Inhibition of autophosphorylation of epidermal growth factor receptor by small peptides in vitro

Abstract

1. Inhibition of uncontrolled epidermal growth factor receptor (EGFR) is one of the approaches for the treatment of breast and lung cancers. We designed oligopeptides consisting of amino-acid sequences of the major (Y1068, Y1148, and Y1173) and minor (Y992) autophosphorylation sites of EGFR. These peptides may be exogenous substrates or pseudosubstrates that interfere with the autophosphorylation of EGFR. The effects of the peptides on autophosphorylation of EGFR were studied. 2. Purified EGFR was phosphorylated in vitro with EGF in the presence of various synthetic peptides. The phosphorylation level of EGFR was then evaluated after SDS-PAGE separation, followed by Western blot analysis with antiphosphotyrosine antibody. 3. Ac-VPEYINQ-NH2 (Y1068) and Ac-DYQQD-NH2 (Y1148) showed the most potent inhibitory effects, followed by Ac-ENAEYLR-NH2 (Y1173). These peptides at 4 mM suppressed phosphorylation to 30-50%. 4. Combination of the three kinds of peptides much more strongly inhibited autophosphorylation. The 50% inhibitory concentration (IC50) value was 0.5 mM as a mixture and was comparable to that of AG1478 (IC50, 0.3 mM) at 0.2 mM ATP. 5. Neither Ac-DIYET-NH2 or Ac-KIYEK-NH2, designed previously based on the amino-acid sequence of an autophosphorylation site of insulin receptor, nor their related (Ac-KIFMK-NH2) or unrelated (Ac-LPFFD-NH2) peptides showed an inhibitory effect. These results suggest that the small peptides that originated from the autophosphorylation sites of EGFR interact solely with EGFR. 6. The peptides containing the sequences surrounding Y1068, Y1148, and Y1173 may be a promising seed for the development of therapeutic agents for breast and lung cancers.

Figure 1

Phosphorylation of purified EGFR in the presence or absence of peptides. (a) Peptides including Y1148 (pY, phosphorylated tyrosine), (b) peptides including Y1173 and the control peptide, Ac-LPFFD-NH₂, (c) a peptide including Y1068, and (d) peptides including Y992. EGFR was incubated with or without peptides for 5 min at 37°C in the buffer containing 0.2 mM of ATP, except open bars shown in (c), where 0.02 mM of ATP was used. Results displayed on the top panels represent typical immunoblots (IB). ^*P<0.05, ^**P<0.01 versus EGF-stimulated tyrosine phosphorylation at 5-min incubation without peptides; n=4 for each lane.

Figure 2

Phosphorylation of purified EGFR in the presence or absence of mixtures of peptides. (a) A mixture of DYQQD and ENAEYLR and that of DYQQD and KYQQK (concentrations indicated are those of each component), (b) comparison of inhibitory effects by 8 mM DYQQD and two kinds of mixtures of peptides with that of 4 mM DYQQD, (c) determination of IC₅₀ value for a mixture of DYQQD and ENAEYLR (concentrations indicated are those of each component), and (d) phosphorylation of purified EGFR in the presence or absence of the mixture of VPEYINQ, DYQQD, and ENAEYLR (concentrations indicated are that of each component). EGFR was incubated in the buffer with or without peptides for 5 min at 37°C. Results displayed on the top panels represent typical immunoblots (IB). ^**P<0.01 versus EGF-stimulated tyrosine phosphorylation at 5 min incubation without peptides; ^#P<0.01 versus EGF-stimulated tyrosine phosphorylation at 5 min incubation with 4 mM DYQQD; ^†P<0.01 versus EGF-stimulated tyrosine phosphorylation at 5 min incubation with 8 mM DYQQD; n=4 for each lane.

Figure 3

Phosphorylation of purified EGFR in the presence or absence of AG1478. EGFR was incubated in the buffer with or without AG1478 for 5 min at 37°C with 0.2 mM of ATP (closed bars) or with 0.02 mM of ATP (open bars). Results displayed on the top panels represent typical immunoblots (IB). ^**P<0.01 versus EGF-stimulated Tyr phosphorylation at 5-min incubation without AG1478; n=4 for each lane.

Figure 4

Phosphorylation of purified EGFR in the presence or absence of peptides, which can suppress phosphorylation of the IR. EGFR was incubated in the buffer with or without peptides for 5 min at 37°C. Results displayed on the top panels represent typical immunoblots (IB). n=4 for each lane.

Figure 5

Effects of peptides, AG1478, or lignocaine on EGF-stimulated Tyr phosphorylation of EGFR at different time points. EGFR was incubated in the buffer for (a): 0, 5, 15, or 25 min at 37°C, respectively and for (b): 0, 5, 15, 25, 35, or 65 min at 37°C, respectively. Each peptide, AG1478, or lignocaine was added at 5 min after EGF stimulation, (a) in the samples of 15- or 25-min incubation and (b) in the samples of 15-, 25-, 35- or 65-min incubation. The ‘control' sample included no peptide, AG1478, or lignocaine. One point was made for each time, resulting in different incubations with or without peptides, AG1478, or lignocaine. Results displayed on the top panel represent typical immunoblots (IB). ^**P<0.01 versus EGF-stimulated Tyr phosphorylation at 5-min incubation without peptides, AG1478 or lignocaine; n=4 for each lane.

Figure 6

Mass spectra of the solutions for mixtures of DYQQD, ENAEYLR, and VPEYINQ incubated with EGFR, EGF, and ATP. (a) Positive-ion mode mass spectrum for a mixture of DYQQD, ENAEYLR, and VPEYINQ, (b) negative-ion mode mass spectrum for a mixture of DYQQD, ENAEYLR, and VPEYINQ, (c) positive-ion mode mass spectrum for a mixture of ENAEYLR and VPEYINQ.

Figure 7

Effects of tyrosine to alanine mutation in DYQQD, ENAEYLR, and VPEYINQ on the inhibitory potencies for autophosphorylation of EGFR. EGFR was incubated in the buffer with or without peptides for 5 min at 37°C. Results displayed on the top panels represent typical immunoblots (IB). ^**P<0.01 versus EGF-stimulated tyrosine phosphorylation at 5-min incubation without peptides; n=4 for each lane.

Figure 8

Detection of phosphorylation using site-specific antiphosphotyrosine antibodies against pY1068, pY1148, or pY1173. EGFR was incubated in the buffer with 4 mM of DYQQD, ENQEYLR, or VPEYINQ for 5 min at 37°C, followed by immunoblotting with three antibodies which recognize phosphorylated Y1068, Y1148, or Y1173 specifically. The EGF-stimulated responses of EGFR without peptides at 5 min were taken as the control and considered to be 100%. ^*P<0.05, ^**P<0.01 versus EGF-stimulated tyrosine phosphorylation at 5-min incubation without peptides; n=4 for each lane.