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Phosphorylation of FE65 Ser610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing.

Chow WN, Ngo JC, Li W, Chen YW, Tam KM, Chan HY, Miller CC, Lau KF - Biochem. J. (2015)

Bottom Line: We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser(610) phosphorylation inhibits this effect.Furthermore, we found that the effect of FE65 Ser(610) phosphorylation on APP processing is linked to a role of FE65 in metabolic turnover of APP via the proteasome.Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser(610) by SGK1 regulates binding of FE65 to APP, APP turnover and processing.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR.

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Phosphorylation status of FE65 Ser610 regulates APP and FE65 turnover(A) CHX chase assay was performed on cells transfected with APP + FE65, FE65 S610A or FE65 S610D. Cell lysates were immunoblotted for APP, FE65 and α-tubulin. (B and C) Densitometric analysis of APP and FE65 levels that are shown in (A). FE65 and APP levels were obtained from four independent experiments with n=5 (total number of samples analysed was 20). Results are means ± S.E.M. FE65 S610A but not S610D prolongs the half-life of APP. (D) Cells co-transfected with FE65, FE65 S610A or FE65 S610D + control or APP siRNA were incubated with 2.5 μM MG132 or DMSO vehicle for 16 h. APP knockdown-mediated reduction in FE65 and FE65 S610A levels is rescued by MG132. Protein level of FE65 S610D is unaffected by APP knockdown. (E) Cells co-transfected with APP + FE65, FE65 S610A or FE65 S610D were subjected to 16 h MG132 treatment. The protein levels of APP and FE65 in transfected cell lysates was compared by Western blotting. Accelerated APP and FE65 turnover in APP + FE65 S610D-transfected cells is rescued by MG132. (F) Cells were co-transfected with APP + FE65, FE65 S610A or FE65 S610D + control or FBL2 siRNA. The transfected cell lysate was immunoblotted for APP, FE65 and FBL2. FBL2 knockdown partially restores APP level in APP + FE65 S610D-transfected cells. Data for graphs in (D–F) were obtained from three independent experiments with n=3 (total number of samples analysed=9) and analysed by one-way ANOVA. **P<0.01; ***P<0.05; ns–P>0.05. Results are means ± S.D.
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Figure 6: Phosphorylation status of FE65 Ser610 regulates APP and FE65 turnover(A) CHX chase assay was performed on cells transfected with APP + FE65, FE65 S610A or FE65 S610D. Cell lysates were immunoblotted for APP, FE65 and α-tubulin. (B and C) Densitometric analysis of APP and FE65 levels that are shown in (A). FE65 and APP levels were obtained from four independent experiments with n=5 (total number of samples analysed was 20). Results are means ± S.E.M. FE65 S610A but not S610D prolongs the half-life of APP. (D) Cells co-transfected with FE65, FE65 S610A or FE65 S610D + control or APP siRNA were incubated with 2.5 μM MG132 or DMSO vehicle for 16 h. APP knockdown-mediated reduction in FE65 and FE65 S610A levels is rescued by MG132. Protein level of FE65 S610D is unaffected by APP knockdown. (E) Cells co-transfected with APP + FE65, FE65 S610A or FE65 S610D were subjected to 16 h MG132 treatment. The protein levels of APP and FE65 in transfected cell lysates was compared by Western blotting. Accelerated APP and FE65 turnover in APP + FE65 S610D-transfected cells is rescued by MG132. (F) Cells were co-transfected with APP + FE65, FE65 S610A or FE65 S610D + control or FBL2 siRNA. The transfected cell lysate was immunoblotted for APP, FE65 and FBL2. FBL2 knockdown partially restores APP level in APP + FE65 S610D-transfected cells. Data for graphs in (D–F) were obtained from three independent experiments with n=3 (total number of samples analysed=9) and analysed by one-way ANOVA. **P<0.01; ***P<0.05; ns–P>0.05. Results are means ± S.D.

Mentions: Next, we sought to determine the effect of FE65 Ser610 phosphorylation on the turnover of APP. The turnover rate of APP in the presence of FE65/FE65 S610A/FE65 S610D was compared by CHX chase assay followed by densitometric analysis (Figures 6A and 6B). Our data show that FE65 S610A but not FE65 S610D prolongs the half-life of APP. Of note, it was found that FE65 S610D shows a higher turnover rate than FE65 and FE65 S610A (Figures 6A and 6C). We asked whether APP plays a role in regulating FE65 turnover. To address this issue, cells transfected with FE65/FE65 S610A/FE65 S610D + control or APP siRNA were either treated with proteasome inhibitor MG132 or vehicle control (DMSO) and the protein level of FE65 was analysed by Western blotting. As shown in Figure 6(D), APP knockdown results in reduction in FE65 and FE65 S610A levels (lane 1 compared with lane 2; lane 5 compared with lane 6) and the depletion is blocked upon MG132 treatment (lane 3 compared with lane 4; lane 7 compared with lane 8). This indicates that APP stabilizes FE65 and FE65 S610A by preventing their degradation through UPS. On the other hand, the protein level of FE65 S610D, which is unable to bind to APP, is not affected by endogenous APP level (lane 9 compared with lane 10). In a complementary approach, we performed an MG132 study in APP + FE65/FE65 S610A/S610D-transfected cells to evaluate the effect of FE65 Ser610 phosphorylation on APP and FE65 proteasomal degradation. As shown in Figure 6(E), depletion of APP and FE65 in APP + FE65 S610D-transfected cells is rescued by proteasome inhibition by MG132. A previous report provided evidence that APP is ubiquitinated by the FBL2, a component of the SCF (Skp1–cullin1–F-box protein) E3 ubiquitin ligase complex, resulting in enhanced proteasomal degradation [40]. We enquired whether FE65 precludes FBL2-mediated APP degradation. To do this, cells were co-transfected with APP + FE65, FE65 S610A or FE65 S610D + control or FBL2 siRNA and APP protein level was compared. As shown in Figure 6(F), loss of FBL2 partially restores APP level in APP + FE65 S610D-transfected cells. Taken together, the current findings suggest that the interaction between APP and FE65 protects APP from proteasomal degradation through FBL2 and this process is regulated by the phosphorylation status of FE65 Ser610.


Phosphorylation of FE65 Ser610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing.

Chow WN, Ngo JC, Li W, Chen YW, Tam KM, Chan HY, Miller CC, Lau KF - Biochem. J. (2015)

Phosphorylation status of FE65 Ser610 regulates APP and FE65 turnover(A) CHX chase assay was performed on cells transfected with APP + FE65, FE65 S610A or FE65 S610D. Cell lysates were immunoblotted for APP, FE65 and α-tubulin. (B and C) Densitometric analysis of APP and FE65 levels that are shown in (A). FE65 and APP levels were obtained from four independent experiments with n=5 (total number of samples analysed was 20). Results are means ± S.E.M. FE65 S610A but not S610D prolongs the half-life of APP. (D) Cells co-transfected with FE65, FE65 S610A or FE65 S610D + control or APP siRNA were incubated with 2.5 μM MG132 or DMSO vehicle for 16 h. APP knockdown-mediated reduction in FE65 and FE65 S610A levels is rescued by MG132. Protein level of FE65 S610D is unaffected by APP knockdown. (E) Cells co-transfected with APP + FE65, FE65 S610A or FE65 S610D were subjected to 16 h MG132 treatment. The protein levels of APP and FE65 in transfected cell lysates was compared by Western blotting. Accelerated APP and FE65 turnover in APP + FE65 S610D-transfected cells is rescued by MG132. (F) Cells were co-transfected with APP + FE65, FE65 S610A or FE65 S610D + control or FBL2 siRNA. The transfected cell lysate was immunoblotted for APP, FE65 and FBL2. FBL2 knockdown partially restores APP level in APP + FE65 S610D-transfected cells. Data for graphs in (D–F) were obtained from three independent experiments with n=3 (total number of samples analysed=9) and analysed by one-way ANOVA. **P<0.01; ***P<0.05; ns–P>0.05. Results are means ± S.D.
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Figure 6: Phosphorylation status of FE65 Ser610 regulates APP and FE65 turnover(A) CHX chase assay was performed on cells transfected with APP + FE65, FE65 S610A or FE65 S610D. Cell lysates were immunoblotted for APP, FE65 and α-tubulin. (B and C) Densitometric analysis of APP and FE65 levels that are shown in (A). FE65 and APP levels were obtained from four independent experiments with n=5 (total number of samples analysed was 20). Results are means ± S.E.M. FE65 S610A but not S610D prolongs the half-life of APP. (D) Cells co-transfected with FE65, FE65 S610A or FE65 S610D + control or APP siRNA were incubated with 2.5 μM MG132 or DMSO vehicle for 16 h. APP knockdown-mediated reduction in FE65 and FE65 S610A levels is rescued by MG132. Protein level of FE65 S610D is unaffected by APP knockdown. (E) Cells co-transfected with APP + FE65, FE65 S610A or FE65 S610D were subjected to 16 h MG132 treatment. The protein levels of APP and FE65 in transfected cell lysates was compared by Western blotting. Accelerated APP and FE65 turnover in APP + FE65 S610D-transfected cells is rescued by MG132. (F) Cells were co-transfected with APP + FE65, FE65 S610A or FE65 S610D + control or FBL2 siRNA. The transfected cell lysate was immunoblotted for APP, FE65 and FBL2. FBL2 knockdown partially restores APP level in APP + FE65 S610D-transfected cells. Data for graphs in (D–F) were obtained from three independent experiments with n=3 (total number of samples analysed=9) and analysed by one-way ANOVA. **P<0.01; ***P<0.05; ns–P>0.05. Results are means ± S.D.
Mentions: Next, we sought to determine the effect of FE65 Ser610 phosphorylation on the turnover of APP. The turnover rate of APP in the presence of FE65/FE65 S610A/FE65 S610D was compared by CHX chase assay followed by densitometric analysis (Figures 6A and 6B). Our data show that FE65 S610A but not FE65 S610D prolongs the half-life of APP. Of note, it was found that FE65 S610D shows a higher turnover rate than FE65 and FE65 S610A (Figures 6A and 6C). We asked whether APP plays a role in regulating FE65 turnover. To address this issue, cells transfected with FE65/FE65 S610A/FE65 S610D + control or APP siRNA were either treated with proteasome inhibitor MG132 or vehicle control (DMSO) and the protein level of FE65 was analysed by Western blotting. As shown in Figure 6(D), APP knockdown results in reduction in FE65 and FE65 S610A levels (lane 1 compared with lane 2; lane 5 compared with lane 6) and the depletion is blocked upon MG132 treatment (lane 3 compared with lane 4; lane 7 compared with lane 8). This indicates that APP stabilizes FE65 and FE65 S610A by preventing their degradation through UPS. On the other hand, the protein level of FE65 S610D, which is unable to bind to APP, is not affected by endogenous APP level (lane 9 compared with lane 10). In a complementary approach, we performed an MG132 study in APP + FE65/FE65 S610A/S610D-transfected cells to evaluate the effect of FE65 Ser610 phosphorylation on APP and FE65 proteasomal degradation. As shown in Figure 6(E), depletion of APP and FE65 in APP + FE65 S610D-transfected cells is rescued by proteasome inhibition by MG132. A previous report provided evidence that APP is ubiquitinated by the FBL2, a component of the SCF (Skp1–cullin1–F-box protein) E3 ubiquitin ligase complex, resulting in enhanced proteasomal degradation [40]. We enquired whether FE65 precludes FBL2-mediated APP degradation. To do this, cells were co-transfected with APP + FE65, FE65 S610A or FE65 S610D + control or FBL2 siRNA and APP protein level was compared. As shown in Figure 6(F), loss of FBL2 partially restores APP level in APP + FE65 S610D-transfected cells. Taken together, the current findings suggest that the interaction between APP and FE65 protects APP from proteasomal degradation through FBL2 and this process is regulated by the phosphorylation status of FE65 Ser610.

Bottom Line: We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser(610) phosphorylation inhibits this effect.Furthermore, we found that the effect of FE65 Ser(610) phosphorylation on APP processing is linked to a role of FE65 in metabolic turnover of APP via the proteasome.Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser(610) by SGK1 regulates binding of FE65 to APP, APP turnover and processing.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR.

Show MeSH
Related in: MedlinePlus