Abstract
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.
Highlights
(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
The cause of this increase in the assortment of phosphorylated proteins has not yet been completely explored; 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 [10, 13]
1 The 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 ϭ 1000 mP units); Y1173 peptide, 13-residue peptide mimic for the minal truncated EGFR [9], whether the mutated EGFR directly phosphorylates this substrate remains unclear
Summary
Materials—EGFR was purified from A431 cells as described previously [20], and the ct1022 mutant was purified from B82L fibroblasts [9, 21]. We inserted it into a pET vector (Novagen) to produce protein with an NH2-terminal His tag to facilitate purification. Unphosphorylated forms of angiotensin II and the Y1173 peptide were purchased from the same vendors at Ն98 and 99% purity, respectively Aqueous samples of these peptides were quantified by amino acid analysis at the Medical College of Wisconsin Protein/Nucleic Acid Facility. FP Assay Conditions—Each experiment (except the [␥-32P]ATP time courses) was performed using PanVera’s tyrosine kinase assay kit, green. EGFR was preincubated for 15 min with the indicated concentration of ATP to autophosphorylate the kinase. Assays performed in endpoint mode were quenched by the addition of protein-tyrosine kinase quench buffer (12 mM EDTA, pH 7.5) and antibody and tracer to 1ϫ, for final concentrations of 6 mM EDTA, 1ϫ antibody, and 1ϫ tracer. [␥-32P]ATP Phosphate Incorporation Assay—The amount of phosphate incorporated into the substrate was quantified using radiolabeled
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