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-gamma 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 [gamma-32P]ATP/filtration assay.
Beebe
Jane A
JA
PanVera, Madison, Wisconsin 53719, USA. jane_beebe@panvera.com
Wiepz
Gregory J
GJ
Guadarrama
Arturo G
AG
Bertics
Paul J
PJ
Burke
Thomas J
TJ
D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D011994:Recombinant Proteins; D066246:ErbB Receptors