EGF regulates the interaction of Tks4 with Src through its SH2 and SH3 domains

Abstract

The non-receptor tyrosine kinase Src is a central component of the EGF signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues: 466-474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues: 508-511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to synergistic enhancement of binding strength and a highly stable, "bidentate"-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least three hours in living cells. We conclude that the interaction of Tks4 with Src may result in the long-term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation. © 2018 American Chemical Society.