The beta-amyloid precursor protein functions as a cytosolic anchoring sitethat prevents Fe65 nuclear translocation

In this study we addressed the question of the intracellular localization o f Fe65, an adaptor protein interacting with the beta -amyloid precursor pro tein (APP) and with the transcription factor CP2/LSF/BP1. By using tagged F e65 expression vectors, we observed that a significant fraction of Fe65 is localized in the nucleus of transfected COS7 cells. Furthermore, the isolat ion of nuclei from untransfected PC12 cells allowed us to observe that a pa rt of the endogenous Fe65 is present in the nuclear extract. The analysis o f Fe65 mutant constructs demonstrated that the region of the protein requir ed for its nuclear translocation includes the WW domain, and that, on the o ther hand, a small fragment of 100 residues, including this WW domain, cont ains enough structural information to target a reporter protein (green fluo rescent protein (GFP)-GFP) to the nucleus. To evaluate whether the Fe65-APP interaction could affect Fe65 intracellular trafficking, COS7 cells were c otransfected with APP(695) or APP(751) and with GFP-Fe65 expression vectors . These experiments demonstrated that Fe65 is no longer translocated to the nucleus when the cells overexpress APP, whereas the nuclear targeting of G FP-Fe65 mutants, unable to interact with APP, is unaffected by the coexpres sion of APP, thus suggesting that the interaction with APP anchors Fe65 in the cytosol.