Phosphorylation of a Tyrosine in the Amyloid-beta Protein Precursor Intracellular Domain Inhibits Fe65 Binding and Signaling
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. Journal of Alzheimer's disease: JAD
(Impact Factor: 4.15).
03/2009; 16(2):301-7. DOI: 10.3233/JAD-2009-0970
The phosphorylation of Tyr-682 residue in the intracellular domain (AID) of amyloid-beta protein precursor (AbetaPP) is significantly enhanced in Alzheimer's disease patients' brain. The role of this phosphotyrosine, however, remains elusive. Here we report that phosphorylation of Tyr-682 inhibits the interactions between AbetaPP and Fe65, which is the main regulatory mechanism controlling Fe65 nuclear signaling. Furthermore, we show that tyrosine phosphorylation of AbetaPP also inhibits interaction of the two other Fe65 family members, Fe65L1 and Fe65L2. Likewise, docking of Fe65, Fe65L1 and Fe65L2 to APLP1 and APLP2, the two other members of the AbetaPP-gene family, is abolished by analogous phosphorylation events. Our results indicate that phosphorylation of the cytoplasmic tail of AbetaPP on Tyr-682 represents a second mechanism, alternative to AbetaPP processing by secretases, that regulates AbetaPP/Fe65 downstream signaling pathways.
Available from: Florian Riese
- "Tyrosine 682 in the Y682ENPTY motif is crucial for the binding of Fe65 and other adaptor proteins. Phosphorylation of this residue by src, abl, trkA, and EGFR tyrosine kinases was reported to enhance the binding of SH2-domain containing adaptor proteins while abolishing binding of Fe65 [34,41-44]. Consequently, our BiFC assay revealed increased nuclear signaling when Tyrosine 682 was mutated to phenylalanine to prevent phosphorylation. "
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ABSTRACT: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation.
To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells.
We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC).
Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer's disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals.
We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence.
Available from: Luciano D'adamio
- "Phosphorylation of Tyr682 is consequential. Some proteins, such as Grb2 , Shc , , Grb7 and Crk  interact with APP only when Tyr682 is phosphorylated; others, like Fe65, Fe65L1 and Fe65L2 only when this tyrosine is not phosphorylated , suggesting that phosphorylation–dephosphorylation on Tyr682 modulates APP functions. To test the in vivo function of Tyr682 we have created mice with Tyr682 replaced by a Gly. "
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ABSTRACT: Mutations in ß and genes cause Familial Alzheimer and Danish Dementias (FAD/FDD), respectively. APP processing by BACE1, which is inhibited by BRI2, yields sAPPß and ß-CTF. ß-CTF is cleaved by gamma-secretase to produce Aß. A knock-in mouse model of FDD, called FDD, shows deficits in memory and synaptic plasticity, which can be attributed to sAPPß/ß-CTF but not Aß. We have investigated further the pathogenic function of ß-CTF focusing on Thr of ß-CTF because phosphorylation of Thr is increased in AD cases. We created a knock-in mouse bearing a ThrAla mutation ( mice) that prevents phosphorylation at this site. This mutation prevents the development of memory and synaptic plasticity deficits in FDD mice. These data are consistent with a role for the carboxyl-terminal APP domain in the pathogenesis of dementia and suggest that averting the noxious role of Thr is a viable therapeutic strategy for human dementias.
Available from: Siro Luvisetto
- "Phosphorylation of Y 682 is consequential. Some proteins, such as Grb2 (Russo et al., 2002; Zhou et al., 2004), Shc (Russo et al., 2002; Tarr et al., 2002), Grb7, and Crk (Tamayev et al., 2009), interact with APP only when Y 682 is phosphorylated, whereas others, like Fe65, Fe65L1, and Fe65L2, only when this tyrosine is not phosphorylated (Zhou et al., 2009), suggesting that phosphorylation–dephosphorylation on Y 682 modulates APP functions. "
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ABSTRACT: Processing of Aβ-precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the (682) YENPTY(687) motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Y(682) to Gly (APP(YG/YG) mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP-like protein 2 KO background. This evidence prompted us to characterize further the APP(YG/YG) mice. Here, we show that APP(YG/YG) mice develop aging-dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP(YG/YG) mice. The profound deficits caused by the Y(682) mutation underscore the biological importance of APP and indicate that APP(YG/YG) are a valuable mouse model to study APP functions in physiological and pathological processes.
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