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. 2019 Nov 22;294(47):17768-17776.
doi: 10.1074/jbc.RA119.008310. Epub 2019 Oct 11.

Phosphorylation of the amyloid precursor protein (APP) at Ser-675 promotes APP processing involving meprin β

Preeti Kumaran Menon  1 , Niina Anneli Koistinen  1 , Kerstin Iverfeldt  1 , Anna-Lena Ström  2
Affiliations

Phosphorylation of the amyloid precursor protein (APP) at Ser-675 promotes APP processing involving meprin β

Preeti Kumaran Menon et al. J Biol Chem. .

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of β-amyloid (Aβ) peptides. Aβ is a cleavage product of the amyloid precursor protein (APP), and aberrant posttranslational modifications of APP can alter APP processing and increase Aβ generation. In the AD brain, seven different residues, including Ser-675 (APP695 numbering) in the APP cytoplasmic domain has been found to be phosphorylated. Here, we show that expression of a phosphomimetic variant of Ser-675 in APP (APP-S675E), in human neuroblastoma SK-N-AS cells, reduces secretion of the soluble APP ectodomain (sAPPα), even though the total plasma membrane level of APP was unchanged compared with APP levels in cells expressing APPwt or APP-S675A. Moreover, the level of an alternative larger C-terminal fragment (CTF) increased in the APP-S675E cells, whereas the CTF form that was most abundant in cells expressing APPwt or APP-S675A decreased in the APP-S675E cells. Upon siRNA-mediated knockdown of the astacin metalloprotease meprin β, the levels of the alternative CTF decreased and the CTF ratio was restored back to APPwt levels. Our findings suggest that APP-Ser-675 phosphorylation alters the balance of APP processing, increasing meprin β-mediated and decreasing α-secretase-mediated processing of APP at the plasma membrane. As meprin β cleavage of APP has been shown to result in formation of highly aggregation-prone, truncated Aβ2-40/42 peptides, enhanced APP processing by this enzyme could contribute to AD pathology. We propose that it would be of interest to clarify in future studies how APP-Ser-675 phosphorylation promotes meprin β-mediated APP cleavage.

Keywords: ADAM; APP-CTF; Alzheimer's disease; amyloid precursor protein (APP); amyloid-beta (Aβ); meprin β; neurodegeneration; proteolytic processing; β-secretase 1 (BACE1).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
APP–Ser-675 phosphorylation decreases sAPPα secretion while increasing the level of a slower migrating APP-CTF. A, representative Western blot analysis of full-length APP (detected by CT-APP), sAPPα (detected by 6E10), and total sAPP (detected by 22C11) from nontransfected (lanes 1 + 2) or APPwt (lanes 3 + 4), APP-S675A (lanes 5 + 6), or APP-S675E (lanes 7 + 8) transfected SK-N-AS cells, in the absence (lanes 1 + 3 + 5 + 7) or presence (lanes 2 + 4 + 6 + 8) of the γ-secretase inhibitor DAPT. B, representative Western blot analysis of APP-CTFs (detected with CT-APP) from nontransfected (lanes 1 + 2) or APPwt (lanes 3 + 4), APP-S675A (lanes 5 + 6), or APP-S675E (lanes 7 + 8) transfected SK-N-AS cells, in the absence (lanes 1 + 3 + 5 + 7) or presence (lanes 2 + 4 + 6 + 8) of the γ-secretase inhibitor DAPT. C, quantification of the full-length APP level, normalized against the corresponding tubulin level. D and E, relative abundance of secreted sAPPα and total sAPP in culture medium from DAPT-treated APPwt, APP-S675A, and APP-S675E overexpressing SK-N-AS cells. The level of sAPPα and total sAPP were normalized against both the level of corresponding total APP expression and the protein concentration in cell lysate. F, quantification of the total APP-CTF levels, normalized against the corresponding APP full-length level, in cell lysate from DAPT-treated APPwt, APP-S675A, and APP-S675E overexpressing SK-N-AS cells. G, ratio of APP-CTF upper/APP-CTF lower band in cell lysate of DAPT-treated APPwt, APP-S675A, and APP-S675E overexpressing SK-N-AS cells. For quantifications, *, p < 0.05; **, p < 0.01; n.s, not significant; n = 3–4.
Figure 2.
Figure 2.
ADAM10, ADAM17/TACE, and meprin β, but not BACE1, are expressed in SK-N-AS cells. Representative Western blot analysis of BACE1, ADAM10, ADAM17/TACE, and meprin β expression in two extracts from SK-N-AS (lane 1 + 2) and SH-SY5Y (lane 3 + 4) cells, respectively. For ADAM10 arrow indicates immature ADAM10 and arrowhead mature ADAM10.
Figure 3.
Figure 3.
APP cell surface localization is not affected by APP–Ser-675 phosphorylation. A, representative Western blot analysis of total APP in cell lysate and pulled down biotinylated APP from APPwt (lanes 2 and 6), APP-S675A (lanes 3 and 7), and APP-S675E (lanes 4 and 8) overexpressing SK-N-AS cells. B, relative abundance of biotinylated full-length APP from APPwt, APP-S675A, and APP-S675E overexpressing SK-N-AS cells. n.s, not significant; n = 4.
Figure 4.
Figure 4.
Analysis of N-terminal APP fragments. Representative Western blot analysis of total secreted sAPP (detected by 22C11) and N-terminal APP fragments (N-APP20, detected with 22C11) in APPwt (lanes 3 + 4), or APP-S675E (lanes 5 + 6) expressing SK-N-AS cells, in the absence (lanes 3 + 5) or presence (lanes 4 + 6) of co-transfected meprin β.
Figure 5.
Figure 5.
Meprin β knockdown decreases formation of the slower migrating CTF in APP-S675E cells, without affecting the total level of APP-CTFs. A, representative blot showing the expression of full-length APP, APP-CTFs, and meprin β in DAPT (γ-secretase inhibitor)-treated, non–APP-transfected (lanes 1 and 2), APPwt (lanes 3 and 4), or APP-S675E (lanes 5 and 6) overexpressing SK-N-AS cells co-transfected with meprin β (lanes 2, 4, 6) or control (lanes 1, 3, 5) siRNA. B, quantification of meprin β expression in SK-N-AS cells transfected with control or meprin β siRNA. C, quantification of full-length APP, normalized against tubulin, in cells treated as in A. D, quantification of the total APP-CTF levels, normalized against the corresponding APP full-length level, in cell lysate from APPwt and APP-S675E overexpressing SK-N-AS cells treated with meprin β or control siRNA. E, quantification of the upper slower C99 CTF, normalized against the corresponding APP full-length level, in cell lysate from APPwt and APP-S675E overexpressing SK-N-AS cells treated with meprin β or control siRNA. F, ratio of APP-CTF upper/APP-CTF lower band in cell lysate of APPwt and APP-S675E overexpressing SK-N-AS cells, treated with meprin β or control siRNA. For quantifications, *, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 4.
Figure 6.
Figure 6.
Metalloproteinase inhibitors decrease the levels of both APP-CTFs generated in APP-S675E expressing cells. A, representative Western blot analysis of full-length APP and APP-CTFs from APPwt (lanes 1 + 2 + 3), APP-S675A (lanes 4 + 5 + 6), and APP-S675E (lanes 7 + 8 + 9) overexpressing SK-N-AS cells, in the presence of the γ-secretase inhibitor DAPT together with either GI254023X (A10inh) or batimastat. UT, untransfected. B, relative abundance of total APP-CTFs in cell lysate from APPwt, APP-S675A, and APP-S675E overexpressing SK-N-AS cells in the presence of the γ-secretase inhibitor DAPT and either GI254023X (A10inh) or batimastat. C, relative abundance of full-length APP in cell lysate from APPwt, APP-S675A, and APP-S675E overexpressing SK-N-AS cells in the presence of the γ-secretase inhibitor DAPT together with either the GI254023X (A10inh) or the batimastat inhibitor. ***, p < 0.001; n = 4.
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