P) residue bind to a groove on the SH2 domain surface and confer the specificity of interaction that is necessary to avoid aberrant signaling (
      • Piccione E.
      • Case R.D.
      • Domchek S.M.
      • Hu P.
      • Chaudhuri M.
      • Backer J.M.
      • Schlessinger J.
      • Shoelson S.E.
      ,
      • Panayotou G.
      • Gish G.
      • End P.
      • Truong O.
      • Gout I.
      • Dhand R.
      • Fry M.J.
      • Hiles I.
      • Pawson T.
      • Waterfield M.D.
      ). The role of SH2 domains in Tyr(P)-dependent protein recruitment is critical for the assembly of active complexes of signaling proteins (
      • Pawson T.
      ,
      • Panayotou G.
      • Waterfield M.D.
      ).
      The p85α/p110α Class IA phosphoinositide 3-OH kinase (PI 3-kinase) contains two SH2 domains in its regulatory p85α subunit (
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ). Upon cell stimulation, the SH2 domains bind to tyrosine-phosphorylated, membrane-bound growth factor receptors. As a result, p85α/p110α is recruited to the vicinity of its phosphoinositide substrates (
      • Kapeller R.
      • Cantley L.C.
      ). The p110α PI 3-kinase activity then produces 3′-phosphorylated phosphoinositides. In this manner, p85α/p110α mediates a dramatic increase in the basal concentration of phosphatidylinositol 3,4,5-trisphosphate (PtdIns (3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate in the plasma membrane shortly after cell stimulation (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ,
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ).
      It is now clear that p85α/p110α phosphorylates phosphoinositides to produce second messengers, which control the membrane recruitment and activation of numerous signaling proteins, notably including regulators of apoptosis (
      • Downward J.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Toker A.
      • Cantley L.C.
      ). Many of the target proteins of these second messengers contain pleckstrin homology (PH) domains and have been shown to bind specifically to PtdIns (3,4,5)P3 and/or phosphatidylinositol 3,4-bisphosphate in vitro and/orin vivo (
      • Frech M.
      • Andjelkovic M.
      • Ingley E.
      • Reddy K.K.
      • Falck J.R.
      • Hemmings B.A.
      ,
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.J.
      • Reese C.B.
      • Cohen P.
      ,
      • Isakoff S.J.
      • Cardozo T.
      • Andreev J.
      • Li Z.
      • Ferguson K.M.
      • Abagyan R.
      • Lemmon M.A.
      • Aronheim A.
      • Skolnik E.Y.
      ,
      • Falasca M.
      • Logan S.K.
      • Lehto V.P.
      • Baccante G.
      • Lemmon M.A.
      • Schlessinger J.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • James S.R.
      • Downes C.P.
      • Gigg R.
      • Grove S.J.
      • Holmes A.B.
      • Alessi D.R.
      ,
      • Stephens L.
      • Anderson K.
      • Stokoe D.
      • Erdjument-Bromage H.
      • Painter G.F.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • McCormick F.
      • Tempst P.
      • Coadwell J.
      • Hawkins P.T.
      ). Indeed, numerous interactions between distinct phosphoinositides and PH domains have now been demonstrated and appear to be essential for the function of various cytoskeletal or signal transduction proteins (reviewed in Ref.
      • Bottomley M.J.
      • Salim K.
      • Panayotou G.
      ).
      However, a few reports have suggested that PH domains are not unique targets of second messenger phosphoinositides produced by PI 3-kinase. It has been proposed that PtdIns (3,4,5)P3 can also bind to SH2 domains. These proposals followed the observation of an inverse correlation between the amount of p85α/p110α associated with tyrosine-phosphorylated proteins and the level of PI 3-kinase lipid products present in the cell (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ). Consequently, a model was proposed in which the PtdIns (3,4,5)P3 produced by PI 3-kinase activation could compete for Tyr(P)-bound p85α SH2 domains and directly result in the relocalization of p85α/p110α at the plasma membrane. Similarly, the production of PtdIns (3,4,5)P3 may regulate additional proteins such as the tyrosine kinase Src and phospholipase C γ-1 (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ,
      • Bae Y.S.
      • Cantley L.G.
      • Chen C.S.
      • Kim S.R.
      • Kwon K.S.
      • Rhee S.G.
      ). It has been shown that in vitro the p85α C-terminal SH2 (C-SH2) domain can bind to PtdIns (3,4,5)P3 (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ). However, it was not demonstrated that recombinant p85α C-SH2 domain can act as a faithful model of p85α activity. Indeed, we noted with intrigue that the reported interaction of PtdIns (3,4,5)P3 with the p85α C-SH2 domain could be significantly inhibited by phenyl phosphate, but that such inhibition was not observed in the case of the reported interaction between PtdIns (3,4,5)P3 and full-length p85α (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ). The work presented herein arose from our attempts to clarify these apparently conflicting observations and to resolve certain issues central to these models describing distinct phosphoinositide-SH2 domain interactions.
      Prerequisites for the models above are that the SH2 domains that interact with phosphoinositides must (a) demonstrate a significant binding affinity for these ligands and (b) discriminate between the numerous phosphoinositides present in the plasma membrane. Because we had access to appropriate reagents and assay techniques, we set out to determine whether the p85α SH2 domains indeed display clear binding specificity and affinity for distinct phosphoinositides. We report the first high resolution structural studies of model phosphoinositide-SH2 domain interactions, which we performed by nuclear magnetic resonance (NMR) spectroscopy. We also employed two sensitive biosensor assays; one to measure interactions between proteins and phospholipid bilayers containing phosphoinositides and another to measure directly the competition between Tyr(P)-containing ligands and phosphoinositides for binding to SH2 domains. In addition, we report in vivo studies in which we sought a correlation between the association of p85α with activated growth factor receptors or tyrosine-phosphorylated proteins and the intracellular level of PI 3-kinase products.

      EXPERIMENTAL PROCEDURES

      Protein Expression and Purification

      The p85α C-SH2 domain (amino acids Glu-614–Arg-724) was expressed and purified as described previously (
      • Siegal G.
      • Davis B.
      • Kristensen S.M.
      • Sankar A.
      • Linacre J.
      • Stein R.C.
      • Panayotou G.
      • Waterfield M.D.
      • Driscoll P.C.
      ). A pGEX-2T plasmid encoding glutathione S-transferase (GST) (Amersham Pharmacia Biotech) fused to the p85α N-terminal SH2 (N-SH2) domain (amino acids Pro-314—Tyr-431) was kindly provided by Dr. R. Stein (Ludwig Institute for Cancer Research, London), and the protein was prepared and purified essentially as described previously (
      • Booker G.W.
      • Breeze A.L.
      • Downing A.K.
      • Panayotou G.
      • Gout I.
      • Waterfield M.D.
      • Campbell I.D.
      ,
      • Smith D.B.
      • Johnson K.S.
      ). TransformedEscherichia coli BL21 (DE3) cells were grown at 37 °C to a culture density A 600 ∼ 0.8. Protein expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a concentration of 0.2 mm. Cells were harvested after 4 h, resuspended in phosphate-buffered saline (PBS), and lysed by a French press. For NMR spectroscopy and biosensor Tyr(P) competition assays, the GST moiety was removed by thrombin cleavage. In contrast, intact fusion protein was used in the liposome binding assays. Further protein purification was accomplished by gel filtration in 50 mmTris-HCl, pH 7.5, 50 mm NaCl, 0.02% NaN3.15N-isotopically enriched samples for NMR spectroscopy were prepared as above except that cells were grown in a minimal M9 medium using 15NH4Cl (Isotec Inc.) as the sole nitrogen source. NMR samples were prepared in 50 mmdeuterated Tris-HCl (Cambridge Isotope Laboratories), pH 7.5, 50 mm NaCl, 2 mm dithiothreitol (for C-SH2 only), 10% (v/v) D2O.

      Ligands Tested for Binding to p85α SH2 Domains

      The water-soluble ligands tested includedd-myo-inositol 1,4,5-trisphosphate (d-Ins (1,4,5)P3), l-Ins (1,4,5)P3,l-α-glycerophospho-d-myo-inositol 4,5-bisphosphate, and phenyl phosphate obtained from Sigma;d-Ins (1,3,4,5)P4 and l-Ins (1,3,4,5)P4, synthesized and purified by ion-exchange chromatography as published (
      • Riley A.M.
      • Mahon M.F.
      • Potter B.V.L.
      ,
      • Potter B.V.L.
      • Lampe D.
      );rac-dihexanoylphosphatidyl-d/l-myo-inositol 3,4,5-trisphosphate (di-C6-PIP3), synthesized using published techniques (
      • Gaffney P.R.J.
      • Reese C.B.
      ); and the nine-residue phosphopeptide SVDY(P)VPMLD (Y(P) is phosphotyrosine) (Genosys Ltd.). The phospholipids tested included PtdIns, PtdIns (
      • Pawson T.
      )P, and PtdIns (
      • Pawson T.
      ,
      • Panayotou G.
      • Waterfield M.D.
      )P2 (obtained from Lipid Products, Redhill, Surrey, UK) and PtdIns (3,4,5)P3, which was prepared as described previously (
      • Desai T.
      • Gigg J.
      • Gigg R.
      • Martin-Zamora E.
      ) and kindly provided by Professor R. Gigg. The additional liposome components described were purchased from Sigma.

      NMR Spectroscopy Experiments

      For NMR spectroscopy, SH2 domain samples were prepared at 0.5 mm concentration in 600 μl. Interactions were monitored via spectra recorded during titration of the SH2 domain with 1.5-μl aliquots of test ligand (prepared at 20 mm in 20 mm Tris-HCl, pH 7.5, 50 mm NaCl). NMR experiments were performed at 15 °C on a Varian UNITY-plus spectrometer operating at a 1H frequency of 600 MHz. Two-dimensional gradient enhanced sensitivity15N-1H heteronuclear single quantum coherence (HSQC) experiments were performed using a pulse sequence kindly provided by Professor L. E. Kay (
      • Zhang O.W.
      • Kay L.E.
      • Olivier J.P.
      • Forman-Kay J.D.
      ). Sign discrimination in t1 was achieved using the States-time-proportional phase incrementation method. The HSQC spectra were acquired with 16 scans, 64 increments in t1, and sweep widths of 10000 Hz (1H) and 2400 Hz (15N). Three-dimensional15N-1H HSQC total correlation spectroscopy and nuclear Overhauser effect spectroscopy experiments were recorded to verify the published resonance assignments for the N-SH2 domain (
      • Hensmann M.
      • Booker G.W.
      • Panayotou G.
      • Boyd J.
      • Linacre J.
      • Waterfield M.
      • Campbell I.D.
      ,
      • Gunther U.L.
      • Liu Y.
      • Sanford D.
      • Bachovchin W.W.
      • Schaffhausen B.
      ).
      NMR data were processed using NMRpipe software (
      • Delaglio F.
      • Grzesiek S.
      • Vuister G.W.
      • Zhu G.
      • Pfeifer J.
      • Bax A.
      ). Phase-shifted, sine-squared shaped weighting functions and zero-filling were applied before Fourier transformation. NMR spectra were analyzed using XEASY (
      • Bartels C.H.
      • Xia T.
      • Billeter M.
      • Güntert P.
      • Wüthrich K.
      • Bartels T.-H.X.
      • Billeter M.
      • Güntert P.
      • Wüthrich K.
      ) and AZARA software (AZARA v.II, W. Boucher, Department of Biochemistry, University of Cambridge, UK).

      Biosensor Experiments

      Preparation of Liposomes for Biosensor Studies

      Large unilamellar liposomes with a phospholipid composition approximating the inner leaflet of the plasma membrane were prepared as described previously (
      • Salim K.
      • Bottomley M.J.
      • Querfurth E.
      • Zvelebil M.J.
      • Gout I.
      • Scaife R.
      • Margolis R.L.
      • Gigg R.
      • Smith C.I.
      • Driscoll P.C.
      • Waterfield M.D.
      • Panayotou G.
      ). By weight, the liposomes contained 30% phosphatidylcholine, 15% sphingomyelin, 20% cholesterol, 15% phosphatidylethanolamine, 10% phosphatidylserine, and 10% of the phosphoinositide to be tested. Liposomes were used in 10 mmHEPES, pH 7.4, 80 mm KCl, 15 mm NaCl, 0.7 mm NaH2PO4, 1 mm EGTA, 0.466 mm CaCl2, 2.1 mmMgCl2.

      Liposome Binding Studies Using the Biosensor

      The basic operating procedures of the surface plasmon resonance BIAcore biosensor (BIACORE AB, Uppsala) have been published (
      • Jonsson U.
      • Fagerstam L.
      • Ivarsson B.
      • Johnsson B.
      • Karlsson R.
      • Lundh K.
      • Lofas S.
      • Persson B.
      • Roos H.
      • Ronnberg I.
      • Sjolander S.
      • Stenberg E.
      • Stahlberg R.
      • Urbaniczky C.
      • Ostlin H.
      • Malmqvist M.
      ). The ability of immobilized GST fusion SH2 domains to bind to phosphoinositides in liposomes was examined using the method described previously (
      • Salim K.
      • Bottomley M.J.
      • Querfurth E.
      • Zvelebil M.J.
      • Gout I.
      • Scaife R.
      • Margolis R.L.
      • Gigg R.
      • Smith C.I.
      • Driscoll P.C.
      • Waterfield M.D.
      • Panayotou G.
      ).

      Phosphoinositide Phosphotyrosine Peptide Competition Studies Using the Biosensor

      A precoated streptavidin biosensor chip (SA-5, BIACORE AB) was used to immobilize the N-terminal-biotinylated, Tyr(P) peptide N-biotinyl-DMSKDESVDY(P)VPMLDMK (Y(P) is phosphotyrosine). The Tyr(P) peptide was loaded in the buffer used throughout the assay: 20 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, 0.005% Tween 20, and 4 mm dithiothreitol. Solutions of 0.5 μm N- or C-SH2 domain were injected over the surface at a flow rate of 5 μl/min at 25 °C, and the maximum response was recorded. Competition experiments were performed by incubating the SH2 domains with a competitor ligand before injection. Efficacious competition resulted in a diminished response. Between injections, protein remaining bound to the biosensor was removed by a 5-μl pulse of 0.05% SDS solution.
      Data analysis of the competition measurements was performed with the BIAcore-2000 software package (BIACORE AB). In calculations of the half-maximal inhibitory constants (IC50), the control response from injection of SH2 domain over the biosensor surface lacking the Tyr(P) peptide was subtracted from the experimental response to yield the corrected response, R. Data was plotted as corrected response units versus concentration of competitor and were fitted to the following equation using a nonlinear least-squares analysis: R =R max/[1 + (C/IC50) P], whereR max is the response for SH2 binding in the absence of competitor, C is the concentration of competitor, and P is the Hill coefficient.

      In Vivo Assays

      Cell Culture

      Mouse NIH3T3 fibroblasts were grown at 37 °C in a humidified atmosphere containing 10% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Life Technologies, Inc.) and penicillin/streptomycin (Life Technologies, Inc.). The cells were grown to confluence in 150-mm dishes and serum-starved in Dulbecco's modified Eagle's medium containing 0.5% (v/v) heat-inactivated fetal calf serum for 16 h.

      Immunoprecipitations

      Cells grown on 150-mm dishes were incubated for 15 min at 37 °C with wortmannin (100 nm in Me2SO) or an equivalent volume of Me2SO and subsequently stimulated with recombinant PDGF-β (100 nm) for 10 min at 37 °C. The dishes were then placed on ice, washed once in ice-cold PBS buffer (Life Technologies, Inc.), and lysed for 20 min on ice in 1 ml of lysis buffer (20 mm HEPES/NaOH, pH 7.4, 150 mm NaCl, 1%(w/v) Triton X-100, 2 mm EDTA, 10 mm NaF, 10 mmNa2HPO4, 10% (w/v) glycerol, 1 mmphenylmethylsulfonyl fluoride, 5 mm benzamidine, 7 mm diisopropylphosphofluoridate, 1 mm1-chloro-3-tosylamido-7-amino-2-heptanone, 20 μmleupeptin, 18 μm pepstatin, 21 μg/ml aprotinin, 2 mm dithiothreitol, 1 mmNa3VO4, 10 mm β-glycerophosphate, 1 mm tetrasodium pyrophosphate, 1 mm sodium molybdate). The cells were then scraped from the dishes and centrifuged for 20 min at 15,000 × g and 4 °C. The supernatant was collected and incubated with the relevant antibody with constant agitation at 4 °C for 2 h. Protein G-Sepharose CL-4B (Amersham Pharmacia Biotech) at 10 μl of bead slurry/sample was then added, and the incubation continued for 1 h at 4 °C on a wheel. The immunoprecipitates were washed three times in lysis buffer and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting or assayed for PI 3-kinase activity.

      PI 3-Kinase Assays

      PI 3-kinase activity was assayed on immunoprecipitates resuspended in 25 μl of 2× kinase buffer (40 mm Tris-HCl, pH 7.4, 200 mm NaCl, 2 mm dithiothreitol). PtdIns stored in CHCl3solution was dried, sonicated for 15 min in 50 mm Tris-HCl, pH 7.4, and added to a concentration of 0.2 mg/ml. The reactions (50-μl final volume) were started by the addition of 40 mm ATP, 10 μCi of [γ-32P ]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech), and 3.5 mmMgCl2. Kinase reactions were stopped by the addition of 100 μl of 1 m HCl. For phospholipid extraction, 200 μl of 1:1 (v/v) CHCl3/CH3OH was added. The organic phase was collected and re-extracted with 40 μl of 1:1 (v/v) 1n HCl/CH3OH. The samples were then dried, resuspended in 30 μl of CHCl3/ CH3OH 1:1 (v/v), and spotted onto prechanneled silica gel 60 TLC plates (Whatman) that had been pretreated in 1% (w/v) oxalic acid, 1 mmEDTA, H2O/CH3OH (60:40 (v/v) and baked for 15 min at 110 °C. The plates were developed in propanol, 2m acetic acid 65:35 (v/v), and the radioactive spots were quantified using a PhosphorImager (Molecular Dynamics).

      Western Blotting

      After SDS- polyacrylamide gel electrophoresis, polyacrylamide gels (7.5%) were transferred onto polyvinylidene difluoride membranes (Gelman Sciences) using a semi-dry blotter (Amersham Pharmacia Biotech). The membranes were then blocked for 1 h in PBS buffer containing 3% (w/v) nonfat dry milk, 0.1% (w/v) polyethylene glycol 20000. The relevant primary antibodies were diluted in PBS buffer and 0.05% (w/v) Tween 20 (PBS/Tween) and incubated with the membranes for 2 h. After extensive washing in PBS/Tween, the blots were incubated for 1 h with goat anti-mouse or anti-rabbit antibodies coupled to horseradish peroxidase (Dako) at 1:2000 dilution. The membranes were then washed in PBS/Tween, and the bands were detected using ECL (Amersham Pharmacia Biotech).

      DISCUSSION

      We sought to verify whether the products of PI 3-kinase activity, 3′-phosphorylated phosphoinositides, can interact with the SH2 domains derived from the p85α regulatory subunit of PI 3-kinase itself. Such interactions have been proposed as competitors of the association of PI 3-kinase with tyrosine-phosphorylated proteins and as regulators of other SH2 domain-containing proteins, e.g. Src and phospholipase C γ-1 (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ,
      • Bae Y.S.
      • Cantley L.G.
      • Chen C.S.
      • Kim S.R.
      • Kwon K.S.
      • Rhee S.G.
      ). Although the SH2 domain-mediated phosphoinositide-dependent regulation of p85α/p110α, Src, or phospholipase C activity has interesting implications, it is a model yet to be clearly established.
      Therefore, our primary experimental aim was to discover whether the p85α N- and C-SH2 domains can bind to distinct phosphoinositides with high affinity, specificity, and stereoselectivity. Furthermore, we investigated whether the well defined interactions between the p85α SH2 domains and Tyr(P)-containing ligands could be competed by phosphoinositides or inositol polyphosphates. We tested both phosphoinositides and inositol polyphosphates as potential ligands of SH2 domains. Inositol polyphosphates were in part used as conveniently water-soluble analogues of the head groups of phosphoinositides but may also represent physiological ligands. In our opinion this usage of inositol polyphosphates is valid because protein-phosphoinositide interactions appear to be predominantly governed by the charge status, phosphorylation positions, and stereochemistry of the inositol ring (
      • Rameh L.E.
      • Arvidsson A.
      • Carraway III, K.L.
      • Couvillon A.D.
      • Rathbun G.
      • Crompton A.
      • VanRenterghem B.
      • Czech M.P.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Wang D.S.
      • Chen C.S.
      • Cantley L.C.
      ,
      • Wang D.-S.
      • Hsu A.-L.
      • Song X.
      • Chiou C.-M.
      • Chen C.-S.
      ,
      • Kavran J.M.
      • Klein D.E.
      • Lee A.
      • Falasca M.
      • Isakoff S.J.
      • Skolnik E.Y.
      • Lemmon M.A.
      ). For example, it has been demonstrated thatd-Ins (1,4,5)P3 can functionally replace PtdIns (
      • Pawson T.
      ,
      • Panayotou G.
      • Waterfield M.D.
      )P2 in the activation of the dynamin GTPase (
      • Salim K.
      • Bottomley M.J.
      • Querfurth E.
      • Zvelebil M.J.
      • Gout I.
      • Scaife R.
      • Margolis R.L.
      • Gigg R.
      • Smith C.I.
      • Driscoll P.C.
      • Waterfield M.D.
      • Panayotou G.
      ) and that the Btk PH domain binds specifically to both d-Ins (1,3,4,5)P4 and PtdIns (3,4,5)P3 (
      • Salim K.
      • Bottomley M.J.
      • Querfurth E.
      • Zvelebil M.J.
      • Gout I.
      • Scaife R.
      • Margolis R.L.
      • Gigg R.
      • Smith C.I.
      • Driscoll P.C.
      • Waterfield M.D.
      • Panayotou G.
      ,
      • Fukuda M.
      • Kojima T.
      • Kabayama H.
      • Mikoshiba K.
      )in vitro and to PtdIns (3,4,5)P3 in vivo (
      • Isakoff S.J.
      • Cardozo T.
      • Andreev J.
      • Li Z.
      • Ferguson K.M.
      • Abagyan R.
      • Lemmon M.A.
      • Aronheim A.
      • Skolnik E.Y.
      ).
      In search of specific phosphoinositide binding preferences of the p85α SH2 domains, we chose to compare their interactions withd-Ins (1,4,5)P3 and d-Ins (1,3,4,5)P4 or PtdIns (4,5)P2 and PtdIns (3,4,5)P3. This choice was based on the knowledge that PtdIns (4,5)P2 is abundant in the plasma membrane of resting cells, whereas PtdIns (3,4,5)P3 is only present in appreciable quantities after cell stimulation (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ,
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ). We also compared the binding of the p85α SH2 domains to the physiologicald- and nonphysiological l-enantiomers of the inositol polyphosphates, because stereoselectivity should be exhibited in the case of true, biological interactions.
      We observed that numerous different phosphoinositides and inositol polyphosphates can bind to the p85α SH2 domains, albeit weakly. Using NMR spectroscopy, we found that di-C6-PIP3 and all the inositol polyphosphates tested bound to the SH2 domains in the Tyr(P) binding pockets that accommodate protein ligands. However, the SH2 domains failed to display clear preferences for distinct phosphoinositides or inositol polyphosphates presented in solution. Similarly, the C-SH2 domain did not demonstrate a distinct binding specificity for phosphoinositides presented in phospholipid bilayers. Surface representations of the SH2 domain structures that display their calculated electrostatic potentials show that the Tyr(P) binding pockets of the N- and particularly of the C-SH2 domains are highly positively charged. Thus, the lack of binding specificity or stereoselectivity shown by the SH2 domains for the test ligands may reflect the likelihood that their interaction is largely based on electrostatic interactions that have little dependence on distinct structural features. Such interactions are thus very different from those of high specificity observed between SH2 domains and physiological Tyr(P)-containing ligands.
      In addition, in a competition assay we observed that despite an overlap of binding sites, phosphoinositides and inositol polyphosphates only poorly displaced SH2 domains from a Tyr(P) peptide ligand. Furthermore, among the ligands tested, there was no significant variation in the efficacy of competition. From this assay, it also emerged that the N-SH2 domain bound to di-C6-PIP3 and inositol polyphosphates similarly to, but even more weakly than, the C-SH2 domain. This observation may perhaps be explained by two factors. First, the Tyr(P) binding pocket produces a greater density of positive charge on the surface of the C-SH2 domain compared with the surface of the N-SH2 domain (see Fig. 2), thus favoring interactions of the former with negatively charged ligands. Second, it has been observed that the unoccupied Tyr(P) binding pocket of the C-SH2 domain is relatively exposed, whereas that of the N-SH2 domain is not fully formed in the absence of a peptide ligand (
      • Nolte R.T.
      • Eck M.J.
      • Schlessinger J.
      • Shoelson S.E.
      • Harrison S.C.
      ) and may therefore be less accessible to phosphoinositides.
      However, the similar patterns of ligand binding observed for both p85α SH2 domains suggest that all the ligands contact the SH2 domains in the same, rather nonspecific manner. We conclude that althoughin vitro both p85α SH2 domains may interact weakly with PtdIns (3,4,5)P3 and inositol polyphosphates, the lack of specificity of these interactions and their inability to compete effectively with Tyr(P) peptide ligands suggest that they do not represent physiologically significant interactions. Rather, it seems that in vitro the SH2 domain Tyr(P) binding pocket has a tendency to bind somewhat indiscriminately to negatively charged ligands. This promiscuity is further witnessed in a crystal form of the p85α C-SH2 domain in which the Tyr(P) binding pocket accommodates an aspartate side chain.
      F. Hoedemaker, G. Siegal, S. M. Roe, P. C. Driscoll, and J. P. Abrahams, manuscript submitted.
      Although the mode of interaction is highly reminiscent of Tyr(P) binding (coordination of the aspartate carboxylate group with both R36 and R18), the aspartate side chain is clearly not a consensus ligand.
      Finally, we demonstrated that although in vivo, wortmannin blocks the activity of PI 3-kinase, it does not affect the ability of activated PDGF receptors (or other tyrosine-phosphorylated proteins) to bind the p85α regulatory subunit. These results are in agreement with characterizations of wortmannin activity (
      • Wymann M.
      • Arcaro A.
      ) but contrast with previous reports of an inverse correlation between the level of 3′-phosphorylated phosphoinositides in the cell and the association of PI 3-kinase with tyrosine-phosphorylated proteins (insulin receptor and insulin receptor substrate) (
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ). Therefore we suggest the levels of PI 3-kinase products in the plasma membrane are unlikely to regulate signal transduction events through interactions with SH2 domains. Rather, we consider that the immediate targets of PI 3-kinase activity are represented by those proteins that display high affinity, distinct binding specificity, and stereoselectivity for 3′-phosphorylated phosphoinositides, such as the PH domain-containing proteins Akt, Btk, PDK-1, and phospholipase C γ-1.

      REFERENCES

        • Pawson T.
        • Schlessinger J.
        Curr. Biol. 1993; 3: 434-442
        • Piccione E.
        • Case R.D.
        • Domchek S.M.
        • Hu P.
        • Chaudhuri M.
        • Backer J.M.
        • Schlessinger J.
        • Shoelson S.E.
        Biochemistry. 1993; 32: 3197-3202
        • Panayotou G.
        • Gish G.
        • End P.
        • Truong O.
        • Gout I.
        • Dhand R.
        • Fry M.J.
        • Hiles I.
        • Pawson T.
        • Waterfield M.D.
        Mol. Cell. Biol. 1993; 13: 3567-3576
        • Pawson T.
        Nature. 1995; 373: 573-580
        • Panayotou G.
        • Waterfield M.D.
        Bioessays. 1993; 15: 171-177
        • Otsu M.
        • Hiles I.
        • Gout I.
        • Fry M.J.
        • Ruiz-Larrea F.
        • Panayotou G.
        • Thompson A.
        • Dhand R.
        • Hsuan J.
        • Totty N.
        • Smith A.D.
        • Morgan S.J.
        • Courtneidge S.A.
        • Parker P.J.
        • Waterfield M.D.
        Cell. 1991; 65: 91-104
        • Kapeller R.
        • Cantley L.C.
        Bioessays. 1994; 16: 565-576
        • Carpenter C.L.
        • Duckworth B.C.
        • Auger K.R.
        • Cohen B.
        • Schaffhausen B.S.
        • Cantley L.C.
        J. Biol. Chem. 1990; 265: 19704-19711
        • Auger K.R.
        • Serunian L.A.
        • Soltoff S.P.
        • Libby P.
        • Cantley L.C.
        Cell. 1989; 57: 167-175
        • Downward J.
        Science. 1998; 279: 673-674
        • Vanhaesebroeck B.
        • Leevers S.J.
        • Panayotou G.
        • Waterfield M.D.
        Trends Biochem. Sci. 1997; 22: 267-272
        • Toker A.
        • Cantley L.C.
        Nature. 1997; 387: 673-676
        • Frech M.
        • Andjelkovic M.
        • Ingley E.
        • Reddy K.K.
        • Falck J.R.
        • Hemmings B.A.
        J. Biol. Chem. 1997; 272: 8474-8481
        • Alessi D.R.
        • James S.R.
        • Downes C.P.
        • Holmes A.B.
        • Gaffney P.R.J.
        • Reese C.B.
        • Cohen P.
        Curr. Biol. 1997; 7: 261-269
        • Isakoff S.J.
        • Cardozo T.
        • Andreev J.
        • Li Z.
        • Ferguson K.M.
        • Abagyan R.
        • Lemmon M.A.
        • Aronheim A.
        • Skolnik E.Y.
        EMBO J. 1998; 17: 5374-5387
        • Falasca M.
        • Logan S.K.
        • Lehto V.P.
        • Baccante G.
        • Lemmon M.A.
        • Schlessinger J.
        EMBO J. 1998; 17: 414-422
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        • Toker A.
        Science. 1997; 275: 665-668
        • James S.R.
        • Downes C.P.
        • Gigg R.
        • Grove S.J.
        • Holmes A.B.
        • Alessi D.R.
        Biochem. J. 1996; 315: 709-713
        • Stephens L.
        • Anderson K.
        • Stokoe D.
        • Erdjument-Bromage H.
        • Painter G.F.
        • Holmes A.B.
        • Gaffney P.R.
        • Reese C.B.
        • McCormick F.
        • Tempst P.
        • Coadwell J.
        • Hawkins P.T.
        Science. 1998; 279: 710-714
        • Bottomley M.J.
        • Salim K.
        • Panayotou G.
        Biochim. Biophys. Acta. 1998; 1436: 165-183
        • Rameh L.E.
        • Chen C.S.
        • Cantley L.C.
        Cell. 1995; 83: 821-830
        • Bae Y.S.
        • Cantley L.G.
        • Chen C.S.
        • Kim S.R.
        • Kwon K.S.
        • Rhee S.G.
        J. Biol. Chem. 1998; 273: 4465-4469
        • Siegal G.
        • Davis B.
        • Kristensen S.M.
        • Sankar A.
        • Linacre J.
        • Stein R.C.
        • Panayotou G.
        • Waterfield M.D.
        • Driscoll P.C.
        J. Mol. Biol. 1998; 276: 461-478
        • Booker G.W.
        • Breeze A.L.
        • Downing A.K.
        • Panayotou G.
        • Gout I.
        • Waterfield M.D.
        • Campbell I.D.
        Nature. 1992; 358: 684-687
        • Smith D.B.
        • Johnson K.S.
        Gene. 1988; 67: 31-40
        • Riley A.M.
        • Mahon M.F.
        • Potter B.V.L.
        Angew. Chem. Int. Ed. Engl. 1997; 36: 1472-1474
        • Potter B.V.L.
        • Lampe D.
        Angew. Chem. Int. Ed. Engl. 1995; 34: 1933-1972
        • Gaffney P.R.J.
        • Reese C.B.
        Bioorg. Med. Chem. Lett. 1997; 7: 3171-3176
        • Desai T.
        • Gigg J.
        • Gigg R.
        • Martin-Zamora E.
        Tyman J.H.P. Synthesis in Lipid Chemistry. Royal Society of Chemistry, London1996: 67-92
        • Zhang O.W.
        • Kay L.E.
        • Olivier J.P.
        • Forman-Kay J.D.
        J. Biomol. NMR. 1994; 4: 845-858
        • Hensmann M.
        • Booker G.W.
        • Panayotou G.
        • Boyd J.
        • Linacre J.
        • Waterfield M.
        • Campbell I.D.
        Protein Sci. 1994; 3: 1020-1030
        • Gunther U.L.
        • Liu Y.
        • Sanford D.
        • Bachovchin W.W.
        • Schaffhausen B.
        Biochemistry. 1996; 35: 15570-15581
        • Delaglio F.
        • Grzesiek S.
        • Vuister G.W.
        • Zhu G.
        • Pfeifer J.
        • Bax A.
        J. Biomol. NMR. 1995; 6: 277-293
        • Bartels C.H.
        • Xia T.
        • Billeter M.
        • Güntert P.
        • Wüthrich K.
        • Bartels T.-H.X.
        • Billeter M.
        • Güntert P.
        • Wüthrich K.
        J. Biomol. NMR. 1995; 5: 1-10
        • Salim K.
        • Bottomley M.J.
        • Querfurth E.
        • Zvelebil M.J.
        • Gout I.
        • Scaife R.
        • Margolis R.L.
        • Gigg R.
        • Smith C.I.
        • Driscoll P.C.
        • Waterfield M.D.
        • Panayotou G.
        EMBO J. 1996; 15: 6241-6250
        • Jonsson U.
        • Fagerstam L.
        • Ivarsson B.
        • Johnsson B.
        • Karlsson R.
        • Lundh K.
        • Lofas S.
        • Persson B.
        • Roos H.
        • Ronnberg I.
        • Sjolander S.
        • Stenberg E.
        • Stahlberg R.
        • Urbaniczky C.
        • Ostlin H.
        • Malmqvist M.
        Biotechniques. 1991; 11: 620-627
        • Otting G.
        Curr. Opin. Struct. Biol. 1993; 3: 760-768
        • Van Nuland N.A.
        • Kroon G.J.
        • Dijkstra K.
        • Wolters G.K.
        • Scheek R.M.
        • Robillard G.T.
        FEBS Lett. 1993; 315: 11-15
        • Nolte R.T.
        • Eck M.J.
        • Schlessinger J.
        • Shoelson S.E.
        • Harrison S.C.
        Nat. Struct. Biol. 1996; 3: 364-374
        • Breeze A.L.
        • Kara B.V.
        • Barratt D.G.
        • Anderson M.
        • Smith J.C.
        • Luke R.W.
        • Best J.R.
        • Cartlidge S.A.
        EMBO J. 1996; 15: 3579-3589
        • Fukuda M.
        • Kojima T.
        • Kabayama H.
        • Mikoshiba K.
        J. Biol. Chem. 1996; 271: 30303-30306
        • Rameh L.E.
        • Arvidsson A.
        • Carraway III, K.L.
        • Couvillon A.D.
        • Rathbun G.
        • Crompton A.
        • VanRenterghem B.
        • Czech M.P.
        • Ravichandran K.S.
        • Burakoff S.J.
        • Wang D.S.
        • Chen C.S.
        • Cantley L.C.
        J. Biol. Chem. 1997; 272: 22059-22066
        • Arcaro A.
        • Wymann M.P.
        Biochem. J. 1993; 296: 297-301
        • Wang D.-S.
        • Hsu A.-L.
        • Song X.
        • Chiou C.-M.
        • Chen C.-S.
        J. Org. Chem. 1998; 63: 5430-5437
        • Kavran J.M.
        • Klein D.E.
        • Lee A.
        • Falasca M.
        • Isakoff S.J.
        • Skolnik E.Y.
        • Lemmon M.A.
        J. Biol. Chem. 1998; 273: 30497-30508
        • Wymann M.
        • Arcaro A.
        Biochem. J. 1994; 298: 517-520
        • Kraulis P.J.
        J. Appl. Crystallogr. 1991; 24: 946-950
        • Merritt E.A.
        • Murphy M.E.P.
        Acta Crystallogr. Sec. D. 1994; 50: 869-873
        • Nicholls A.
        • Sharp K.A.
        • Honig B.
        Proteins Struct. Funct. Genet. 1991; 11: 281-296