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Biochemistry. 1995 Dec 19;34(50):16456-66.

In vitro phosphorylation of the epidermal growth factor receptor autophosphorylation domain by c-src: identification of phosphorylation sites and c-src SH2 domain binding sites.

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Department of Protein Biochemistry, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.


During epidermal growth factor mediated signal transduction, intracellular receptor autophosphorylation on tyrosine residues results in the localization of several SH2 domain bearing proteins, including c-src, to the plasma membrane. This process is part of a complex pathway of specific protein associations that culminates in the regulation of cell growth and mitogenesis. The SH2 domain-mediated interaction of c-src with the EGF receptor has been demonstrated, yet the precise function of c-src in EGF receptor signaling remains unclear. The phosphorylation of EGFR by c-src was studied in order to evaluate the molecular basis for this interaction. The C-terminal autophosphorylation domain of EGFR was extensively phosphorylated by c-src and EGFR kinase activities in vitro as determined by electrospay ionization mass spectrometry. The sites of phosphorylation within the autophosphorylation domain (residues 976-1186) were identified by LC/MS, LC/MS/MS, and Edman sequencing. The majority of the sites identified corresponded to the known autophosphorylation sites of EGFR. Kinetic analyses of site-specific phosphorylation were made combining very fast enzyme digests (< = or 2 min) and high-speed, perfusion chromatography. These studies revealed that Y1086 was phosphorylated to a significantly higher extent by c-src than by EGFR. Additionally, Y1101 was identified as a unique c-src phosphorylation site. The function of these phosphorylation sites with respect SH2 domain interactions was investigated by affinity chromatography/mass spectrometry. A subset of peptides corresponding to the eight possible tyrosine phosphorylation sites within the EGFR autophosphorylation domain was demonstrated to bind to the SH2 domain of c-src. Those which bound to the SH2 domain included peptides derived from EGFR sequences flanking Y992, Y1086, Y1101, and Y1148. These data indicate that specific EGF receptor c-src phosphorylation sites are also ligands for the SH2 domain of c-src. Finally, extensive c-src phosphorylation of EGFR promoted its conversion to a form that exhibits high-affinity (KD = 380 nM) and cooperative (Hill coefficient; n = 2) binding to the SH2 domain of c-src as measured by surface plasmon resonance. The identification of c-src phosphorylation sequences on EGFR as c-src SH2 binding sites supports the notion that this interaction plays a significant role in the regulation of growth factor receptor function and signal transduction.

[Indexed for MEDLINE]

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