A presenilin-1/gamma-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

Abstract

E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a gamma-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PS1/gamma-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/gamma-secretase cleavage dissociates E-cadherins, beta-catenin and alpha-catenin from the cytoskeleton, thus promoting disassembly of the E-cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble beta- and alpha-catenins. Thus, the PS1/gamma-secretase system stimulates disassembly of the E-cadherin- catenin complex and increases the cytosolic pool of beta-catenin, a key regulator of the Wnt signaling pathway.

Fig. 1. A PS1-mediated γ-secretase activity controls E-cadherin processing. (A) Extracts from PS1+/+ or PS1–/– mouse embryos were probed on western blots with either anti-cytoplasmic E-cadherin C36 (upper panel) or anti-cytoplasmic APP R1 (middle panels) antibodies. E-Cad/FL denotes full-length E-cadherin. The asterisk identifies mouse IgGs. Lower panel: extract probed with anti-PS1/CTF antibody 33B10. (B) Extracts from E-cadherin-transfected PS1+/+ or PS1–/– mouse fibroblasts were probed with anti-E-cadherin C36 (upper panel) or 33B10 (lower panel) antibodies. (C) PS1+/+ fibroblasts were treated for 6 h either with the γ-secretase inhibitor L-685,458 (0.5 µM) or with dimethylsulfoxide. Extracts from these cell cultures were then probed with anti-E-cadherin C36. The asterisk indicates a non-specific band.

Fig. 2. A PS1-mediated γ-secretase activity cleaves E-cadherin. (A) A431 cells were treated for the indicated times with 1 µM of staurosporine (STS) to induce apoptosis (Steinhusen et al., 2001), solubilized in RIPA and blotted with anti-E-cadherin C36 antibody. (B) A431 cells were pre-incubated for 30 min in the absence (–) or presence (+) of GM6001 (2.5 µM), Z-DEVD-FMK (DEVD, 50 µM), an inactive analogue of L-685,458 (Control, 0.5 µM) or L-685,458 (0.5 µM). Cells were then treated with STS for 6 h to induce apoptosis, and cell extracts were probed with C36 antibody. Middle and lower panels: the filter was exposed to X-ray films for either 5 or 0.5 min. (C) Conditioned media (20 µl) from A431 cells cultured in the absence (–) or presence (+) of GM6001 and treated with STS as above were probed on western blots with anti-E-cadherin ectodomain antibody H108. E-Cad/NTF1 indicates the secreted E-cadherin ectodomain. (D) Extracts from HEK293 cells stably transfected with either wild-type (WT) PS1 or vector alone were immunoprecipitated and probed with C36 antibody (upper panels). Lower panel: extracts from the above cells were probed with anti-PS1/NTF antibody R222. (E) A431 cells were pre-incubated for 30 min in the absence (–) or presence (+) of GM6001 (2.5 µM). Cells were then treated with STS for 6 h and cell extracts were probed on western blots with either H108 (left panel) or C36 (right panel) antibodies.

Fig. 3. Amino acid sequence of human E-cadherin (SWISS-PROT accession No. P12830) indicating the N-termini of E-Cad/CTF1 and E-Cad/CTF2. Arrows identify the cleavage sites of MMP and PS1/γ-secretase. The sequence mediating E-cadherin–PS1 binding is underlined (Baki et al., 2001). Caspase-3 cleavage was reported recently (Steinhusen et al., 2001). EC1–5 denote the extracellular E-cadherin repeats. TM, transmembrane domain. H108 and C36 are immunogenic regions recognized by the respective antibodies.

Fig. 4. Released E-Cad/CTF2 dissociates from PS1 but remains bound to β-catenin. (A) Extracts from STS-treated A431 cells were immunoprecipitated with antibodies against PS1 (I-R222), pre-immune serum (PI-R222), β-catenin or desmoglein, and the immunoprecipitates (IPs) obtained were probed on western blots with anti-E-cadherin antibody C36. For reference, cell lysate was also probed; the asterisk shows IgGs. (B) A431 cells treated for 6 h with STS were fractionated into membrane, soluble cytosolic and Triton X-100-insoluble (TX100- insoluble) fractions, and the fractions obtained were then probed on western blots with C36 antibody.

Fig. 5. A PS1/γ-secretase cleavage promotes disassembly of the E-cadherin–catenin adhesion complex. (A and B) A431 cell cultures were pre-incubated for 30 min in the absence (–) or presence (+) of L-685,458 and then treated for the indicated times with ionomycin (10 µM). Cell extracts were fractionated, and the Triton X-100-insoluble and cytosolic soluble fractions were analyzed on western blots with antibodies against cytoplasmic E-cadherin C36 (A) or β- and α-catenins (B). The immunoblots are representative of three independent experiments. (C) Signals from cytosolic β-catenin (upper graph) or α-catenin (lower graph) obtained from ionomycin-treated cultures in the presence (+L-685,458) or absence (–L-685,458) of γ-secretase inhibitor L-685,458 were quantified by densitometric analysis. The graphs show the averaged immunoreactivities observed in three independent experiments.

Fig. 6. Immunostaining and LSCM analysis of ionomycin- and L-685,458-treated A431 cultures. A431 cells were pre-incubated for 30 min in the presence or absence of L-685,458 and then treated for 45 min with ionomycin. Control cells were not treated. Following the ionomycin-induced cell–cell dissociation, the distribution of PS1, E-cadherin, β-catenin and α-catenin in all cultures was analyzed by LSCM using the constant detector setting. Cells were double labeled either with anti-PS1/NTF antibody R222 (A–C) and anti-cytoplasmic E-cadherin antibody C36 (D–F), or with anti-ectodomain E-cadherin antibody H108 (G–I) and anti-β-catenin antibody (J–L). Cells were also labeled for α-catenin (M–O). Arrows indicate a cell population showing β-catenin immunoreactivity at the cell surface without ectodomain E-cadherin labeling (I and L). Scale bar = 30 µm.

Fig. 7. E-cadherin mutation GGG759-761AAA prevents binding to PS1 and inhibits γ-secretase cleavage of E-cadherin and cytosolic release of catenins. (A) Right panel: extracts from A431D cells stably transfected with vector, wild-type E-cadherin (WT E-Cad) or E-cadherin mutant GGG759-761AAA (761AAA) were immunoprecipitated with anti-PS1/NTF antibody R222 (PS1 IP) and the IPs were probed with either anti-E-cadherin antibody C36 (upper panel) or anti-PS1/CTF antibody 33B10 (lower panel). The left panel shows relative E-cadherin levels in transfectants. (B) A431D cells transfected with either wild-type E-cadherin or E-cadherin mutant 761AAA were incubated in the absence (–) or presence (+) of ionomycin for 45 min, and RIPA extracts were probed on western blots with C36 antibody (upper panel). Cytosolic fractions of the above cultures were probed on western blots with antibodies against E-cadherin (C36, second panel), β-catenin (third panel) or α-catenin (lower panel). (C) Schematic representation of the PS1/γ-secretase-mediated disassembly of CAJs. An MMP-mediated proteolytic activity cleaves the extracellular domain of cytoskeletal E-cadherin and releases E-Cad/NTF1 to the extracellular medium (a). Fragment E-Cad/CTF1 containing the transmembrane and cytoplasmic sequence of E-cadherin remains bound to PS1, β-catenin, α-catenin and the actin cytoskeleton. E-Cad/CTF1 is then cleaved by a PS1/γ-secretase activity at the membrane–cytosol interface to produce E-Cad/CTF2, which dissociates from both PS1 and F-actin and is released to the cytosol in a complex with β-catenin (b). Full-length E-cadherin bound to the cytoskeleton can also be cleaved by the PS1/γ-secretase activity (c). No E-Cad/CTF2–α-catenin complex was detected, suggesting that α-catenin dissociates from E-Cad/CTF2 (unpublished observations). α, α-catenin; β, β-catenin.