Membrane-Microdomain Localization of Amyloid β-Precursor Protein (APP) C-terminal Fragments Is Regulated by Phosphorylation of the Cytoplasmic Thr668 Residue

Matsushima, Taijiro; Saito, Yuhki; Elliott, James I.; Iijima-Ando, Kanae; Nakagawa, Masaki; Kimura, Naoko; Hata, Satoshi; Yamamoto, Tohru; Nakaya, Tadashi; Suzuki, Toshiharu

doi:10.1074/jbc.m111.334847

Cited by 22 publications

(16 citation statements)

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“…In the current study we investigated whether, similar to ATP7A, copper-responsive APP trafficking (37) is also regulated by phosphorylation at specific sites. Previous studies have reported that APP is phosphorylated at several sites within its intracellular domain, including Thr-668, which is phosphorylated both in vivo and in vitro and has been widely studied (22,32,36,(45)(46)(47).…”

Section: Resultsmentioning

confidence: 99%

“…To investigate whether phosphorylation of Thr-668 is required for copper-responsive APP trafficking, the Thr-668 residue was substituted to an alanine to create a phospho-deficient mutant at this site. Previous studies have shown that a threonine to alanine substitution at APPThr-668 effectively mimics the non-phosphorylated state with respect to the helical structure of the cytoplasmic domain (35,36). To determine whether the APP T668A mutant traffics in response to copper in comparison to wild type APP (APP WT ), SH-SY5Y stable cell lines were generated that expressed APP T668A and APP WT with a C-terminal cherry fluorescent tag.…”

Section: Resultsmentioning

confidence: 99%

“…Some studies based their finding on the phosphomimetic APP protein, whereby the threonine is mutated to an aspartate or glutamate. It has been reported that replacing threonine with either of these amino acids does not accurately mimic the phosphorylated state of Thr-668 (36). In contrast, NMR studies have shown that mutating Thr-668 to an alanine accurately simulates the non-phosphorylated state (36).…”

Section: Discussionmentioning

confidence: 99%

“…A recent study has shown that non-phosphorylated forms (at Thr-668) of C-terminal APP fragments are associated with lipid raft-like microdomains where the ␥-secretase complex (amyloidogenic) resides, whereas Thr-668-phosphorylated C-terminal fragments reside predominantly in cytoplasmic fractions (36). Hence phosphorylation regulates the localization of APP and thus affects its processing by ␥-secretases (36).…”

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Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells

Acevedo

Opazo

Norrish

et al. 2014

Journal of Biological Chemistry

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Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells

Acevedo

Opazo

Norrish

et al. 2014

Journal of Biological Chemistry

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“…For example, Thr 668 phosphorylation impairs APP/Fe65 interaction [20], [21] but promotes Pin1 binding [22]. In addition, this phosphorylation regulates trafficking of APP and APP derived metabolites [26]. Previous studies in mice suggested a protective role for phosphorylation of Thr 668 in the pathogenesis of AD by showing that Pin1 decreases APP processing and Aß production by binding APP phosphorylated on Thr 668 [27].…”

Section: Discussionmentioning

confidence: 99%

An Intracellular Threonine of Amyloid-β Precursor Protein Mediates Synaptic Plasticity Deficits and Memory Loss

et al. 2013

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Mutations in Amyloid-ß Precursor Protein (APP) and BRI2/ITM2b 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 FDDKI, 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 Thr668 of ß-CTF because phosphorylation of Thr668 is increased in AD cases. We created a knock-in mouse bearing a Thr668Ala mutation (APPTA mice) that prevents phosphorylation at this site. This mutation prevents the development of memory and synaptic plasticity deficits in FDDKI 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 Thr668 is a viable therapeutic strategy for human dementias.

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The cytoplasmic region of the amyloid β‐protein precursor (APP) is necessary and sufficient for the enhanced fast velocity of APP transport by kinesin‐1

et al. 2018

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Amyloid β-protein precursor (APP) is transported mainly by kinesin-1 and at a higher velocity than other kinesin-1 cargos, such as Alcadein α (Alcα); this is denoted by the enhanced fast velocity (EFV). Interaction of the APP cytoplasmic region with kinesin-1, which is essential for EFV transport, is mediated by JNK-interacting protein 1 (JIP1). To determine the roles of interactions between the APP luminal region and cargo components, we monitored transport of chimeric cargo receptors, Alcα (luminal)-APP (cytoplasmic) and APP (luminal)-Alcα (cytoplasmic). Alcα-APP is transported at the EFV, whereas APP-Alcα is transported at the same velocity as wild-type Alcα. Thus, the cytoplasmic region of APP is necessary and sufficient for the EFV of APP transport by kinesin-1.

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Membrane-Microdomain Localization of Amyloid β-Precursor Protein (APP) C-terminal Fragments Is Regulated by Phosphorylation of the Cytoplasmic Thr668 Residue

Cited by 22 publications

References 50 publications

Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells

Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells

An Intracellular Threonine of Amyloid-β Precursor Protein Mediates Synaptic Plasticity Deficits and Memory Loss

The cytoplasmic region of the amyloid β‐protein precursor (APP) is necessary and sufficient for the enhanced fast velocity of APP transport by kinesin‐1

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