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Published on: May 20, 2020
Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and
Ying-Xin Fan1, Lily Wong, Tushar B Deb
1Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Abstract:
The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.
Insights
Epidermal growth factor receptor (EGFR) kinase specificity is altered by epidermal growth factor (EGF) binding. EGF increases EGFR
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- The epidermal growth factor receptor (EGFR) is a key regulator of cell growth and survival.
- EGFR kinase activity is central to its signaling function, involving phosphorylation of tyrosine residues.
- Understanding how EGFR kinase activity and specificity are regulated is crucial for comprehending cellular responses to growth factors.
Purpose of the Study:
- To investigate whether ligand binding modulates the substrate specificity of the EGFR kinase.
- To determine the effect of epidermal growth factor (EGF) on the binding affinity and catalytic activity of EGFR towards different peptide substrates.
- To elucidate the role of specific amino acid residues in EGF-mediated regulation of EGFR kinase specificity.
Main Methods:
- Kinetic analysis of EGFR kinase activity using peptides derived from autophosphorylation sites and known substrates.
- Measurement of steady-state kinetic parameters (K(m) and k(cat)) with and without EGF stimulation.
- Assessment of EGFR binding affinity to purified GST fusion proteins of adaptor proteins (Gab1, Shc) in the presence and absence of EGF.
- Analysis of substrate phosphorylation in cell lysates containing full-length adaptor proteins.
Main Results:
- EGF binding did not alter EGFR peptide binding but increased binding affinity for peptides mimicking major phosphorylation sites on Gab1 and Shc (3-7-fold).
- EGF stimulation increased the catalytic rate (k(cat)) approximately 5-fold for all tested peptides.
- EGF significantly enhanced EGFR's specificity constant (k(cat)/K(m)) for physiological substrates like Gab1 Tyr-627 (15-40-fold increases).
- EGF increased EGFR binding to Gab1 and Shc sites and enhanced their phosphorylation relative to EGFR autophosphorylation.
- Residues C-terminal to the target tyrosine were identified as critical for EGF-mediated specificity regulation.
Conclusions:
- Ligand binding by EGF alters the substrate specificity of the EGFR kinase.
- EGFR kinase exhibits increased preference for specific cellular substrates upon EGF stimulation.
- This represents the first demonstration of ligand-induced specificity changes in a receptor tyrosine kinase towards relevant targets.
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