Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites.
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.
Fan
Ying-Xin
YX
Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Wong
Lily
L
Deb
Tushar B
TB
Johnson
Gibbes R
GR
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D020134:Catalytic Domain; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D066246:ErbB Receptors / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018076:DNA, Complementary; D000939:Epitopes; C490897:Gab1 protein, mouse; D008024:Ligands; D010455:Peptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases