RESUMEN
Scaffold proteins play important roles in regulating signalling network fidelity, the absence of which is often the basis for diseases such as cancer. In the present work, we show that the prototypical scaffold protein Shc is phosphorylated by the extracellular signal-regulated kinase, Erk. In addition, Shc threonine phosphorylation is specifically up-regulated in two selected triple-negative breast cancer (TNBC) cell lines. To explore how Erk-mediated threonine phosphorylation on Shc might play a role in the dysregulation of signalling events, we investigated how Shc affects pathways downstream of EGF receptor. Using an in vitro model and biophysical analysis, we show that Shc threonine phosphorylation is responsible for elevated Akt and Erk signalling, potentially through the recruitment of the 14-3-3 ζ and Pin-1 proteins.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Cross-Talk , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Ligandos , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/química , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Treonina/metabolismo , Neoplasias de la Mama Triple Negativas/enzimología , Regulación hacia ArribaRESUMEN
The three isoforms of the adaptor protein Shc play diverse roles in cell signalling. For example, the observation of p46 Shc in the nuclei of hepatocellular carcinoma cells suggests a function quite distinct from the better characterised cytoplasmic role. Ligands responsible for the transport of various Shc isoforms into organelles such as the nucleus have yet to be reported. To identify such ligands a far western approach was used to determine the p52 Shc interactome. The Ran-GTPase nuclear transport protein was identified and found to bind to p52 Shc in vitro with low micromolar affinity. Co-immunoprecipitation, pull down and fluorescence lifetime imaging microscopy experiments in stable cells confirmed cellular interaction and nuclear localisation. The nuclear transport factor protein NTF2, which functions in cohort with Ran, was shown to form a complex with both RAN and Shc, suggesting a mechanism for Shc entry into the nucleus as part of a tertiary complex.