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Binding of a Diphosphotyrosine-containing Peptide That Mimics Activated Platelet-derived Growth Factor Receptor β Induces Oligomerization of Phosphatidylinositol 3-Kinase*

Open AccessPublished:December 11, 1998DOI:https://doi.org/10.1074/jbc.273.50.33379
      Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric enzyme comprising a p110 catalytic subunit and a p85 regulatory subunit. We have recently shown that the isolated p85 subunit exists as a dimer; therefore, we examined whether the heterodimeric enzyme was capable of further self-association. Size-exclusion chromatography demonstrated that PI3K was a 1:1 complex of p85 and p110 under native conditions. However, binding of a diphosphotyrosine-containing peptide that mimics an activated platelet-derived growth factor receptor β induced an increase in the apparent molecular mass of PI3K. This increase was due to dimerization of PI3K and was dependent on PI3K concentration but not diphosphopeptide concentration. Dimer formation was also observed directly using fluorescence resonance energy transfer. Diphosphopeptide-induced activation of PI3K (Carpenter, C. L., Auger, K. R., Chanudhuri, M., Yoakim, M., Schaffhausen, B., Shoelson, S., and Cantley, L. C. (1993) J. Biol. Chem. 268, 9478–9483; Rordorf-Nikolic, T., Van Horn, D. J., Chen, D., White, M. F., and Backer, J. M. (1995)J. Biol. Chem. 270, 3662–3666) was not a direct result of dimerization and occurred only when phosphatidylinositol, and not phosphatidylinositol-4,5-diphosphate, was the phosphorylation substrate. Binding of the tandem SH2 domains of the p85 regulatory subunit to activated receptor tyrosine kinases therefore induces dimerization of PI3K, which may be an early step in inositol lipid-mediated signal transduction.
      Propagation of extracellular signals to the nucleus is mediated by a wide range of second messengers, which employ a number of common signaling mechanisms, such as dimerization or oligomerization, phosphorylation of tyrosine, serine, or threonine residues, the binding of SH2
      The abbreviations used are: SH, src homology; BH, BCR homology; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; HP-SEC, high performance size-exclusion chromatography; IR, insulin receptor; IRS, insulin receptor substrate; MALDI, matrix-assisted laser desorption and ionization; PDGFβ-R, platelet-derived growth factor receptor β; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; pY, phosphotyrosine; RTK, receptor tyrosine kinase; 2-ME, 2-mercaptoethanol; Bicine, N,N-bis(2- hydroxyethyl)glycine.
      1The abbreviations used are: SH, src homology; BH, BCR homology; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; HP-SEC, high performance size-exclusion chromatography; IR, insulin receptor; IRS, insulin receptor substrate; MALDI, matrix-assisted laser desorption and ionization; PDGFβ-R, platelet-derived growth factor receptor β; PI, phosphatidylinositol; PI3K, phosphatidylinositol 3-kinase; pY, phosphotyrosine; RTK, receptor tyrosine kinase; 2-ME, 2-mercaptoethanol; Bicine, N,N-bis(2- hydroxyethyl)glycine.
      or phosphotyrosine binding domains to phosphotyrosine-containing sequences, or binding of SH3 domains to proline-rich sequence motifs.
      Phosphatidylinositol 3-kinase (PI3K) is a dual-specificity kinase that has been implicated in a wide range of cellular events, including proliferation and cell migration (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). In response to a number of cell stimuli, this two-subunit enzyme phosphorylates the 3′ position of inositol lipids to produce the second messengers phosphatidylinositol 3,4-diphosphate (PI-3,4-P2) and phosphatidylinositol 3,4,5-triphosphate (PI-3,4,5-P3), which have been shown to bind to and activate plekstrin homology domain-containing proteins such as protein kinase B (AKT) and PDK1 (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ). PI3K is also a serine/threonine kinase that can autophosphorylate either subunit (
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Katsuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ,
      • Vanhaesebroeck B.
      • Welham M.J.
      • Kotani K.
      • Stein R.
      • Warne P.H.
      • Zvelebil M.J.
      • Higashi K.
      • Volinia S.
      • Downward J.
      • Waterfield M.D.
      ) and has been reported to phosphorylate the intracellular docking protein, IRS-1 (
      • Freund G.G.
      • Wittig J.G.
      • Mooney R.A.
      ).
      A number of PI3K isoforms are known, which can be classified on the basis of their subunit composition and sequence homology. The class IA PI3Ks are heterodimers of a p110 catalytic subunit, of which there are three isoforms, p110α, p110β, and p110δ (
      • Zvelebil M.J.
      • MacDougall L.
      • Leevers S.
      • Volinia S.
      • Vanhaesebroeck B.
      • Gout I.
      • Panayotou G.
      • Domin J.
      • Stein R.
      • Pages F.
      • Koga H.
      • Salim K.
      • Linacre J.
      • Das P.
      • Panaretou C.
      • Wetzker R.
      • Waterfield M.
      ), and a p85 adapter or regulatory subunit, of which there are five isoforms, p85α, p85β, p55γ, p55α, and p50α (
      • Inukai K.
      • Funaki M.
      • Ogihara T.
      • Katagiri H.
      • Kanda A.
      • Anai M.
      • Fukushima Y.
      • Hosaka T.
      • Suzuki M.
      • Shin B.C.
      • Takata K.
      • Yazaki Y.
      • Kikuchi M.
      • Oka Y.
      • Asano T.
      ). The 85-kDa isoforms, p85α and p85β (
      • 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.
      ), contain several homology domains, including two SH2 domains, a BCR homology (BH) domain and an SH3 domain, whereas the lower molecular mass isoforms, p55γ, p55α, and p50α, lack the BH and SH3 domains. The catalytic subunit, p110, also contains several homology domains, including the kinase domain, a PIK domain whose sequence is conserved in PI3-kinases and PI4-kinases, as well as putative N-terminal p85-binding and Ras binding domains (
      • Zvelebil M.J.
      • MacDougall L.
      • Leevers S.
      • Volinia S.
      • Vanhaesebroeck B.
      • Gout I.
      • Panayotou G.
      • Domin J.
      • Stein R.
      • Pages F.
      • Koga H.
      • Salim K.
      • Linacre J.
      • Das P.
      • Panaretou C.
      • Wetzker R.
      • Waterfield M.
      ).
      A number of regulatory mechanisms for PI3K have been described, but no single event has been demonstrated to result in full activation of this enzyme. It is likely that multiple events are required to transduce a signal via this lipid/protein kinase. Binding of the tandem SH2 domains in the regulatory subunit to specific phosphotyrosine-containing motifs present on a range of molecules, such as receptor tyrosine kinases (RTKs) and cytoplasmic signaling proteins, including IRS-1, has been reported to increase the intrinsic activity of PI3K (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Rordorf-Nikolic T.
      • Van Horn D.J.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ) and trigger a conformational change in the regulatory subunit (
      • Panayotou G.
      • Bax B.
      • Gout I.
      • Federwisch M.
      • Wroblowski B.
      • Dhand R.
      • Fry M.J.
      • Blundell T.L.
      • Wollmer A.
      • Waterfield M.D.
      ). Binding of a range of other intracellular ligands, such as SH3 domains and small GTPases, has also been reported to activate PI3K (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ,
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ,
      • Rodriguez Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). Alternatively, binding of these ligands may result in activation simply by translocating PI3K to the plasma membrane, where its lipid substrates are located (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ). For example, activation of PI3K by binding of Ha-RasV12 has been most convincingly demonstrated when Ras was immobilized in phosphatidylinositol-containing lipid vesicles (
      • Rodriguez Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ), suggesting that the role of Ras may be to recruit PI3K to its membrane substrate, in a mechanism analogous to that of c-Raf activation by Ras (
      • Leevers S.J.
      • Paterson H.F.
      • Marshall C.J.
      ).
      Another common theme in activation of intracellular signaling proteins is dimerization or oligomerization, which can be induced by ligand binding and, in the case of RTKs, lead to auto- or trans-phosphorylation of the RTK and up-regulation of catalytic activity. Several intracellular protein kinases, such as c-Raf, have also been shown to be activated by dimerization (
      • Farrar M.A.
      • Alberol I.
      • Perlmutter R.M.
      ,
      • Luo Z.
      • Tzivion G.
      • Belshaw P.J.
      • Vavvas D.
      • Marshall M.
      • Avruch J.
      ). We have recently demonstrated that the 85-kDa subunits of PI3K exist as dimers in the absence of the catalytic subunit, whereas the low molecular mass isoforms of the regulatory subunit are monomeric.
      A. G. Harpur, M. J. Layton, P. Das, M. J. Bottomley, G. Panayotou, P. C. Driscoll, and M. D. Waterfield, submitted for publication.
      2A. G. Harpur, M. J. Layton, P. Das, M. J. Bottomley, G. Panayotou, P. C. Driscoll, and M. D. Waterfield, submitted for publication.
      In contrast, PI3K activity in unstimulated cells has been reported to have an apparent molecular mass of approximately 200 kDa (
      • Shibasaki F.
      • Fukui Y.
      • Takenawa T.
      ,
      • Woscholski R.
      • Dhand R.
      • Fry M.J.
      • Waterfield M.D.
      • Parker P.J.
      ), suggesting the whole enzyme comprises one of each of the p110 and p85 subunits. In the present study, we have demonstrated that the binding of a peptide that mimics an activated PDGFβ receptor to the tandem SH2 domains of the regulatory subunit induces dimerization of the 200-kDa enzyme via an interface that includes regions of the regulatory subunits.

      DISCUSSION

      One of the first events in RTK-mediated signal transduction is growth factor-induced receptor dimerization or oligomerization (
      • Schlessinger J.
      • Ullrich A.
      ). Receptor dimerization triggers the intrinsic kinase activity, resulting in trans- and auto-phosphorylation of tyrosine residues and creation of specific binding sites for SH2 domain-containing proteins. SH2 domains bind phosphotyrosine-containing sequence motifs with a high degree of specificity (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ,
      • Songyang Z.
      • Shoelson S.E.
      • McGlade J.
      • Olivier P.
      • Pawson T.
      • Bustelo X.R.
      • Barbacid M.
      • Sabe H.
      • Hanafusa H.
      • Yi T.
      • Baltimore D.
      • Ratnofsky S.
      • Feldman R.A.
      • Cantley L.C.
      ), and the consensus sequence motifs that bind a number of SH2 domains have been defined, including that of the p85 subunit of PI3K (
      • Songyang Z.
      • Shoelson S.E.
      • McGlade J.
      • Olivier P.
      • Pawson T.
      • Bustelo X.R.
      • Barbacid M.
      • Sabe H.
      • Hanafusa H.
      • Yi T.
      • Baltimore D.
      • Ratnofsky S.
      • Feldman R.A.
      • Cantley L.C.
      ). Both SH2 domains in p85α bind a linear epitope with the sequence pYXXM (where X is any amino acid), and this sequence motif is present in a number of RTKs and intracellular docking proteins. Some signaling proteins, such as IRS-1 and Erb B3 (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Kim H.H.
      • Sierke S.L.
      • Koland J.G.
      ), have more than one sequence pYXXM motif, and the PDGFβ-R has two pYXXM motifs with seven amino acid residues between them (
      • Claesson Welsh L.
      • Eriksson A.
      • Moren A.
      • Severinsson L.
      • Ek B.
      • Ostman A.
      • Betsholtz C.
      • Heldin C.H.
      ).
      The binding of a peptide containing both pYXXM motifs from the PDGFβ-R (Tyr-751/Tyr-740) (
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ) and Fig. 4 A) or from IRS-1 (
      • Rordorf-Nikolic T.
      • Van Horn D.J.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ) has been shown to activate the intrinsic lipid kinase activity of PI3K when PI was the phosphorylation substrate, whereas peptides containing only one p85-binding site bind with lower affinity and activate the enzyme to a lesser extent. A mechanism for PI3K activation has been postulated in which both SH2 domains in p85 are simultaneously occupied by both phosphotyrosine residues in a diphosphopeptide, which causes a conformational change within the inter-SH2 region of the p85 subunit and activates p110 (
      • Rordorf-Nikolic T.
      • Van Horn D.J.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ). Binding of Tyr-740/Tyr-751 has been shown to induce a conformational change in the p85α regulatory subunit (
      • Panayotou G.
      • Bax B.
      • Gout I.
      • Federwisch M.
      • Wroblowski B.
      • Dhand R.
      • Fry M.J.
      • Blundell T.L.
      • Wollmer A.
      • Waterfield M.D.
      ), which may be the same conformational change that is involved in the self-association of the PI3K heterodimer (Figs. Figure 1, Figure 2, Figure 3). Binding of the two phosphotyrosine residues in the diphosphopeptide to SH2 domains in different PI3K molecules did not seem to be the mechanism by which dimerization occurs, as the formation of higher molecular mass complexes of PI3K was independent of diphosphopeptide concentration (Fig. 2 C), suggesting that the diphosphopeptide does not participate directly in the dimerization event.
      Binding of a diphosphopeptide to the SH2 domains of PI3K therefore seems to affect another region of the protein, resulting in self-association of the enzyme (Figs. 1 and 3). In contrast, binding of singly phosphorylated peptides did not induce dimerization of PI3K (Fig. 1 B), even though Tyr-751 binds both SH2 domains of p85 with similar affinities (
      • Panayotou G.
      • Gish G.
      • End P.
      • Truong O.
      • Gout I.
      • Dhand R.
      • Fry M.J.
      • Hiles I.
      • Pawson T.
      • Waterfield M.D.
      ) and can presumably occupy both SH2 domains simultaneously. Occupation of SH2 domains is therefore not sufficient to induce dimerization, and a subsequent event must be required which, given the distance constraints imposed by the seven residues between the two pYXXM motifs in this peptide, may involve the SH2 domains moving closer together. This would suggest that the conformational change occurs within the inter-SH2 region, which may act as a flexible linker between the two SH2 domains. The inter-SH2 region is the binding site for the p110 subunit (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ), which may explain how an SH2 domain-mediated event could be transmitted to the p110 subunit. The variable length of the sequence between phosphotyrosine residues in proteins with more than one pYXXM motif suggests that not all binding events involving both SH2 domains would lead to dimerization. The insulin receptor (IR) has only 6 residues between tandem pYXXM motifs, whereas IRS-1 has 20 residues. PI3K is activated by a peptide derived from the IRS-1 sequence (
      • Rordorf-Nikolic T.
      • Van Horn D.J.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ) but not the IR sequence (
      • Van Horn D.J.
      • Myers Jr., M.G.
      • Backer J.M.
      ), supporting the idea that it is the precise distance constraint imposed on the two SH2 domains that is important for inducing a conformational change in the inter-SH2 region. Different arrangements pYXXM motifs in three-dimensional space may therefore mediate signal specificity in PI3K signaling pathways.
      The conformational change in the inter-SH2 region that is transmitted to the p110 subunit results in the unmasking of a binding site for another diphosphopeptide-bound PI3K. This binding site must be obscured when the SH2 domains are not peptide-bound or when they are bound to singly phosphorylated peptides. It has previously been observed that immunoprecipitation of PI3K using a polyclonal antibody against p85α was less efficient in the presence of Tyr-740/Tyr-751 (
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ), suggesting that some surfaces on PI3K are less accessible when the SH2 domains are bound to diphosphopeptide. We have previously shown that p85α forms a homodimer and that the dimerization interface resides in the N-terminal half of the protein, which contains the SH3 and BH domains and two proline-rich motifs.2 Combinations of these N-terminal domains have also been shown to be dimers (
      • Musacchio A.
      • Cantley L.C.
      • Harrison S.C.
      ,
      • Chen J.K.
      • Schreiber S.L.
      ). The SH3 and BH domains are therefore free to form a dimeric interface in p85α alone but not in the p110α-EE tag-p85α complex. This implies that p110 may interact with the SH3 and BH domains, preventing their interaction with each other, and that this interaction is released when the SH2 domains bind diphosphopeptide. This may also explain how the binding of SH3 or BH domain ligands to PI3K could regulate the catalytic activity (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ,
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ).
      Dimerization of receptor tyrosine kinases (RTKs) (
      • Redemann N.
      • Holzmann B.
      • von Ruden T.
      • Wagner E.F.
      • Schlessinger J.
      • Ullrich A.
      ,
      • Honegger A.M.
      • Schmidt A.
      • Ullrich A.
      • Schlessinger J.
      ), transmembrane phosphatases (
      • Mourey R.J.
      • Dixon J.E.
      ), and cytoplasmic protein kinases (
      • Farrar M.A.
      • Alberol I.
      • Perlmutter R.M.
      ,
      • Luo Z.
      • Tzivion G.
      • Belshaw P.J.
      • Vavvas D.
      • Marshall M.
      • Avruch J.
      ) is often an early step in signal transduction. Another emerging theme in receptor-mediated signaling is the formation of multiprotein complexes, some of which bind to dimeric receptors, and may also contain dimeric components. Formation of these complexes can result in the activation of one or more components of the complex, in recruitment to the juxtamembrane region of the cell, or in the creation of novel protein-binding surfaces. New binding sites can be generated by heterotypic or homotypic protein-protein interactions, which may allow recruitment of other components of the complex. Dimerization of PI3K does not directly activate the intrinsic kinase activity in vitro; therefore, the role of dimer formation may be to generate novel binding surfaces for recruitment of other cellular signaling proteins that may be the factors that moderate PI3K activity in vivo. The differences in the three-dimensional arrangements of pYXXM motifs in signaling proteins and their ability or inability to induce PI3K dimerization increases the repertoire of PI3K-containing multiprotein complexes and may be a mechanism that directs specificity in PI3K-mediated signal transduction downstream of different cell stimuli.

      Acknowledgments

      We thank Akunna Akpan and Krishna Pitrola for assistance with Sf9 cell culture; Elaine Stimson for MALDI mass spectrometry analysis; and Zeiss Evotec for the loan of the ConfoCor instrument.

      REFERENCES

        • Vanhaesebroeck B.
        • Leevers S.J.
        • Panayotou G.
        • Waterfield M.D.
        Trends Biochem. Sci. 1997; 22: 267-272
        • Alessi D.R.
        • James S.R.
        • Downes C.P.
        • Holmes A.B.
        • Gaffney P.R.
        • Reese C.B.
        • Cohen P.
        Curr. Biol. 1997; 7: 261-269
        • Dhand R.
        • Hiles I.
        • Panayotou G.
        • Roche S.
        • Fry M.J.
        • Gout I.
        • Totty N.F.
        • Truong O.
        • Vicendo P.
        • Yonezawa K.
        • Katsuga M.
        • Courtneidge S.A.
        • Waterfield M.D.
        EMBO J. 1994; 13: 522-533
        • Vanhaesebroeck B.
        • Welham M.J.
        • Kotani K.
        • Stein R.
        • Warne P.H.
        • Zvelebil M.J.
        • Higashi K.
        • Volinia S.
        • Downward J.
        • Waterfield M.D.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4330-4335
        • Freund G.G.
        • Wittig J.G.
        • Mooney R.A.
        Biochem. Biophys. Res. Commun. 1995; 206: 272-278
        • Zvelebil M.J.
        • MacDougall L.
        • Leevers S.
        • Volinia S.
        • Vanhaesebroeck B.
        • Gout I.
        • Panayotou G.
        • Domin J.
        • Stein R.
        • Pages F.
        • Koga H.
        • Salim K.
        • Linacre J.
        • Das P.
        • Panaretou C.
        • Wetzker R.
        • Waterfield M.
        Philos. Trans. R. Soc. Lond. Biol. Sci. 1996; 351: 217-223
        • Inukai K.
        • Funaki M.
        • Ogihara T.
        • Katagiri H.
        • Kanda A.
        • Anai M.
        • Fukushima Y.
        • Hosaka T.
        • Suzuki M.
        • Shin B.C.
        • Takata K.
        • Yazaki Y.
        • Kikuchi M.
        • Oka Y.
        • Asano T.
        J. Biol. Chem. 1997; 272: 7873-7882
        • 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
        • Backer J.M.
        • Myers Jr., M.G.
        • Shoelson S.E.
        • Chin D.J.
        • Sun X.J.
        • Miralpeix M.
        • Hu P.
        • Margolis B.
        • Skolnik E.Y.
        • Schlessinger J.
        • White M.F.
        EMBO J. 1992; 11: 3469-3479
        • Rordorf-Nikolic T.
        • Van Horn D.J.
        • Chen D.
        • White M.F.
        • Backer J.M.
        J. Biol. Chem. 1995; 270: 3662-3666
        • Carpenter C.L.
        • Auger K.R.
        • Chanudhuri M.
        • Yoakim M.
        • Schaffhausen B.
        • Shoelson S.
        • Cantley L.C.
        J. Biol. Chem. 1993; 268: 9478-9483
        • Panayotou G.
        • Bax B.
        • Gout I.
        • Federwisch M.
        • Wroblowski B.
        • Dhand R.
        • Fry M.J.
        • Blundell T.L.
        • Wollmer A.
        • Waterfield M.D.
        EMBO J. 1992; 11: 4261-4272
        • Pleiman C.M.
        • Hertz W.M.
        • Cambier J.C.
        Science. 1994; 263: 1609-1612
        • Tolias K.F.
        • Cantley L.C.
        • Carpenter C.L.
        J. Biol. Chem. 1995; 270: 17656-17659
        • Rodriguez Viciana P.
        • Warne P.H.
        • Dhand R.
        • Vanhaesebroeck B.
        • Gout I.
        • Fry M.J.
        • Waterfield M.D.
        • Downward J.
        Nature. 1994; 370: 527-532
        • Klippel A.
        • Reinhard C.
        • Kavanaugh W.M.
        • Apell G.
        • Escobedo M.A.
        • Williams L.T.
        Mol. Cell. Biol. 1996; 16: 4117-4127
        • Rodriguez Viciana P.
        • Warne P.H.
        • Vanhaesebroeck B.
        • Waterfield M.D.
        • Downward J.
        EMBO J. 1996; 15: 2442-2451
        • Leevers S.J.
        • Paterson H.F.
        • Marshall C.J.
        Nature. 1994; 369: 411-414
        • Farrar M.A.
        • Alberol I.
        • Perlmutter R.M.
        Nature. 1996; 383: 178-181
        • Luo Z.
        • Tzivion G.
        • Belshaw P.J.
        • Vavvas D.
        • Marshall M.
        • Avruch J.
        Nature. 1996; 383: 181-185
        • Shibasaki F.
        • Fukui Y.
        • Takenawa T.
        Biochem. J. 1993; 289: 227-231
        • Woscholski R.
        • Dhand R.
        • Fry M.J.
        • Waterfield M.D.
        • Parker P.J.
        J. Biol. Chem. 1994; 269: 25067-25072
        • Porfiri E.
        • Evans T.
        • Chardin P.
        • Hancock J.F.
        J. Biol. Chem. 1994; 269: 22672-22677
        • Hiles I.D.
        • Otsu M.
        • Volinia S.
        • Fry M.J.
        • Gout I.
        • Dhand R.
        • Panayotou G.
        • Ruiz Larrea F.
        • Thompson A.
        • Totty N.F.
        • Hsuan J.J.
        • Courtneidge S.A.
        • Parker P.A.
        • Waterfield M.D.
        Cell. 1992; 70: 419-429
        • O'Reilly D.R.
        • Miller L.K.
        • Luckow V.A.
        Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, New York1994
        • Harlow E.
        • Lane D.
        Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988
        • Whitman M.
        • Kaplan D.R.
        • Schaffhausen B.
        • Cantley L.
        • Roberts T.M.
        Nature. 1985; 315: 239-242
        • 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
        • Elgen M.
        • Rigler R.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5740-5747
        • Yu J.
        • Zhang Y.
        • McIlroy J.
        • Rordorf Nikolic T.
        • Orr G.A.
        • Backer J.M.
        Mol. Cell. Biol. 1998; 18: 1379-1387
        • Carpenter C.L.
        • Duckworth B.C.
        • Auger K.R.
        • Cohen B.
        • Schaffhausen B.S.
        • Cantley L.C.
        J. Biol. Chem. 1990; 265: 19704-19711
        • Regnier F.
        Methods Enzymol. 1983; 91: 137-190
        • Southwick P.L.
        • Ernst L.A.
        • Tauriello E.W.
        • Parker S.R.
        • Mujumdar R.B.
        • Mujumdar S.R.
        • Clever H.A.
        • Waggoner A.S.
        Cytometry. 1990; 11: 418-430
        • Dixon M.
        • Webb E.C.
        Enzymes. 3rd Ed. Academic Press, New York1979: 47-137
        • Schlessinger J.
        • Ullrich A.
        Neuron. 1992; 9: 383-391
        • Songyang Z.
        • Shoelson S.E.
        • Chaudhuri M.
        • Gish G.
        • Pawson T.
        • Haser W.G.
        • King F.
        • Roberts T.
        • Ratnofsky S.
        • Lechleider R.J.
        • Neel B.G.
        • Birge R.B.
        • Fajardo J.E.
        • Chou M.M.
        • Hanafusa H.
        • Schaffhausen B.
        • Cantley L.C.
        Cell. 1993; 72: 767-778
        • Songyang Z.
        • Shoelson S.E.
        • McGlade J.
        • Olivier P.
        • Pawson T.
        • Bustelo X.R.
        • Barbacid M.
        • Sabe H.
        • Hanafusa H.
        • Yi T.
        • Baltimore D.
        • Ratnofsky S.
        • Feldman R.A.
        • Cantley L.C.
        Mol. Cell. Biol. 1994; 14: 2777-2785
        • Kim H.H.
        • Sierke S.L.
        • Koland J.G.
        J. Biol. Chem. 1994; 269: 24747-24755
        • Claesson Welsh L.
        • Eriksson A.
        • Moren A.
        • Severinsson L.
        • Ek B.
        • Ostman A.
        • Betsholtz C.
        • Heldin C.H.
        Mol. Cell. Biol. 1988; 8: 3476-3486
        • Dhand R.
        • Hara K.
        • Hiles I.
        • Bax B.
        • Gout I.
        • Panayotou G.
        • Fry M.J.
        • Yonezawa K.
        • Kasuga M.
        • Waterfield M.D.
        EMBO J. 1994; 13: 511-521
        • Van Horn D.J.
        • Myers Jr., M.G.
        • Backer J.M.
        J. Biol. Chem. 1994; 269: 29-32
        • Musacchio A.
        • Cantley L.C.
        • Harrison S.C.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14373-14378
        • Chen J.K.
        • Schreiber S.L.
        BioMed. Chem. Lett. 1994; 4: 1755-1760
        • Redemann N.
        • Holzmann B.
        • von Ruden T.
        • Wagner E.F.
        • Schlessinger J.
        • Ullrich A.
        Mol. Cell. Biol. 1992; 12: 491-498
        • Honegger A.M.
        • Schmidt A.
        • Ullrich A.
        • Schlessinger J.
        Mol. Cell. Biol. 1990; 10: 4035-4044
        • Mourey R.J.
        • Dixon J.E.
        Curr. Opin. Genet. Dev. 1994; 4: 31-39