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. 2002 Mar 19;99(6):4014-9.
doi: 10.1073/pnas.052017699. Epub 2002 Mar 12.

Notch receptor cleavage depends on but is not directly executed by presenilins

Yoshihito Taniguchi  1 , Helena Karlström, Johan Lundkvist, Tomohiko Mizutani, Akira Otaka, Monica Vestling, Alan Bernstein, Dorit Donoviel, Urban Lendahl, Tasuku Honjo
Affiliations

Notch receptor cleavage depends on but is not directly executed by presenilins

Yoshihito Taniguchi et al. Proc Natl Acad Sci U S A. .

Abstract

Notch receptors undergo three distinct proteolytic cleavages during maturation and activation. The third cleavage occurs within the plasma membrane and results in the release and translocation of the intracellular domain into the nucleus to execute Notch signaling. This so-called gamma-secretase cleavage is under the control of presenilins, but it is not known whether presenilins themselves carry out the cleavage or whether they act by means of yet-unidentified gamma-secretase(s). In this article, we show that Notch intracellular cleavage in intact cells completely depends on presenilins. In contrast, partial purification of the Notch cleavage activity reveals an activity, which is present only in protein extracts from presenilin-containing cells, and which does not comigrate with presenilin. This finding provides evidence for the existence of a specific Notch-processing activity, which is physically distinct from presenilins. We conclude from these experiments that presenilins are critically required for Notch intracellular cleavage but are not themselves directly mediating the cleavage.

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Figures

Figure 1
Figure 1
Analysis of Notch ΔEGVP constructs. (A) Schematic illustration of the different constructs used in the study. * denotes the site of mutation in the Notch (N) receptors (V1744G in N1 and GVM1662–1664AAA in N3). Gal4VP16 is denoted GVP. HA tag, hemagglutinin tag. (B) Analysis of the effects of mutations in the cleavage site 3 of N1 ΔEGVP and N3 ΔEGVP (compare N1 ΔEGVPmut versus N1 ΔEGVP and N3 ΔEGVPmut versus N3 ΔEGVP). A strong reduction of cleavage is observed with both mutants. The N1 LNR GVP protein, which lacks the EGF repeats at the extracellular side, is not processed at detectable levels. Below is shown a Western blot examining the expression levels of the different constructs. The experiment was repeated at least three times with triplicates in each assay. ***, P < 0 001; **, P <0.01; *, P < 0.05 vs. Mock. (C) Analysis of the effects of γ-secretase inhibitors on Notch processing. Cleavage of both N1 ΔEGVP and N3 ΔEGVP was reduced by the addition of L-685,458 and MW167, but not by DMSO and L-405,484, which is structurally related to L-685,458, but does not inhibit γ-secretase activity. ###, P < 0.001 vs. N1 ΔEGVP; §§§, P < 0.001 vs. N3 ΔEGVP. (D) Analysis of N ΔEGVP processing in the presence and absence of presenilins. Cleavage of N1 ΔEGVP and N3 ΔEGVP was analyzed in transiently transfected cells deficient for PS1 and PS2 (BD8 cells). No cleavage of the N ΔEGVP-encoding constructs is observed in BD8 cells in the absence of presenilin, whereas substantial processing is recorded after cotransfection of PS1 cDNA, PS2 cDNA, or both PS1 and PS2 cDNAs. The experiment was performed in triplicate and repeated three times. ***, P < 0.001 vs. Mock; ###, P < 0.001 vs. N1 ΔEGVP; §§§, P < 0.001 vs. N3 ΔEGVP.
Figure 2
Figure 2
Specific cleavage of a Notch 1 fluorogenic peptide. (A) Depicted is the amino acid sequence encompassing the mouse N1 site 3 cleavage site. The wild-type (GCGVLL) and mutant V→K (GCGKLL) fluorogenic peptides are outlined below. The position at which the site 3 cleavage occurs is underlined. (B) Cleavage of the fluorogenic peptide depends on addition of membrane fraction. The fluorescence signal emitted from the cleaved peptide increased linearly with time when incubated with the membrane fraction for up to 6 h. No signal was observed when the peptide was incubated in the absence of membrane fraction. (C) Cleavage of the peptide is sequence-specific. The conversion rate measured after 120 min of incubation increased proportionally to the amount of membrane fraction added. The wild-type peptide (WT, ◊) was more frequently cleaved than the mutant (MUT, V→K) peptide (■) at all concentrations of membrane fraction. (D) Challenge of peptide cleavage with protease inhibitors. Cleavage of the wild-type peptide was inhibited by MW167 and MG132 but to a much lesser extent by clasto-lactacystin β-lactone.
Figure 3
Figure 3
A detergent solubilized Notch intracellular cleavage activity. Fluorogenic conversion of the wild-type Notch peptide measured in the supernatant (Sup, filled bars) or pellet (empty bars) from membrane fractions solubilized at different concentrations of CHAPS. Maximal solubilized activity was obtained at 0.3% CHAPS.
Figure 4
Figure 4
Purification of the detergent-solubilized Notch intracellular cleavage activity based on charge distribution. (A) CHAPS-solubilized protein fractions of HEK293T cells were passed through a Sepharose Q column in the presence of 1 mM EDTA and eluted at various NaCl concentrations (0–500 mM NaCl). The eluates were investigated for fluorogenic conversion and two peaks were identified: peak A (fraction 9–13) and peak B (fraction 16–22). (B) Comparison of cleavage activities from presenilin-containing and presenilin-deficient cells. The fluorogenic conversion profiles from presenilin-containing BD3 (□) and presenilin-deficient BD8 (⧫) cells after separation over a Sepharose Q column. Note that peak A is present only in extracts from BD3 cells, whereas peak B is retained in extracts from both BD3 and BD8 cells. A Western blot analysis of the protein fractions from the BD3 and BD8 cells probed with an antibody to PS1 is shown at the bottom. Note that PS1 protein is detected only in BD3 cells, where it coincides with peak B.
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References

    1. Artavanis-Tsakonas S, Rand M D, Lake R J. Science. 1999;284:770–776. - PubMed
    1. Frisén J, Lendahl U. BioEssays. 2001;23:3–7. - PubMed
    1. Kidd S, Lieber T, Young M. Genes Dev. 1998;12:3728–3740. - PMC - PubMed
    1. Lecourtois M, Schweisguth F. Curr Biol. 1998;8:771–774. - PubMed
    1. Schroeter E H, Kisslinger J A, Kopan R. Nature (London) 1998;393:382–385. - PubMed

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