Assuntos
Antineoplásicos/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores Tumorais , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Mutação , Células Tumorais CultivadasRESUMO
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.
Assuntos
Mutação , Proteínas Proto-Oncogênicas c-ets/química , Receptor trkC/química , Proteínas Repressoras/química , Animais , Calorimetria , Transformação Celular Neoplásica , Humanos , Lisina/química , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Nus , Células NIH 3T3 , Polímeros/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ets/genética , Receptor trkC/genética , Proteínas Repressoras/genética , Sais/química , Transdução de Sinais , Eletricidade Estática , Tirosina/química , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Receptor tyrosine kinases are integral components of cellular signaling pathways and are frequently deregulated in malignancies. The NTRK family of neurotrophin receptors mediate neuronal cell survival and differentiation, but altered NTRK signaling has also been implicated in oncogenesis. The ETV6-NTRK3 (EN) gene fusion occurs in human pediatric spindle cell sarcomas and secretory breast carcinoma, and encodes the oligomerization domain of the ETV6 transcription factor fused to the protein-tyrosine kinase domain of NTRK3. The EN protein functions as a constitutively active protein-tyrosine kinase with potent transforming activity in multiple cell lineages, and EN constitutively activates both the Ras-MAPK and phosphatidylinositol 3-kinase-Akt pathways. EN transformation is associated with constitutive tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Further, IRS-1 functions as the adaptor protein linking EN to downstream signaling pathways. However, the exact nature of the EN-IRS-1 interaction remains unknown. We now demonstrate that EN specifically binds the phosphotyrosine binding domain of IRS-1 via an interaction at the C terminus of EN. An EN mutant lacking the C-terminal 19 amino acids does not bind IRS-1 and lacks transforming ability. Moreover, expression of an IRS-1 polypeptide containing the phosphotyrosine binding domain acts in a dominant negative manner to inhibit EN transformation, and overexpression of IRS-1 potentiates EN transforming activity. These findings indicate that EN.IRS-1 complex formation through the NTRK3 C terminus is essential for EN transformation.