title: Nerve growth factor receptor TrkA signaling in breast cancer cells involves Ku70 to prevent apoptosis.
abstract: The nerve growth factor (NGF)-tyrosine kinase receptor TrkA plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation, and proliferation. In breast cancer cells, TrkA stimulation results in the activation of cellular growth, but downstream signaling largely remains to be described. Here we used a proteomics-based approach to identify partners involved in TrkA signaling in breast cancer cells. Wild type and modified TrkA chimeric constructs with green fluorescent protein were transfected in MCF-7 cells, and co-immunoprecipitated proteins were separated by SDS-PAGE before nano-LC-MS/MS analysis. Several TrkA putative signaling partners were identified among which was the DNA repair protein Ku70, which is increasingly reported for its role in cell survival and carcinogenesis. Physiological interaction of Ku70 with endogenous TrkA was induced upon NGF stimulation in non-transfected cells, and co-localization was observed with confocal microscopy. Mass spectrometry analysis and Western blotting of phosphotyrosine immunoprecipitates demonstrated the induction of Ku70 tyrosine phosphorylation upon NGF stimulation. Interestingly no interaction between TrkA and Ku70 was detected in PC12 cells in the absence or presence of NGF, suggesting that it is not involved in the initiation of neuronal differentiation. In breast cancer cells, RNA interference indicated that whereas Ku70 depletion had no direct effect on cell survival, it induced a strong potentiation of apoptosis in TrkA-overexpressing cells. In conclusion, TrkA signaling appears to be proapoptotic in the absence of Ku70, and this protein might therefore play a role in the long time reported ambivalence of tyrosine kinase receptors that can exhibit both anti- and eventually proapoptotic activities.
authors.affiliation: INSERM ERI-8 (JE-2488), Growth factor signaling in breast cancer. Functional proteomics, University of Sciences and Technologies Lille, 59655 Villeneuve d'Ascq, France .
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Com
authors.forename: Chann
authors.initials: C
authors.lastname: Lagadec
authors.forename: Adeline
authors.initials: A
authors.lastname: Page
authors.forename: Ikram
authors.initials: I
authors.lastname: El Yazidi-Belkoura
authors.forename: Christian
authors.initials: C
authors.lastname: Slomianny
authors.forename: Ambre
authors.initials: A
authors.lastname: Spencer
authors.forename: Djilali
authors.initials: D
authors.lastname: Hammache
authors.forename: Brian B
authors.initials: BB
authors.lastname: Rudkin
authors.forename: Hubert
authors.initials: H
authors.lastname: Hondermarck
mesh_terms: D000595:Amino Acid Sequence; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000378:metabolism*; D034622:RNA Interference; D020917:Receptor, trkA / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D020932:Nerve Growth Factor; D020917:Receptor, trkA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen