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. 1997 May 13;94(10):5090-4.
doi: 10.1073/pnas.94.10.5090.

Evidence for phosphorylation and oligomeric assembly of presenilin 1

M Seeger  1 , C Nordstedt, S Petanceska, D M Kovacs, G K Gouras, S Hahne, P Fraser, L Levesque, A J Czernik, P S George-Hyslop, S S Sisodia, G Thinakaran, R E Tanzi, P Greengard, S Gandy
Affiliations

Evidence for phosphorylation and oligomeric assembly of presenilin 1

M Seeger et al. Proc Natl Acad Sci U S A. .

Abstract

Pathogenic mutations in presenilin 1 (PS1) are associated with approximately 50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12, 13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20-23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage lambda protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.

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Figures

Figure 1
Figure 1
Effect of PDBu on phosphorylation of PS1 CTF. (Left) COS-huPS1 cells were incubated for 3.5 hr in the presence of [32P]orthophosphate. Vehicle or 1 μM PDBu was added for an additional 45 min. Cells were lysed and immunoprecipitation was performed with α PS1-loop antiserum as described. (Right) COS-huPS1, human PS1-transfected N2a, and PC12 cells were treated with PDBu, lysed, and subjected to immunoprecipitation with α PS1-loop antiserum as described. Immunoblot analysis was performed using α PS1-loop antiserum.
Figure 2
Figure 2
Regulation of phosphorylation of PS1 CTF by various protein kinase activators and phosphoprotein phosphatase inhibitors. COS-huPS1 cells were incubated in the absence or presence of PDBu (PBu; 1 μM), calyculin A (Cal; 10 nM), cyclosporin A (Cyc; 100 nM), thapsigargin (THP; 100 nM), or forskolin (FSK; 5 μM). Cells were lysed and subjected to immunoprecipitation with α PS1-loop antiserum followed by immunoblot analysis using α PS1-loop antiserum.
Figure 3
Figure 3
Effect of phosphoprotein phosphatase on electrophoretic migration of PS1 CTF. COS-huPS1 cells were treated with vehicle or PDBu, lysed, and subjected to immunoprecipitation. Sepharose-bound immune complexes were treated with λ protein phosphatase. Proteins were eluted from the Sepharose beads with Laemmli sample buffer and applied to SDS/12% PAGE, and immunoblot analysis was performed using α PS1-loop antiserum.
Figure 4
Figure 4
Phosphoamino acid analysis of 20-kDa and 23-kDa PS1 CTFs. COS-huPS1 cells were labeled with [32P]orthophosphate, treated with PDBu, and subjected to immunoprecipitation. The two major phosphorylated CTF species were subjected to phosphoamino acid analysis and were visualized by phosphorimager.
Figure 5
Figure 5
Gel filtration of proteins from COS-huPS1 cells. Nonidet P-40 extracts of cells were loaded onto a Superose 12 column and protein was eluted with buffer A containing 0.15 M NaCl. The indicated column fractions were analyzed by immunoblotting. Elution patterns of PS1 NTF (Ab 14) (A) and PS1 CTF (α PS1-loop) (B) are shown. The elution positions of molecular mass standards (kDa) are indicated by arrows.
Figure 6
Figure 6
Sucrose gradient fractionation of COS-huPS1 cells. Cells were treated with vehicle or PDBu and lysed using a steel-to-steel homogenizer, and extracts were applied to discontinuous sucrose gradients. Fractions were collected, and proteins were separated by SDS/12% PAGE and immunoblotted with antisera against the PS1 NTF (Ab 14) and PS1 CTF (α PS1-loop). Fractions are numbered from top to bottom of gradient.
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