STRUCTURAL REQUIREMENTS OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR FOR TYROSINE PHOSPHORYLATION OF EPS8 AND EPS15, SUBSTRATES LACKING SRC SH2 HOMOLOGY DOMAINS

Phosphorylation of two newly identified epidermal growth factor (EG;F) receptor substrates, eps8 and eps15, which do not possess Src homolog y (SH2) domains, was investigated using EGF receptor mutants of the au tophosphorylation sites and deletion mutants of the carboxyl terminal region. Two mutants, F5, in which all five tyrosine autophosphorylatio n sites substituted by phenylalanine, and Dc 123F, in which four tyros ines were removed by deletion and the fifth (Tyr-992) was mutated into phenylalanine, phosphorylated eps8 and eps15 as efficiently as the wi ld-type receptor. In contrast, SH2-containing substrates, phospholipas e C gamma, the GTPase-activating protein of Ras, the p85 subunit of ph osphatidylinositol 3 kinase, and the Src and collagen homology protein , are not phosphorylated by the FS and Dc 123F mutants. A longer EGF r eceptor deletion mutant, Dc 214, lacking all five autophosphorylation sites, was unable to phosphorylate eps15 but phosphorylated eps8 13-fo ld more than the wild type receptor. To determine the EGF receptor reg ion important for phosphorylation of eps8 and eps15, progressive delet ion mutants lacking the final 123, 165, 196, and 214 COOH-terminal res idues were used. eps8 phosphorylation was progressively increased in D c 165, Dc 196, and Dc 214 EGF receptor mutants, indicating that remova l of the final 214 COOH-terminal residues increases the phosphorylatio n of this substrate by the EGF receptor. In contrast, eps15 was phosph orylated by Dc 123 and Dc 165 EGF receptor mutants but not by Dc 196 a nd Dc 214 mutants. This indicates that a region of 30 residues located between Dc 165 and Dc 196 is essential for eps15 phosphorylation. Thi s is the first demonstration of structural requirements in the EGF rec eptor COOH terminus for efficient phosphorylation of non SH2-containin g substrates. In addition, enhanced eps8 phosphorylation correlates we ll with the increased transforming potential of EGF receptor deletion mutants Dc 196 and Dc 214, suggesting that this substrate may be invol ved in mitogenic signaling.