Mutation of the salt bridge-forming residues in the ETV6-SAM domain interface blocks ETV6-NTRK3-induced cellular transformation.

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.