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Proteolytic processing of the amyloid-β protein precursor (AβPP) occurs via alternative pathways, culminating with the production of the AβPP intracellular domain (AICD). AICD can translocate to the nucleus and regulate transcription, but its activity is modulated by interactions with other proteins. In the nucleus, AICD, FE65, and Tip60 associate into AFT complexes, which are targeted to nuclear spots which correspond to transcription factories. Here we report that RanBP9 interacts with the cytoplasmic domain of AβPP, through the NPXY internalization motif. Moreover, RanBP9 interaction with Tip60 is also described. The RanBP9-Tip60 interaction dramatically relocated RanBP9 from a widespread cellular distribution to nuclear speckles. AβPP processing is a central aspect in determining the protein's function and that of its resulting proteolytic fragments, among them AICD. The latter results from the amyloidogenic pathway and is the peptidic species predominantly involved in nuclear signaling. Of note RanBP9 transfection was previously demonstrated to increase amyloid-β generation. Here we show that RanBP9 relocates AICD to the Tip60-enriched nuclear speckles, and prevented the formation of nuclear spots formation, having therefore a negative effect on AICD mediated nuclear signaling and consequently AFT complex formation. Furthermore, by transfecting cells with increasing amounts of RanBP9, the expression of AICD-regulated genes, including AβPP itself, was reduced. Given the data presented, one can deduce that RanBP9 has an inhibitory regulatory effect on AICD-mediated transcription and the effect is mediated by relocating AICD away from transcription factories.

The amyloid-β protein precursor (AβPP) binds several proteins determining metabolism, processing, and the physiological fate of the former. Among these is Fe65, a protein with specific functional significance for AβPP, in particular conferring stability when the latter is dephosphorylated on Thr668. Thus, it follows that phosphatases like protein phosphatase 1 (PP1) are relevant to AβPP processing. Consequently, the identification of AβPP binding proteins, which can be modulated directly or indirectly by PP1, take on added relevance in terms of biological significance. Using the yeast tri-hybrid system and co-immunoprecipitation assays, we describe a novel tri-complex comprising AβPP, Fe65 and PP1. We show that the trimeric complex (AβPP:Fe65:PP1γ) occurs in COS-7 cells, rat hippocampal and cortical primary neurons, and in adult rat hippocampus and cortex. Using overlay assays, we demonstrate that Fe65 is in fact the bridging protein in the complex formed and thus we simultaneously describe another PP1 binding protein. This is singularly important given that PP1 binding proteins determine and confer subcellular localization, as well as substrate specificity, thus regulating the phosphatase activity and subsequent intracellular events. Additionally, we show that this interaction correlates with AβPP Thr668 phosphorylation state, consistent with the role of protein (de)phosphorylation as a key mechanism in regulating cellular events.

Fe65 is a multimodular adaptor protein that interacts with the cytosolic domain of the β-amyloid precursor protein (APP), the major component of Alzheimer's disease (AD) senile plaques. In the work here presented, we describe the existence of a new Fe65 transcript variant (GenBank Accession EF103274). A unique 5' sequence of 69 nucleotides, spanning a region between exons 2 and 3 of the FE65 gene, was present in a yeast two-hybrid (YTH) clone from a human brain cDNA library. In silico analysis and RT-PCR revealed the presence of a novel exon of 133 bp, and we redefined the structure of the human FE65 gene. The novel exon 3a-inclusive transcript generates a shorter isoform, p60Fe65. The migration pattern of the p60Fe65 isoform was observed previously and attributed to an alternative translation initiation site within the p97Fe65 transcript. Here, we provide evidence for the origin of the previously unexplained p60Fe65 isoform. Moreover, Fe65E3a is expressed preferentially in the brain and the p60Fe65 protein levels increased during PC12 cell differentiation. This novel Fe65 isoform and the regulation of the splicing events leading to its production, may contribute to elucidating neuronal specific roles of Fe65 and its contribution to AD pathology., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)