It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for ␤-amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr 743 by APP751 numbering, Tyr 682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, we mutated all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737-743). Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impaired APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (⌬C), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr 743 regulates rapid APP turnover because half-life increased by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, A␤, was tightly correlated with APP internalization, such that A␤ secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the A␤42 isoform was also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic A␤ species. APP1 is a transmembrane protein with homology to glycosylated cell surface receptors (1), can reside at the cell surface (2-4) and is reinternalized via clathrin-coated pits (5, 6) to the endosomal-lysosmal pathway (7,8). Some internalized APP remains intact to be recycled to the cell surface plasma membrane (9, 10). However, internalized APP can also be proteolytically processed into several distinct secreted fragments, which include the large secreted N-terminal APP ectodomain (APP s ), and A␤, the major protein component of senile plaques in Alzheimer's disease (AD; reviewed in Ref. 11).Because A␤ deposition may be central to AD pathogenesis, the mechanism by which A␤ is generated from the precursor is an important focus of AD research. At least two species of A␤, differing by two amino acids at the C terminus (A␤40 and A␤42), are released from cells during normal cellular metabolism (12-14). A␤42, which readily aggregates in vitro (reviewed in Ref. 15) appears to be more pathogenic and may serve as a seed for plaque formation in individuals with AD (16), hereditary cerebral hemorrhage with amyloidosis Dutch type (17), and Down's syndrome (18). The source of A␤ deposited in brain tissues is still uncertain. However, cell lines expressing wild type APP can produce and release A␤ primarily after internalization of APP from the cell surface (19,20). Although familial mutations in APP can enhance A␤ secretion (e.g. the Swedish KM 3 NL mutation; Refs. 20 -23), almost all huma...

The Alzheimer's disease-linked gene presenilin 1 (PS1) is required for intramembrane proteolysis of APP and Notch. In addition, recent observations strongly implicate PS1 as a negative regulator of the Wnt/beta-catenin signaling pathway, although the mechanism underlying this activity is unknown. Here, we show that presenilin functions as a scaffold that rapidly couples beta-catenin phosphorylation through two sequential kinase activities independent of the Wnt-regulated Axin/CK1alpha complex. Thus, presenilin deficiency results in increased beta-catenin stability in vitro and in vivo by disconnecting the stepwise phosphorylation of beta-catenin, both in the presence and absence of Wnt stimulation. These findings highlight an aspect of beta-catenin regulation outside of the canonical Wnt-regulated pathway and a function of presenilin separate from intramembrane proteolysis.

Presenilin 1 (PS1) is required for the proteolytic processing of Notch and the ␤-amyloid precursor protein (APP), molecules that play pivotal roles in cell-fate determination during development and Alzheimer's disease pathogenesis, respectively. In addition, PS1 interacts with ␤-catenin and promotes its turnover through independent mechanisms. Consistent with this activity, we report here that PS1 is important in controlling epidermal cell proliferation in vivo. PS1 knockout mice that are rescued through neuronal expression of human PS1 transgene develop spontaneous skin cancers. PS1-null keratinocytes exhibit higher cytosolic ␤-catenin and ␤-catenin͞lymphoid enhancer factor-1͞T cell factor (␤-catenin͞ LEF)-mediated signaling. This effect can be reversed by reintroducing wild-type PS1, but not a PS1 mutant active in Notch processing but defective in ␤-catenin binding. Nuclear ␤-catenin protein can be detected in tumors. Elevated ␤-catenin͞LEF signaling is correlated with activation of its downstream target cyclin D1 and accelerated entry from G 1 into S phase of the cell cycle. This report demonstrates a function of PS1 in adult tissues, and our analysis suggests that deregulation of ␤-catenin pathway contributes to the skin tumor phenotype.

In addition to its documented role in the proteolytic processing of Notch-1 and the β-amyloid precursor protein, presenilin 1 (PS1) associates with β-catenin. In this study, we show that this interaction plays a critical role in regulating β-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic β-catenin, leading to a β-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle. Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner. The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind β-catenin (PS1Δcat) or by two different familial Alzheimer's disease mutants. In contrast, PS1Δcat restores Notch-1 proteolytic cleavage and Aβ generation in PS1-deficient cells, indicating that PS1 function in modulating β-catenin levels can be separated from its roles in facilitating γ-secretase cleavage of β-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of β-catenin in the absence of PS1, a phenotype that may account for the increased stability in PS1-deficient cells. Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of β-catenin.