A Carboxyl-terminal Mutation of the Epidermal Growth Factor Receptor Alters Tyrosine Kinase Activity and Substrate Specificity as Measured by a Fluorescence Polarization Assay

The expression of certain COOH-terminal truncation mutants of the epidermal growth factor receptor (EGFR) can lead to cell transformation, and with ligand stimulation, a broader spectrum of phosphorylated proteins appears compared with EGF-treated cells expressing wild-type EGFR. Accordingly, it has been proposed that elements within the COOH terminus may determine substrate specificity of the EGFR tyrosine kinase (Decker, S. J., Alexander, C., and Habib, T. (1992) J. Biol. Chem. 267, 1104–1108; Walton, G. M., Chen, W. S., Rosenfeld, M. G., and Gill, G. N. (1990) J. Biol. Chem. 265, 1750–1754). To address this hypothesis, we analyzed in vitro the steady-state kinetic parameters for phosphorylation of several substrates by both wild-type EGFR and an oncogenic EGFR mutant (the ct1022 mutant) truncated at residue 1022. The substrates included: (i) a phospholipase C-γ fragment (residues 530–850); (ii) the 46-kDa isoform of the Shc adapter protein; (iii) a 13-residue peptide mimic for the region around the major autophosphorylation tyrosine and the Shc binding site (the Y1173 peptide); (iv) a poly(Glu,Tyr) 4:1 copolymer; and (v) the 8-residue peptide, angiotensin II. Our data demonstrate that the steady-state kinetic parameters for the ct1022 mutant differ from those of the wild-type enzyme, and the differences are substrate-dependent. These results support the concept that this oncogenic truncation/mutation alters EGFR substrate specificity, rather than causing a general alteration of activity. We performed the experiments using a non-radioactive fluorescence polarization assay that quantifies the degree of phosphorylation of peptide as well as natural substrates. The results are consistent with those from the traditional [γ-32P]ATP/filtration assay. The expression of certain COOH-terminal truncation mutants of the epidermal growth factor receptor (EGFR) can lead to cell transformation, and with ligand stimulation, a broader spectrum of phosphorylated proteins appears compared with EGF-treated cells expressing wild-type EGFR. Accordingly, it has been proposed that elements within the COOH terminus may determine substrate specificity of the EGFR tyrosine kinase (Decker, S. J., Alexander, C., and Habib, T. (1992) J. Biol. Chem. 267, 1104–1108; Walton, G. M., Chen, W. S., Rosenfeld, M. G., and Gill, G. N. (1990) J. Biol. Chem. 265, 1750–1754). To address this hypothesis, we analyzed in vitro the steady-state kinetic parameters for phosphorylation of several substrates by both wild-type EGFR and an oncogenic EGFR mutant (the ct1022 mutant) truncated at residue 1022. The substrates included: (i) a phospholipase C-γ fragment (residues 530–850); (ii) the 46-kDa isoform of the Shc adapter protein; (iii) a 13-residue peptide mimic for the region around the major autophosphorylation tyrosine and the Shc binding site (the Y1173 peptide); (iv) a poly(Glu,Tyr) 4:1 copolymer; and (v) the 8-residue peptide, angiotensin II. Our data demonstrate that the steady-state kinetic parameters for the ct1022 mutant differ from those of the wild-type enzyme, and the differences are substrate-dependent. These results support the concept that this oncogenic truncation/mutation alters EGFR substrate specificity, rather than causing a general alteration of activity. We performed the experiments using a non-radioactive fluorescence polarization assay that quantifies the degree of phosphorylation of peptide as well as natural substrates. The results are consistent with those from the traditional [γ-32P]ATP/filtration assay. Binding of EGF 1The abbreviations used are: EGF, epidermal growth factor; CR, cysteine-rich domain; ct1022 mutant, EGFR mutant possessing a truncation in the COOH terminus after residue 1022; EGFR, EGF receptor; FP, fluorescence polarization; mP, millipolarization unit (1 polarization unit = 1000 mP units); Y1173 peptide, 13-residue peptide mimic for the region around the major autophosphorylation tyrosine (tyrosine 1173) and the Shc binding site; v-erbB, avian erythroblastosis virus; PLC-γ, phospholipase C-γ.1The abbreviations used are: EGF, epidermal growth factor; CR, cysteine-rich domain; ct1022 mutant, EGFR mutant possessing a truncation in the COOH terminus after residue 1022; EGFR, EGF receptor; FP, fluorescence polarization; mP, millipolarization unit (1 polarization unit = 1000 mP units); Y1173 peptide, 13-residue peptide mimic for the region around the major autophosphorylation tyrosine (tyrosine 1173) and the Shc binding site; v-erbB, avian erythroblastosis virus; PLC-γ, phospholipase C-γ. and other ligands to EGFR and its relatives (ErbB2, ErbB3, and ErbB4) triggers a highly complicated signaling network (1Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5474) Google Scholar, 2Salomon D. Gullick W. Signal. 2001; 2: 4-11Google Scholar). Overexpression as well as oncogenic deletions and truncations of EGFR have been observed in many cancer types, including glioblastomas and non-small cell lung, pancreatic, breast, head and neck, colon, prostate, ovarian, and cervical tumors (3Wells A. Signal. 2000; 1: 4-10Google Scholar, 4Ekstrand A.J. Sugawa N. James C.D. Collins V.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4309-4313Crossref PubMed Scopus (495) Google Scholar, 5Frederick L. Wang X.Y. Eley G. James C.D. Cancer Res. 2000; 60: 1383-1387PubMed Google Scholar). The most commonly found mutation (EGFRvIII) involves a deletion in the NH2-terminal portion of EGFR, from residues 6 to 273 (5Frederick L. Wang X.Y. Eley G. James C.D. Cancer Res. 2000; 60: 1383-1387PubMed Google Scholar, 6Wikstrand C.J. Sampson J.H. Bigner D.D. Expert Opin. Ther. Targets. 2000; 4: 497-514Google Scholar, 7Moscatello D.K. Holgado-Madruga M. Emlet D.R. Montgomery R.B. Wong A.J. J. Biol. Chem. 1998; 273: 200-206Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Several other types of rearrangements have been found, however, including even a tandem duplication of the tyrosine kinase and calcium internalization domains (4Ekstrand A.J. Sugawa N. James C.D. Collins V.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4309-4313Crossref PubMed Scopus (495) Google Scholar, 5Frederick L. Wang X.Y. Eley G. James C.D. Cancer Res. 2000; 60: 1383-1387PubMed Google Scholar, 8Fenstermaker R.A. Ciesielski M.J. Castiglia G.J. Oncogene. 1998; 16: 3435-3443Crossref PubMed Scopus (27) Google Scholar). Half of all glioblastomas exhibit EGFR amplification, and of these, 15% possess COOH-terminal truncated EGFR (at residue 958) (5Frederick L. Wang X.Y. Eley G. James C.D. Cancer Res. 2000; 60: 1383-1387PubMed Google Scholar). Furthermore, expressing certain COOH-terminal truncation mutants in cell culture results in dramatic EGF-induced increases in the variety of phosphorylated proteins present compared with those observed in cells expressing wild-type EGFR (9Kim Y.N. Wiepz G.J. Guadarrama A.G. Bertics P.J. J. Biol. Chem. 2000; 275: 7481-7491Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Decker S.J. Alexander C. Habib T. J. Biol. Chem. 1992; 267: 1104-1108Abstract Full Text PDF PubMed Google Scholar, 11Walton G.M. Chen W.S. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1990; 265: 1750-1754Abstract Full Text PDF PubMed Google Scholar, 12Sasaoka T. Langlois W.J. Bai F. Rose D.W. Leitner J.W. Decker S.J. Saltiel A. Gill G.N. Kobayashi M. Draznin B. Olefsky J.M. J. Biol. Chem. 1996; 271: 8338-8344Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The cause of this increase in the assortment of phosphorylated proteins has not yet been completely explored; however, EGF-induced receptor internalization and degradation occurred at a slower rate with an oncogenic EGFR mutant possessing a COOH-terminal truncation at residue 973 rather than with wild-type EGFR (10Decker S.J. Alexander C. Habib T. J. Biol. Chem. 1992; 267: 1104-1108Abstract Full Text PDF PubMed Google Scholar, 13Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Crossref PubMed Scopus (342) Google Scholar). Cells expressing this mutant develop a transformed phenotype when grown in the presence of low EGF concentrations (13Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Crossref PubMed Scopus (342) Google Scholar). Moreover, data suggest that Δ973-EGFR and ErbB2 heterodimers form in cells expressing this mutant. The ErbB2 in these cells is tyrosyl phosphorylated in the absence of added ligand (12Sasaoka T. Langlois W.J. Bai F. Rose D.W. Leitner J.W. Decker S.J. Saltiel A. Gill G.N. Kobayashi M. Draznin B. Olefsky J.M. J. Biol. Chem. 1996; 271: 8338-8344Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Interestingly, the oncogene product of the avian erythroblastosis virus (v-erbB) is an NH2- and COOH-terminal truncated version of EGFR (ErbB1) (14Lax I. Kris R. Sasson I. Ullrich A. Hayman M.J. Beug H. Schlessinger J. EMBO J. 1985; 4: 3179-3182Crossref PubMed Scopus (17) Google Scholar, 15Maihle N.J. Kung H.J. Biochim. Biophys. Acta. 1989; 948: 287-304PubMed Google Scholar). Cellular changes in phosphotyrosine levels may occur not only as a result of changes in the catalytic properties of the enzyme, but may also reflect alterations in the regulation of other kinases and/or phosphatases in addition to the possible changes in receptor internalization and dimerization already mentioned. For example, although cellular phosphorylation of caveolin-1 is more pronounced in cells expressing COOH-terminal truncated EGFR (9Kim Y.N. Wiepz G.J. Guadarrama A.G. Bertics P.J. J. Biol. Chem. 2000; 275: 7481-7491Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), whether the mutated EGFR directly phosphorylates this substrate remains unclear. Therefore, to precisely isolate the effects of an EGFR mutation on the tyrosine kinase activity, it was necessary to purify the mutant and compare its activity to that of wild-type enzyme in a biochemical assay. In this study, an in vitro fluorescence polarization (FP) assay (described below) has been used quantitatively to compare steady-state kinetic parameters of wild-type EGFR with those of the ct1022 mutant. FP measures molecular rotation occurring during the fluorescence lifetime, the period between excitation and emission of a fluorophore. Relatively small molecules tumble quickly in solution. When excited by plane-polarized light, these molecules emit light that is relatively depolarized. In contrast, larger molecules and complexes tumble more slowly in solution, and emit more highly polarized light (16Nasir M.S. Jolley M.E. Comb. Chem. High Throughput Screen. 1999; 2: 177-190PubMed Google Scholar). The FP competitive immunoassay involves the addition of a fluorescently labeled phosphopeptide tracer (a relatively small molecule) and an antibody (a relatively large molecule) specific for phosphorylated tyrosine to a kinase reaction. Initially, the phosphopeptide tracer binds to the antibody. As the kinase reaction proceeds, however, phosphorylated substrate binds to the antibody and displaces the tracer. Free fluorescent tracer possesses a lower polarization than the fluorescent tracer the polarization in polarization can with a FP G.J. J. Screen. 2000; PubMed Scopus Google Scholar, W.J. T. PubMed Scopus Google Scholar). To determine whether the steady-state kinetic parameters for wild-type EGFR and for the ct1022 mutant with the of substrates peptide natural substrates (the Shc adapter and a fragment the and a poly(Glu,Tyr) 4:1 the of EGFR (3Wells A. Signal. 2000; 1: 4-10Google Scholar). of EGFR residues that for this enzyme, of the COOH-terminal substrate with the rather than phosphorylation of the kinase is for cellular J. Sliwkowski M.X. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the that of the dimerization is with the kinase and we that truncations in the COOH terminus may cause changes that substrate In this we used an FP competitive immunoassay to the of phosphorylated substrate in kinase We FP to concentrations of phosphorylated of In we performed phosphorylation with EGFR and the ct1022 mutant for of the EGFR substrates at substrate We the reaction using We the of phosphorylated substrate at using the for We the from these experiments as a of substrate and the data to the We and compared the steady-state kinetic parameters for phosphorylation by both wild-type EGFR and the ct1022 mutant for was from cells as L. Bertics P.J. J. 1992; PubMed Scopus (27) Google Scholar), and the ct1022 mutant was from (9Kim Y.N. Wiepz G.J. Guadarrama A.G. Bertics P.J. J. Biol. Chem. 2000; 275: 7481-7491Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, J. Wiepz G.J. Guadarrama A.G. Bertics P.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The for expressing the 46-kDa isoform of the Shc adapter G. L. F. J. F. G. I. T. Cell. 1992; Full Text PDF PubMed Scopus Google was a from G. of We it a to with an NH2-terminal to was transformed cells Cells grown at with at an = and grown for Cells using a and the Shc adapter was using a The was with a and using FP used tyrosine kinase assay for the FP was using fluorescence polarization the and used with the For the was from and used for angiotensin and the Y1173 peptide, a 13-residue peptide mimic for the region around the Y1173 autophosphorylation site The version of angiotensin was from at The version of the Y1173 peptide was by the of at of angiotensin and the Y1173 peptide from the at and of these by at the of The poly(Glu,Tyr) 4:1 and from and a fragment of (residues domains and was from FP the was performed using tyrosine kinase assay and at in the reaction and EGFR was for with the of to the The reaction was with the addition of and the in polarization was as The for autophosphorylation P.J. Gill G.N. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). Furthermore, we observed that in polarization by autophosphorylation within the not that observed in polarization was of substrate not FP in the presence of antibody and tracer at performed in by the addition of kinase and antibody and tracer to for concentrations of 6 and tracer. of the substrate was using P.J. Gill G.N. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar, W. Bertics P.J. Gill G.N. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the reaction and with of of was and the poly(Glu,Tyr) 4:1 the Shc adapter was from the by The the product was by data with version using the mP = polarization in of (1 mP = to the polarization observed when the of present in the substrate is at the between its and for of is as = of and = the rate for of is as is the is is = of reaction. for EGFR and the ct1022 polarization to the of phosphorylated product using the phosphorylated of the substrates We used these to the of phosphorylated product that a in polarization and of the phosphorylated substrates the antibody and tracer added the used for the assay for phosphorylated of angiotensin and the Y1173 peptide in this phosphorylated of the fragment and poly(Glu,Tyr) 4:1 not to by using a relatively enzyme phosphorylation reaction and EGFR ct1022 mutant as well as fragment poly(Glu,Tyr) 4:1 and was at for of these phosphorylated substrates and analyzed as for the peptide substrates. For we and polarization as a of the phosphorylated substrate to the as in These data by a in of of for the poly(Glu,Tyr) 4:1 of for the peptide, of for the and of for angiotensin II. The a of the data using the for a with = of for the poly(Glu,Tyr) 4:1 of for the peptide, of for the and of for angiotensin II. The for angiotensin may of its J. 1998; PubMed Scopus Google Scholar). The results from experiments concentrations of enzyme and for the fragment and poly(Glu,Tyr) 4:1 that reaction completely phosphorylated substrates not the we performed FP and the of phosphorylated product from the transformed for substrate FP, we substrate phosphorylation directly for substrate at substrate data for both the Y1173 substrate and the fragment are in In the FP product is to fluorescent phosphopeptide tracer and the binding of phosphorylated substrate the in product product displaces all of the fluorescent phosphopeptide tracer. Therefore, of polarization are not As the assay to the in polarization to the polarization of the tracer. the for a not data for the the for a the data with = and for the Y1173 peptide and the fragment We used the data to to the of substrate phosphorylation for with the and the results with to These data and from the of these experiments performed at substrate and data for the Y1173 peptide the of wild-type EGFR ct1022 mutant with by the addition of the Y1173 transformed of of EGFR as and as a of substrate to the A. and W. H. Scholar), and the steady-state kinetic parameters The mutant enzyme a for the phosphorylation of the Y1173 peptide substrate than EGFR. The steady-state kinetic parameters in this for substrate are in kinetic parameters by FP for wild-type EGFR and the ct1022 of are of of 4:1 is the steady-state kinetic for the reaction by the ct1022 mutant by the kinetic for wild-type has been for in wild-type EGFR specific has been for in wild-type EGFR specific adapter has been for in wild-type EGFR specific of are of of is the steady-state kinetic for the reaction by the ct1022 mutant by the kinetic for wild-type has been for in wild-type EGFR specific activity. in a the poly(Glu,Tyr) 4:1 and the fragment more than phosphorylation the kinetic parameters for these substrates are S. B. S. D. G. S. J. 2001; PubMed Scopus Google Scholar). has that are phosphorylated by EGFR and J.W. S. G. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). Therefore, the fragment of we used as a substrate (residues possesses that EGFR can and kinetic parameters for angiotensin and the fragment within of those using the assay W. Bertics P.J. Gill G.N. J. Biol. Chem. Full Text PDF PubMed Google Scholar). J. of for the 4:1 experiments that the poly(Glu,Tyr) 4:1 and fragment used for the been completely we these results by an for a for the poly(Glu,Tyr) 4:1 for and by We analyzed reaction with the traditional kinase assay using and other with the FP that and substrate the for all the of substrate phosphorylation to a in polarization We the phosphorylation of the poly(Glu,Tyr) 4:1 by the ct1022 mutant in the FP assay in both and and by the assay as was and the and that the assay reaction of for the to the for a with = = of and = of For of the FP the of to the substrate was using the that the of = of of was as a of of and data to with assay = of = and assay = of = These to other and to that using a of Therefore, the kinetic parameters by polarization to phosphorylated product with of a Furthermore, the the data a by For the = mP, = of and = For the = mP, = of and = reaction was performed that also of but was analyzed in by FP in the fluorescence polarization within of those in the absence of not a in the reaction was by FP, and the of at was from the data at substrate and steady-state kinetic parameters EGFR the ct1022 mutant with for substrate was added and polarization was transformed of of EGFR as and by the of these as a of substrate and these data to the The steady-state kinetic parameters for the poly(Glu,Tyr) 4:1 as well as the other substrates are in and the for the phosphorylation of this substrate is with the ct1022 mutant. a for the 46-kDa isoform of the Shc adapter was by and ct1022 mutant at analyzed by FP in using the fluorescence polarization and the other by the assay as of was not and to those for the poly(Glu,Tyr) 4:1 with = from the using a and = of not kinetic parameters for of phosphorylation by wild-type and mutant enzyme by of substrate to the and the kinetic parameters for the Shc adapter as well as all of the other substrates. The of the FP to have used to demonstrate that a tyrosine kinase FP assay may used may may a traditional [γ-32P]ATP/filtration as an FP to the of phosphorylated product to the observed polarization For substrate phosphorylation was by FP as a of at substrate We have data and to the of The in this as a of substrate for and data by the in the of steady-state kinetic data are and These steady-state kinetic parameters by FP are in I. The FP assay has the of and to Furthermore, we have that this FP assay can performed at relatively concentrations more with the traditional assay. In the is directly to the of and to the of Therefore, the of to at are in the of concentrations for D. S.J. PubMed Scopus Google Scholar), in cells M.E. G.J. J. 273: PubMed Google Scholar), and in cells C. S.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We found that the for in the for certain substrates. For example, for the poly(Glu,Tyr) 4:1 = using the FP assay at as the = 6 and the mutant) = data not we observed that the for with the fragment EGFR, substrate = using the FP assay at as is than not Therefore, the of in the assay it the for increase Furthermore, for the Shc adapter we found that was than = using the FP assay at the was than = using the FP assay at FP may an for the of the wild-type enzyme for this natural Moreover, we have that this assay is in The of the the assay for the poly(Glu,Tyr) 4:1 for this FP the for phosphorylated product to to with the antibody and tracer is that the results are the assay may performed directly in an FP, rather than by with and for antibody and tracer to to Therefore, during in the absence of to determine the to the reaction with is to in that the of polarization to phosphorylation of substrate is not and and and and that the assay is more to phosphorylation all of the tracer is In to determine kinetic are to PubMed Scopus Google Scholar), and this is also for the FP however, the of polarization as tracer is of for by the ct1022 to for data demonstrate that the steady-state kinetic parameters for the ct1022 mutant differ from those of the wild-type enzyme, and these differences on the of the substrate The increases in for certain substrates with the of internalization and possible with ErbB2 may the broader spectrum of proteins phosphorylated when COOH-terminal truncated mutant of EGFR are (9Kim Y.N. Wiepz G.J. Guadarrama A.G. Bertics P.J. J. Biol. Chem. 2000; 275: 7481-7491Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Decker S.J. Alexander C. Habib T. J. Biol. Chem. 1992; 267: 1104-1108Abstract Full Text PDF PubMed Google Scholar, 11Walton G.M. Chen W.S. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1990; 265: 1750-1754Abstract Full Text PDF PubMed Google Scholar, 12Sasaoka T. Langlois W.J. Bai F. Rose D.W. Leitner J.W. Decker S.J. Saltiel A. Gill G.N. Kobayashi M. Draznin B. Olefsky J.M. J. Biol. Chem. 1996; 271: 8338-8344Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 13Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Crossref PubMed Scopus (342) Google Scholar). the for the increase in the for the Y1173 peptide and the poly(Glu,Tyr) 4:1 is more product rather than an increase in the rate of the catalytic may as product has been found to for the of phosphorylation of a peptide substrate by ErbB2 A.J. 2000; PubMed Scopus Google Scholar, A. and W. H. Scholar). truncated EGFR on the other have been to than wild-type EGFR in vitro with peptide substrates J.M. J. 1999; PubMed Scopus Google Scholar). In addition to the performed with differences in the of specific phosphorylated proteins in the presence of COOH-terminal truncated of EGFR compared with those in the presence of wild-type EGFR have been in The results suggest that phosphorylation of both and was in EGFR mutants truncated at residues however, the truncated EGF phosphorylation of the Shc adapter (10Decker S.J. Alexander C. Habib T. J. Biol. Chem. 1992; 267: 1104-1108Abstract Full Text PDF PubMed Google Scholar, N. A. Y. S. S. T. Y. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, N. M. M. Mol. Cell. Biol. PubMed Scopus Google Scholar, C. L. G. Oncogene. Google Scholar). Furthermore, caveolin-1 phosphorylation occurred in cells expressing truncated EGF but not in cells expressing wild-type EGFR (9Kim Y.N. Wiepz G.J. Guadarrama A.G. Bertics P.J. J. Biol. Chem. 2000; 275: 7481-7491Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). As changes in phosphotyrosine levels observed in may of changes in the regulation of other kinases and/or and not to an alteration in the catalytic properties of the enzyme in cell growth not occur in cells expressing a ct1022 kinase but occur in cells expressing an EGFR mutant truncated at residue the of kinase activity for COOH-terminal truncation mutants (13Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Crossref PubMed Scopus (342) Google Scholar). Interestingly, we observed that the ct1022 mutant phosphorylated the fragment in vitro as as wild-type EGFR. of the tyrosine autophosphorylation remains in the ct1022 mutant and this phosphorylation site has been as the site for binding D. B. M. G. N. Ullrich A. Schlessinger J. EMBO J. 1992; PubMed Scopus Google Scholar). We that the lower for the ct1022 mutant with the wild-type for the Shc adapter may of slower product as product is for ErbB2 A.J. 2000; PubMed Scopus Google Scholar). Furthermore, the lower suggest a between the ct1022 mutant and Shc than between wild-type EGFR and In the absence of data the rate we determine whether the the for enzyme and we that the Shc adapter as well as with EGFR in and in the of the kinase (1Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5474) Google Scholar). In when compared with wild-type EGFR, several substrates are more phosphorylated by the ct1022 mutant, other substrates are well phosphorylated even phosphorylated by the mutant These data that COOH-terminal may in the substrate specificity of EGFR, in addition to as for We for the molecular necessary for expression of the Shc adapter and and for in tyrosine kinase assay We are also to and for the [γ-32P]ATP/filtration assay. We W. for in the and for in the