IDENTIFICATION OF AMINO-ACIDS IN THE N-TERMINAL SH2 DOMAIN OF PHOSPHOLIPASE C-GAMMA-1 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 C gamma 1 (PLC gamm a 1) N-terminal SH2 domain involved in recognition of the activated ep idermal growth factor receptor (EGFR). The photoactive amino acid p-be nzoylphenylalanine (Bpa) was incorporated into phosphotyrosine-contain ing peptides derived from EGFR autophosphorylation sites Tyr992 and Ty r1068. Irradiation of these labels in the presence of SH2 domains show ed cross-linking which was time-dependent and specific; labeling was i nhibited with non-Bpa-containing peptides from EGFR in molar excess. T he phosphotyrosine residue on the peptides was important for SH2 recog nition, as dephosphorylated peptides did not cross-link. Radiolabeled peptides were used to identify sites of cross-linking to the N-termina l SH2 of PLC gamma 1. Bpa-peptide-SH2 complexes were digested with try psin, and radioactive fragments were purified by HPLC and analyzed by Edman sequencing. These experiments showed Arg562 and an additional si te in the alpha(A)-beta(B) region of the SH2 domain, most likely Glu58 7, to be labeled by the Tyr992-derived peptide. Similar analysis of th e reaction with the Tyr1068-derived photoaffinity label identified Leu 653 as the cross-linked site. Mutation of the neighboring residues of Glu587 decreased photo-cross-linking, emphasizing the importance of th is region of the molecule for recognition. These results are consisten t with evidence from the v-Src crystal structure and implicate the loo p spanning residues Gln640-Ser654 of PLC gamma 1 in specific recogniti on of phosphopeptides.