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.
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.
Lombardo
C R
CR
Department of Protein Biochemistry, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.
Consler
T G
TG
Kassel
D B
DB
D000595:Amino Acid Sequence; D001665:Binding Sites; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D018909:src Homology Domains
D010446:Peptide Fragments; D011994:Recombinant Proteins; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src)