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 gamma 1) 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 PLC gamma 1. 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 alpha A-beta B region of the SH2 domain, most likely Glu587, to be labeled by the Tyr992-derived peptide. Similar analysis of the reaction with the Tyr1068-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 PLC gamma 1 in specific recognition of phosphopeptides.
Gergel
J R
JR
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook 11794.
McNamara
D J
DJ
Dobrusin
E M
EM
Zhu
G
G
Saltiel
A R
AR
Miller
W T
WT
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D066246:ErbB Receptors / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D010777:Photochemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism*
D000345:Affinity Labels; D000596:Amino Acids; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D010738:Type C Phospholipases; D051966:Phospholipase C gamma