Identification of Amino Acids in the N-Terminal SH2 Domain of Phospholipase Cγ1 Important in the Interaction with Epidermal Growth Factor Receptor

Photoaffinity labeling and site-directed mutagenesis have been used to identify amino acid residues of the phospholipase Cγ1 (PLCγ1) N-terminal SH2 domain involved in recognition of the activated epidermal growth factor receptor (EGFR). The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into phosphotyrosine-containing peptides derived from EGFR autophosphorylation sites Tyr992 and Tyr1068. Irradiation of these labels in the presence of SH2 domains showed cross-linking which was time-dependent and specific; labeling was inhibited with non-Bpa-containing peptides from EGFR in molar excess. The phosphotyrosine residue on the peptides was important for SH2 recognition, as dephosphorylated peptides did not cross-link. Radiolabeled peptides were used to identify sites of cross-linking to the N-terminal SH2 of PLCγ1. Bpa peptide‒SH2 complexes were digested with trypsin, and radioactive fragments were purified by HPLC and analyzed by Edman sequencing. These experiments showed Arg562 and an additional site in the α_A ‒ β_B region of the SH2 domain, most likely Glu587, to be labeled by the Tyr992-derived peptide. Similar analysis of the reaction with the Tyrl068-derived photoaffinity label identified Leu653 as the cross-linked site. Mutation of the neighboring residues of Glu587 decreased photo-cross-linking, emphasizing the importance of this region of the molecule for recognition. These results are consistent with evidence from the v-Src crystal structure and implicate the loop spanning residues Gln640‒Ser654 of PLCγ1 in specific recognition of phosphopeptides.