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Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing.

Soriano S, Kang DE, Fu M, Pestell R, Chevallier N, Zheng H, Koo EH - J. Cell Biol. (2001)

Bottom Line: PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle.The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants.Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
In addition to its documented role in the proteolytic processing of Notch-1 and the beta-amyloid precursor protein, presenilin 1 (PS1) associates with beta-catenin. In this study, we show that this interaction plays a critical role in regulating beta-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-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 beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants. In contrast, PS1Deltacat restores Notch-1 proteolytic cleavage and Abeta generation in PS1-deficient cells, indicating that PS1 function in modulating beta-catenin levels can be separated from its roles in facilitating gamma-secretase cleavage of beta-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of beta-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 beta-catenin.

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Effects of PS1 on β-catenin stability and signaling require the interaction of the two proteins. (a) Expression of wild-type PS1 and PS1Δcat were comparable in all experiments, and similar to those in PS1+/− cells (not shown). As ΔCTF lacks amino acids 330–360, a domain recognizable by the αPS1Loop antibody, its signal appears to be weaker than wild-type carboxy-terminal fragment. Immunoprecipitation with J27, an antibody against the NH2 terminus of PS1, shows that PS1Δcat does not interact with β-catenin. (b, Top) Retroviral infection of wild-type PS1 or axin, but not PS1Δcat, in PS1  cells results in reduction of cytosolic β-catenin levels. (Bottom) β-Tubulin blot shown as loading control. (c) Expression of wild-type PS1 in PS1  cells restores efficient degradation of β-catenin. In contrast, expression of PS1Δcat has no significant effect. Cells were metabolically labeled with [35S]methionine for 20 min, chased for 0, 10, and 30 min, and immunoprecipitated for cytosolic β-catenin as described in Materials and Methods. Results from a representative experiment performed in duplicate are shown. (d) Decrease in BrdU incorporation in PS1−/− cells after expression of wild-type PS1 and axin, but not PS1Δcat. Cells were infected with the indicated retroviral vectors, and BrdU incorporation was performed as described 48 h after plating when the cells reached 70–80% of confluence.
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Figure 3: Effects of PS1 on β-catenin stability and signaling require the interaction of the two proteins. (a) Expression of wild-type PS1 and PS1Δcat were comparable in all experiments, and similar to those in PS1+/− cells (not shown). As ΔCTF lacks amino acids 330–360, a domain recognizable by the αPS1Loop antibody, its signal appears to be weaker than wild-type carboxy-terminal fragment. Immunoprecipitation with J27, an antibody against the NH2 terminus of PS1, shows that PS1Δcat does not interact with β-catenin. (b, Top) Retroviral infection of wild-type PS1 or axin, but not PS1Δcat, in PS1 cells results in reduction of cytosolic β-catenin levels. (Bottom) β-Tubulin blot shown as loading control. (c) Expression of wild-type PS1 in PS1 cells restores efficient degradation of β-catenin. In contrast, expression of PS1Δcat has no significant effect. Cells were metabolically labeled with [35S]methionine for 20 min, chased for 0, 10, and 30 min, and immunoprecipitated for cytosolic β-catenin as described in Materials and Methods. Results from a representative experiment performed in duplicate are shown. (d) Decrease in BrdU incorporation in PS1−/− cells after expression of wild-type PS1 and axin, but not PS1Δcat. Cells were infected with the indicated retroviral vectors, and BrdU incorporation was performed as described 48 h after plating when the cells reached 70–80% of confluence.

Mentions: We previously reported that the turnover rate of β-catenin in PS1-deficient immortalized fibroblasts was slower when compared with hemizygous PS1+/− cells, resulting in higher levels of cytosolic β-catenin (Kang et al. 1999). Accordingly, based on the recent finding that cyclin D1 transcription is activated by β-catenin/LEF, we predicted comparable alterations in cyclin D1 transcription in primary fibroblasts from early passages. Western blotting showed that PS1−/− cells contained higher amounts of β-catenin and cyclin D1 protein (Fig. 1 a). In contrast, levels of cdc2 and cyclin A, two other related genes not subject to β-catenin–mediated transcription (Tetsu and McCormick 1999), were not elevated in PS1−/− cells. This was confirmed by quantitative RT-PCR, which showed that PS1−/− cells expressed ∼ 2.5-fold higher levels of cyclin D1 mRNA than that of PS1+/− controls (2.53 ± 0.1, mean ± SD, n = 3) while β-catenin mRNA levels were unchanged (Fig. 1 b). Consistent with our previous report using immortalized PS1-deficient cell lines (Kang et al. 1999), pulse-chase experiments in primary fibroblasts showed that β-catenin turnover was reduced in PS1−/− cells (data not shown, see Fig. 3). No difference in cyclin D1 turnover was detected (Fig. 1 c), demonstrating that differences in cyclin D1 levels occurred at the transcriptional level.


Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing.

Soriano S, Kang DE, Fu M, Pestell R, Chevallier N, Zheng H, Koo EH - J. Cell Biol. (2001)

Effects of PS1 on β-catenin stability and signaling require the interaction of the two proteins. (a) Expression of wild-type PS1 and PS1Δcat were comparable in all experiments, and similar to those in PS1+/− cells (not shown). As ΔCTF lacks amino acids 330–360, a domain recognizable by the αPS1Loop antibody, its signal appears to be weaker than wild-type carboxy-terminal fragment. Immunoprecipitation with J27, an antibody against the NH2 terminus of PS1, shows that PS1Δcat does not interact with β-catenin. (b, Top) Retroviral infection of wild-type PS1 or axin, but not PS1Δcat, in PS1  cells results in reduction of cytosolic β-catenin levels. (Bottom) β-Tubulin blot shown as loading control. (c) Expression of wild-type PS1 in PS1  cells restores efficient degradation of β-catenin. In contrast, expression of PS1Δcat has no significant effect. Cells were metabolically labeled with [35S]methionine for 20 min, chased for 0, 10, and 30 min, and immunoprecipitated for cytosolic β-catenin as described in Materials and Methods. Results from a representative experiment performed in duplicate are shown. (d) Decrease in BrdU incorporation in PS1−/− cells after expression of wild-type PS1 and axin, but not PS1Δcat. Cells were infected with the indicated retroviral vectors, and BrdU incorporation was performed as described 48 h after plating when the cells reached 70–80% of confluence.
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Related In: Results  -  Collection

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Figure 3: Effects of PS1 on β-catenin stability and signaling require the interaction of the two proteins. (a) Expression of wild-type PS1 and PS1Δcat were comparable in all experiments, and similar to those in PS1+/− cells (not shown). As ΔCTF lacks amino acids 330–360, a domain recognizable by the αPS1Loop antibody, its signal appears to be weaker than wild-type carboxy-terminal fragment. Immunoprecipitation with J27, an antibody against the NH2 terminus of PS1, shows that PS1Δcat does not interact with β-catenin. (b, Top) Retroviral infection of wild-type PS1 or axin, but not PS1Δcat, in PS1 cells results in reduction of cytosolic β-catenin levels. (Bottom) β-Tubulin blot shown as loading control. (c) Expression of wild-type PS1 in PS1 cells restores efficient degradation of β-catenin. In contrast, expression of PS1Δcat has no significant effect. Cells were metabolically labeled with [35S]methionine for 20 min, chased for 0, 10, and 30 min, and immunoprecipitated for cytosolic β-catenin as described in Materials and Methods. Results from a representative experiment performed in duplicate are shown. (d) Decrease in BrdU incorporation in PS1−/− cells after expression of wild-type PS1 and axin, but not PS1Δcat. Cells were infected with the indicated retroviral vectors, and BrdU incorporation was performed as described 48 h after plating when the cells reached 70–80% of confluence.
Mentions: We previously reported that the turnover rate of β-catenin in PS1-deficient immortalized fibroblasts was slower when compared with hemizygous PS1+/− cells, resulting in higher levels of cytosolic β-catenin (Kang et al. 1999). Accordingly, based on the recent finding that cyclin D1 transcription is activated by β-catenin/LEF, we predicted comparable alterations in cyclin D1 transcription in primary fibroblasts from early passages. Western blotting showed that PS1−/− cells contained higher amounts of β-catenin and cyclin D1 protein (Fig. 1 a). In contrast, levels of cdc2 and cyclin A, two other related genes not subject to β-catenin–mediated transcription (Tetsu and McCormick 1999), were not elevated in PS1−/− cells. This was confirmed by quantitative RT-PCR, which showed that PS1−/− cells expressed ∼ 2.5-fold higher levels of cyclin D1 mRNA than that of PS1+/− controls (2.53 ± 0.1, mean ± SD, n = 3) while β-catenin mRNA levels were unchanged (Fig. 1 b). Consistent with our previous report using immortalized PS1-deficient cell lines (Kang et al. 1999), pulse-chase experiments in primary fibroblasts showed that β-catenin turnover was reduced in PS1−/− cells (data not shown, see Fig. 3). No difference in cyclin D1 turnover was detected (Fig. 1 c), demonstrating that differences in cyclin D1 levels occurred at the transcriptional level.

Bottom Line: PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle.The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants.Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
In addition to its documented role in the proteolytic processing of Notch-1 and the beta-amyloid precursor protein, presenilin 1 (PS1) associates with beta-catenin. In this study, we show that this interaction plays a critical role in regulating beta-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-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 beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants. In contrast, PS1Deltacat restores Notch-1 proteolytic cleavage and Abeta generation in PS1-deficient cells, indicating that PS1 function in modulating beta-catenin levels can be separated from its roles in facilitating gamma-secretase cleavage of beta-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of beta-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 beta-catenin.

Show MeSH
Related in: MedlinePlus