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J. Biol. Chem., Vol. 279, Issue 37, 38143-38150, September 10, 2004

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Ligand Regulates Epidermal Growth Factor Receptor Kinase Specificity

ACTIVATION INCREASES PREFERENCE FOR GAB1 AND SHC VERSUS AUTOPHOSPHORYLATION SITES^*

Ying-Xin Fan, Lily Wong, Tushar B. Deb, and Gibbes R. Johnson¶

From the Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

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 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of 5-fold in the specificity constants (k_cat/K_m) for EGFR peptides, whereas 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 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.

Received for publication, May 24, 2004 , and in revised form, July 1, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Lombardi Cancer Center and Dept. of Oncology, Georgetown University, Washington, D. C. 20007.

To whom correspondence may be addressed: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bldg. 29A, Rm. 3B-20, 8800 Rockville Pike, Bethesda, MD 20892. Tel.: 301-827-1763; Fax: 301-480-3256; E-mail: fany{at}cber.fda.gov. ¶ To whom correspondence may be addressed: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bldg. 29A, Rm. 3B-16, 8800 Rockville Pike, Bethesda, MD 20892. Tel.: 301-827-1770; Fax: 301-480-3256; E-mail: johnsong{at}cber.fda.gov.

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