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Constitutive Activation of Phosphatidylinositol 3-Kinase by a Naturally Occurring Mutant Epidermal Growth Factor Receptor*

Open AccessPublished:January 02, 1998DOI:https://doi.org/10.1074/jbc.273.1.200
      The most frequently found alteration of the epidermal growth factor receptor (EGFR) in human tumors is a deletion of exons 2–7. This receptor, termed EGFRvIII, can transform NIH 3T3 cells, and the frequent expression of this variant implies that it confers a selective advantage upon tumor cells in vivo. Although EGFRvIII is a constitutively activated tyrosine kinase, there is no increase in Ras·GTP levels and low levels of mitogen-activated protein kinase activity in NIH 3T3 cells expressing this variant. We investigated whether phosphatidylinositol (PI) 3-kinase was an effector in transformation by the EGFRvIII. High levels of PI 3-kinase activity were constitutively present in EGFRvIII-transformed cells and were dependent upon the kinase activity of the receptor. While mitogen-activated protein kinase activity was quickly down-regulated to basal levels after 12 h of continuous EGFR activation, there was a 3-fold increase in PI 3-kinase activity in cells expressing normal EGFR and an 8-fold increase in cells expressing EGFRvIII after 48 h. This increased activity may reflect enhanced binding to EGFRvIII and the presence of novel PI 3-kinase isoforms. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 blocked both anchorage-independent growth and growth in low serum media and also resulted in morphological reversion of EGFRvIII-transformed cells. These results support an essential role for PI 3-kinase in transformation by this EGFR variant.
      Overexpression of the EGFR
      The abbreviations used are: EGFR, epidermal growth factor receptor; CS, calf serum; DMEM, Dulbecco's modified Eagle's medium; EGFRvIII, type III EGF receptor variant; GST, glutathione S-transferase; MAP, mitogen-activated protein; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; SH2, Src homology region 2; pTyr, phosphotyrosine.
      1The abbreviations used are: EGFR, epidermal growth factor receptor; CS, calf serum; DMEM, Dulbecco's modified Eagle's medium; EGFRvIII, type III EGF receptor variant; GST, glutathione S-transferase; MAP, mitogen-activated protein; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; SH2, Src homology region 2; pTyr, phosphotyrosine.
      has been implicated in the pathogenesis of many human tumors, including those derived from the brain, breast, lung, ovary, prostate, and skin (
      • Harris A.L.
      • Nicholson S.
      • Sainsbury R.
      • Wright C.
      • Farndon J.
      ,
      • Schlegel J.
      • Merdes A.
      • Stumm G.
      • Albert F.K.
      • Forsting M.
      • Hynes N.
      • Kiessling M.
      ). A number of alterations within the EGF receptor gene that result in aberrant protein products have also been described, primarily in human glial tumors (
      • Wong A.J.
      • Ruppert J.M.
      • Bigner S.H.
      • Grzeschik C.H.
      • Humphrey P.A.
      • Bigner D.S.
      • Vogelstein B.
      ,
      • Yamazaki H.
      • Fukui Y.
      • Ueyama Y.
      • Tamaoki N.
      • Kawamoto T.
      • Taniguchi S.
      • Shibuya M.
      ). The most common alteration of the EGF receptor gene is a deletion encompassing exons 2–7 (
      • Wong A.J.
      • Ruppert J.M.
      • Bigner S.H.
      • Grzeschik C.H.
      • Humphrey P.A.
      • Bigner D.S.
      • Vogelstein B.
      ,
      • Yamazaki H.
      • Fukui Y.
      • Ueyama Y.
      • Tamaoki N.
      • Kawamoto T.
      • Taniguchi S.
      • Shibuya M.
      ,
      • Schwechheimer K.
      • Huang S.
      • Cavenee W.K.
      ) (referred to as EGFRvIII, ΔEGFR, or de2–7EGFR) (
      • Moscatello D.K.
      • Holgado-Madruga M.
      • Godwin A.K.
      • Ramirez G.
      • Gunn G.
      • Zoltick P.W.
      • Biegel J.A.
      • Hayes R.L.
      • Wong A.J.
      ,
      • Nishikawa R.
      • Ji X.-D.
      • Harmon R.C.
      • Lazar C.S.
      • Gill G.N.
      • Cavenee W.K.
      • Su Huang H.-J.
      ,
      • Ekstrand A.J.
      • Longo N.
      • Hamid M.L.
      • Olson J.J.
      • Liu L.
      • Collins V.P.
      • James C.D.
      ). This receptor variant has subsequently been identified in other types of primary human brain tumors as well as breast carcinomas, non-small cell lung carcinomas, and ovarian tumors (
      • Moscatello D.K.
      • Holgado-Madruga M.
      • Godwin A.K.
      • Ramirez G.
      • Gunn G.
      • Zoltick P.W.
      • Biegel J.A.
      • Hayes R.L.
      • Wong A.J.
      ,
      • Garcia de Palazzo I.E.
      • Adams G.P.
      • Sundareshan P.
      • Wong A.J.
      • Testa J.R.
      • Bigner D.D.
      • Weiner L.M.
      ). This deletion results in a receptor with a 267-amino acid deletion in the extracytoplasmic domain near the amino terminus. The frequent expression of this variant in various tumors types suggests a strong selective advantage conferred upon tumor cellsin vivo (
      • Nishikawa R.
      • Ji X.-D.
      • Harmon R.C.
      • Lazar C.S.
      • Gill G.N.
      • Cavenee W.K.
      • Su Huang H.-J.
      ,
      • Nagane M.
      • Coufal F.
      • Lin H.
      • Bögler O.
      • Cavenee W.K.
      • Su Huang H.-J.
      ).
      Because the deletion occurs after the signal sequence, the EGFRvIII can be properly targeted to the membrane, and the remaining extracellular portion is glycosylated (
      • Ekstrand A.J.
      • Liu L.
      • He J.
      • Hamid M.L.
      • Longo N.
      • Collins V.P.
      • James C.D.
      ,
      • Batra S.K.
      • Castelino-Prabhu S.
      • Wikstrand C.J.
      • Zhu X.
      • Humphrey P.A.
      • Friedman H.S.
      • Bigner D.D.
      ). While the EGFRvIII has been detected on the cell surface of both tumor cells in vivo (
      • Humphrey P.
      • Wong A.J.
      • Vogelstein B.
      • Zalutsky M.R.
      • Fuller G.N.
      • Archer G.E.
      • Friedman H.S.
      • Kwatra M.M.
      • Bigner S.H.
      • Bigner D.D.
      ,
      • Wickstrand C.J.
      • Hale L.P.
      • Batra S.K.
      • Hill M.L.
      • Humphrey P.A.
      • Kurpad S.N.
      • McLendon R.E.
      • Moscatello D.K.
      • Pegram C.N.
      • Reist C.J.
      • Traweek S.T.
      • Wong A.J.
      • Zalutsky M.R.
      • Bigner D.D.
      ) and a number of different transfectants (
      • Batra S.K.
      • Castelino-Prabhu S.
      • Wikstrand C.J.
      • Zhu X.
      • Humphrey P.A.
      • Friedman H.S.
      • Bigner D.D.
      ,
      • Wickstrand C.J.
      • Hale L.P.
      • Batra S.K.
      • Hill M.L.
      • Humphrey P.A.
      • Kurpad S.N.
      • McLendon R.E.
      • Moscatello D.K.
      • Pegram C.N.
      • Reist C.J.
      • Traweek S.T.
      • Wong A.J.
      • Zalutsky M.R.
      • Bigner D.D.
      ,
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ), significant accumulations in the perinuclear area have also been observed, which suggests aberrant trafficking of this receptor variant (
      • Ekstrand A.J.
      • Liu L.
      • He J.
      • Hamid M.L.
      • Longo N.
      • Collins V.P.
      • James C.D.
      ,
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ). A number of other functional differences between EGFRvIII and normal EGF receptor have been characterized. Although EGFRvIII fails to bind EGF, the receptors can dimerize, and the tyrosine kinase in the intracellular portion of the receptor is constitutively activated (
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ,
      • Huang H.-J.S.
      • Nagane M.
      • Klingbeil C.K.
      • Lin H.
      • Nishikawa R.
      • Ji X.-D.
      • Huang C.-M.
      • Gill G.N.
      • Wiley H.S.
      • Cavenee W.K.
      ), so that the receptor undergoes autophosphorylation as well as phosphorylating substrates such as Shc (
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ,
      • Huang H.-J.S.
      • Nagane M.
      • Klingbeil C.K.
      • Lin H.
      • Nishikawa R.
      • Ji X.-D.
      • Huang C.-M.
      • Gill G.N.
      • Wiley H.S.
      • Cavenee W.K.
      ,
      • Prigent S.A.
      • Nagane M.
      • Lin H.
      • Huvar I.
      • Boss G.R.
      • Feramisco J.R.
      • Cavenee W.K.
      • Huang H.-J.S.
      ). While EGFRvIII can bind Grb2·mSos complexes, implicating activation of the Ras/Raf/MAP kinase pathway (
      • Buday L.
      • Downward J.
      ,
      • Rozakis-Adcock M.
      • McGlade J.
      • Mbamalu G.
      • Pelicci G.
      • Daly R.
      • Li W.
      • Batzer A.
      • Thomas S.
      • Brugge J.
      • Pelicci P.G.
      • Schlessinger J.
      • Pawson T.
      ), we found no increase in Ras·GTP levels and very low levels of MAP kinase activity (
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ,
      • Montgomery R.B.
      • Moscatello D.K.
      • Wong A.J.
      • Cooper J.A.
      • Stahl W.L.
      ), so this is unlikely to be the primary proliferative and transforming signal propagated by EGFRvIII. Interestingly, there are two points in the signal transduction pathway at which MAP kinase activation is down-regulated. Overexpression of EGFRvIII leads to decreased levels of Shc and Grb2, which could reduce Ras activation, and there is an increase in MAP kinase phosphatase activity in these cells as well (
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ,
      • Montgomery R.B.
      • Moscatello D.K.
      • Wong A.J.
      • Cooper J.A.
      • Stahl W.L.
      ).
      The normal EGF receptor is capable of initiating a variety of signaling cascades upon ligand activation. One such effector whose importance in tumorigenesis is becoming increasingly apparent is phosphatidylinositol 3-kinase (PI 3-kinase). PI 3-kinase was first shown to be important in transformation by the observations that it associates with polyoma virus middle T protein upon phosphorylation by c-Src, and that mutants of middle T which fail to recruit PI 3-kinase activity are impaired in their tumorigenic activity (,
      • Talmadge D.A.
      • Freund R.
      • Young A.T.
      • Dahl J.
      • Dawe C.J.
      • Benjamin T.L.
      ,
      • 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.
      ). In addition, PI 3-kinase activation has been shown to be essential for induction of DNA synthesis by EGF (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ). We therefore investigated the possible role played by this enzyme in transformation by the EGFRvIII, and we now report that PI 3-kinase is constitutively activated in EGFRvIII-transformed cells and is essential for transformation by this receptor variant.

      DISCUSSION

      Work in this and other laboratories has demonstrated that expression of the EGFRvIII results in neoplastic transformation and enhanced tumorigenicity, which is due to its constitutive kinase activity (
      • Nishikawa R.
      • Ji X.-D.
      • Harmon R.C.
      • Lazar C.S.
      • Gill G.N.
      • Cavenee W.K.
      • Su Huang H.-J.
      ,
      • Batra S.K.
      • Castelino-Prabhu S.
      • Wikstrand C.J.
      • Zhu X.
      • Humphrey P.A.
      • Friedman H.S.
      • Bigner D.D.
      ,
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ). While activation of the normal EGF receptor results in activation of MAP kinase via Ras (
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ,
      • Buday L.
      • Downward J.
      ), our studies on EGFRvIII showed only a low level of activation of the Ras-MAP kinase pathway, which was due to decreases in Shc and Grb2 levels and induction of a MAP kinase phosphatase (
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ,
      • Montgomery R.B.
      • Moscatello D.K.
      • Wong A.J.
      • Cooper J.A.
      • Stahl W.L.
      ). The down-regulation of the MAP kinase pathway has also been observed in NIH 3T3 cells transformed by viral oncogenes such as v-src and v-ras, and the evidence supports a role for a MAP kinase phosphatase in these cells as well (
      • Stofega M.R.
      • Yu C.-L.
      • Wu J.
      • Jove R.
      ).
      Because PI 3-kinase is essential for DNA synthesis induced by EGF (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ), we studied this enzyme in cells expressing EGFRvIII for its possible contribution to transformation. We found that EGFRvIII-transformed cells exhibited a high constitutive level of PI 3-kinase activity not shown by cells overexpressing normal EGF receptor. Analysis of the kinetics of PI 3-kinase and MAP kinase activation revealed an important difference in the regulation of these pathways by the EGFRvIII relative to the normal EGF receptor. While the EGF-stimulated PI 3-kinase activity in CO12 20c2/b cells peaked quickly and declined to a moderate level by 12 h, the activity in HC2 20d2/c cells rose more slowly and did not decline throughout the period tested (Fig. 3 C). The slower rate of increase in PI 3-kinase activity in the EGFRvIII-expressing cells was most likely due to the gradual decline in tyrphostin activity, but the lack of subsequent down-regulation of PI 3-kinase activity cannot be so explained, because HC2 20d2/c cells which have never been exposed to the drug exhibit high PI 3-kinase activity (Fig. 1). One possibility is that cells achieve down-regulation of EGF receptor-initiated PI 3-kinase activity primarily by down-regulation of the number of receptors. The EGFRvIII does not bind ligand and is not actively down-regulated despite its constitutive activation (
      • Moscatello D.K.
      • Montgomery R.B.
      • Sundareshan P.
      • McDanel H.
      • Wong M.Y.
      • Wong A.J.
      ,
      • Huang H.-J.S.
      • Nagane M.
      • Klingbeil C.K.
      • Lin H.
      • Nishikawa R.
      • Ji X.-D.
      • Huang C.-M.
      • Gill G.N.
      • Wiley H.S.
      • Cavenee W.K.
      ), and this may account for the lack of decrease in PI 3-kinase activity in HC2 20d2/c cells. In contrast, MAP kinase activity declines to barely above basal levels within about 12 h in both cell lines, indicating that regulation of this pathway primarily occurs downstream of the receptor. It further suggests that while prolonged, high level PI 3-kinase activation is compatible with continuous growth, the prolonged, high level activation of MAP kinase is not essential. Thus, our results are in agreement with the recent report that prolonged MAP kinase activation in NIH 3T3 cells results in growth arrest (
      • Pumiglia K.M.
      • Decker S.J.
      ).
      While we found that p85 can associate with both normal EGF receptor and EGFRvIII, it is not clear that p85 can account for all PI 3-kinase activity associated with EGFRvIII. Although immunoprecipitation with anti-p85α/β antibody reduced the PI 3-kinase activity in anti-pTyr immunoprecipitates from CO12 20c2/b cells, it did not reduce the pTyr-associated activity in HC2 20d2/c cells (data not shown). Furthermore, we found elevated levels of p85α in HC2 20d2/c, as well as bands with molecular masses of 50 and 55 kDa which cross-reacted strongly with antibody to rat p85. This suggests that the constitutive activity of the EGFRvIII may influence the expression of PI 3-K adapter subunits. At least five forms of regulatory subunits have been cloned (
      • Carpenter C.
      • Cantley L.
      ), including 50- and 55-kDa splice variants of p85α. Further studies are necessary to determine whether these molecules are involved in the PI 3-kinase activity detected in EGFRvIII transfectants.
      There are several mechanisms by which PI 3-kinase may contribute to tumorigenesis. Inhibition of PI 3-kinase activation has been shown to block the EGF-dependent transformation of murine JB6 P+ cells (
      • Huang C.
      • Ma W.-Y.
      • Dong Z.
      ). We found that PI 3-kinase inhibitors inhibited both monolayer growth in low serum and anchorage-independent growth of cells expressing normal EGF receptor and EGFRvIII. These inhibitors also caused a partial reversion of the transformed morphology of HC2 20d2/c and blocked the EGF-induced transformed morphology of CO12 20c2/b. PI 3-kinase activity can influence cell morphology, as it has been shown to affect cytoskeletal organization (
      • Kapeller R.
      • Cantley L.C.
      , ,
      • Carpenter C.
      • Cantley L.
      ). For instance, PI 3-kinase interacts with Rac·GTP, a member of the Rho family of small G proteins which regulate the actin cytoskeleton (
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ). Nagane et al. recently reported that EGFRvIII expression reduces apoptosis of glioblastoma cells bothin vitro and in vivo (
      • Nagane M.
      • Coufal F.
      • Lin H.
      • Bögler O.
      • Cavenee W.K.
      • Su Huang H.-J.
      ). As activation of the Raf/MAP kinase pathway by Ras in the absence of PI 3-kinase activity was recently shown to promote apoptosis in fibroblasts (
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ), and PI 3-kinase activity has been shown to be essential for survival of a number of cell types (
      • Yao R.
      • Cooper G.M.
      ,
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ), these data suggest another mechanism by which consitutive PI 3-kinase activity contributes to tumorigenesis. Thus, because PI 3-kinase can play a central role in growth, morphological transformation, and the inhibition of cell death by the both normal EGF receptor and EGFRvIII, it seems likely that enhancement of this activity provides an important selective advantage for EGFRvIII-expressing tumor cells in vivo.

      References

        • Harris A.L.
        • Nicholson S.
        • Sainsbury R.
        • Wright C.
        • Farndon J.
        Natl. Cancer Inst. Monogr. 1992; 11: 181-187
        • Schlegel J.
        • Merdes A.
        • Stumm G.
        • Albert F.K.
        • Forsting M.
        • Hynes N.
        • Kiessling M.
        Int. J. Cancer. 1994; 56: 72-77
        • Wong A.J.
        • Ruppert J.M.
        • Bigner S.H.
        • Grzeschik C.H.
        • Humphrey P.A.
        • Bigner D.S.
        • Vogelstein B.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2965-2969
        • Yamazaki H.
        • Fukui Y.
        • Ueyama Y.
        • Tamaoki N.
        • Kawamoto T.
        • Taniguchi S.
        • Shibuya M.
        Mol. Cell. Biol. 1988; 8: 1816-1820
        • Schwechheimer K.
        • Huang S.
        • Cavenee W.K.
        Int. J. Cancer. 1995; 62: 145-148
        • Moscatello D.K.
        • Holgado-Madruga M.
        • Godwin A.K.
        • Ramirez G.
        • Gunn G.
        • Zoltick P.W.
        • Biegel J.A.
        • Hayes R.L.
        • Wong A.J.
        Cancer Res. 1995; 55: 5536-5539
        • Nishikawa R.
        • Ji X.-D.
        • Harmon R.C.
        • Lazar C.S.
        • Gill G.N.
        • Cavenee W.K.
        • Su Huang H.-J.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731
        • Ekstrand A.J.
        • Longo N.
        • Hamid M.L.
        • Olson J.J.
        • Liu L.
        • Collins V.P.
        • James C.D.
        Oncogene. 1994; 9: 2313-2320
        • Garcia de Palazzo I.E.
        • Adams G.P.
        • Sundareshan P.
        • Wong A.J.
        • Testa J.R.
        • Bigner D.D.
        • Weiner L.M.
        Cancer Res. 1993; 53: 3217-3220
        • Nagane M.
        • Coufal F.
        • Lin H.
        • Bögler O.
        • Cavenee W.K.
        • Su Huang H.-J.
        Cancer Res. 1996; 56: 5079-5086
        • Ekstrand A.J.
        • Liu L.
        • He J.
        • Hamid M.L.
        • Longo N.
        • Collins V.P.
        • James C.D.
        Oncogene. 1995; 10: 1455-1460
        • Batra S.K.
        • Castelino-Prabhu S.
        • Wikstrand C.J.
        • Zhu X.
        • Humphrey P.A.
        • Friedman H.S.
        • Bigner D.D.
        Cell Growth Differ. 1995; 6: 1251-1259
        • Humphrey P.
        • Wong A.J.
        • Vogelstein B.
        • Zalutsky M.R.
        • Fuller G.N.
        • Archer G.E.
        • Friedman H.S.
        • Kwatra M.M.
        • Bigner S.H.
        • Bigner D.D.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4207-4211
        • Wickstrand C.J.
        • Hale L.P.
        • Batra S.K.
        • Hill M.L.
        • Humphrey P.A.
        • Kurpad S.N.
        • McLendon R.E.
        • Moscatello D.K.
        • Pegram C.N.
        • Reist C.J.
        • Traweek S.T.
        • Wong A.J.
        • Zalutsky M.R.
        • Bigner D.D.
        Cancer Res. 1995; 51: 3140-3148
        • Moscatello D.K.
        • Montgomery R.B.
        • Sundareshan P.
        • McDanel H.
        • Wong M.Y.
        • Wong A.J.
        Oncogene. 1996; 13: 85-96
        • Huang H.-J.S.
        • Nagane M.
        • Klingbeil C.K.
        • Lin H.
        • Nishikawa R.
        • Ji X.-D.
        • Huang C.-M.
        • Gill G.N.
        • Wiley H.S.
        • Cavenee W.K.
        J. Biol. Chem. 1997; 272: 2927-2935
        • Prigent S.A.
        • Nagane M.
        • Lin H.
        • Huvar I.
        • Boss G.R.
        • Feramisco J.R.
        • Cavenee W.K.
        • Huang H.-J.S.
        J. Biol. Chem. 1996; 271: 25639-25645
        • Buday L.
        • Downward J.
        Cell. 1993; 73: 611-620
        • Rozakis-Adcock M.
        • McGlade J.
        • Mbamalu G.
        • Pelicci G.
        • Daly R.
        • Li W.
        • Batzer A.
        • Thomas S.
        • Brugge J.
        • Pelicci P.G.
        • Schlessinger J.
        • Pawson T.
        Nature. 1992; 360: 689-692
        • Montgomery R.B.
        • Moscatello D.K.
        • Wong A.J.
        • Cooper J.A.
        • Stahl W.L.
        J. Biol. Chem. 1995; 270: 30562-30566
        • Kapeller R.
        • Cantley L.C.
        BioEssays. 1994; 16: 565-576
        • Hunter T.
        Cell. 1997; 88: 333-346
        • Talmadge D.A.
        • Freund R.
        • Young A.T.
        • Dahl J.
        • Dawe C.J.
        • Benjamin T.L.
        Cell. 1989; 59: 55-65
        • 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
        • Roche S.
        • Koegl M.
        • Courtneidge S.A.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9185-9189
        • Holgado-Madruga M.
        • Emlet D.R.
        • Moscatello D.K.
        • Godwin A.K.
        • Wong A.J.
        Nature. 1996; 379: 560-564
        • Hu P.
        • Margolis B.
        • Skolnik E.Y.
        • Lammers R.
        • Ullrich A.
        • Schlessinger J.
        Mol. Cell. Biol. 1992; 12: 981-990
        • Levitzki A.
        • Gazit A.
        Science. 1995; 267: 1782-1788
        • Wymann M.P.
        • Bulgarelli-Leva G.
        • Zvelebil M.J.
        • Pirola L.
        • Vanhaesebroeck B.
        • Waterfield M.D.
        • Panayotou G.
        Mol. Cell. Biol. 1996; 16: 1722-1733
        • Han Y.
        • Caday C.G.
        • Nanda A.
        • Cavenee W.K.
        • Su Huang H.-J.
        Cancer Res. 1996; 56: 3859-3861
        • Stover D.R.
        • Becker M.
        • Liebetanz J.
        • Lydon N.B.
        J. Biol. Chem. 1995; 270: 15591-15597
        • Antonetti D.A.
        • Algenstaedt P.
        • Kahn C.R.
        Mol. Cell. Biol. 1996; 16: 2195-2203
        • Carpenter C.
        • Cantley L.
        Biochim. Biophys. Acta. 1996; 1288: M11-M16
        • Panaretou C.
        • Domin J.
        • Cockroft S.
        • Waterfield M.D.
        J. Biol. Chem. 1997; 272: 2477-2485
        • Stofega M.R.
        • Yu C.-L.
        • Wu J.
        • Jove R.
        Cell Growth Differ. 1997; 8: 113-119
        • Pumiglia K.M.
        • Decker S.J.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 448-452
        • Huang C.
        • Ma W.-Y.
        • Dong Z.
        Mol. Cell. Biol. 1996; 16: 6427-6435
        • Tolias K.F.
        • Cantley L.C.
        • Carpenter C.L.
        J. Biol. Chem. 1995; 270: 17656-17659
        • Kauffmann-Zeh A.
        • Rodriguez-Viciana P.
        • Ulrich E.
        • Gilbert C.
        • Coffer P.
        • Downward J.
        • Evan G.
        Nature. 1997; 385: 544-548
        • Yao R.
        • Cooper G.M.
        Oncogene. 1996; 13: 343-351
        • Parrizas M.
        • Saltiel A.R.
        • LeRoith D.
        J. Biol. Chem. 1997; 272: 154-161