MAGUKs, scaffolding proteins at cell junctions, are substrates of different proteases during apoptosis

Abstract

A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell-cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein-protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent L-leucyl-L-leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell-cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment.

Figure 1

Cleavage of Dlg1, ZO-1 and ZO-3 in MDCK and HaCaT cells after induction of apoptosis with UV-irradiation and STS. (a) MDCK cells 12 h after UV irradiation and HaCaT cells 12 h after STS treatment were viewed under light microscopy. zVAD-fmk was added 2 h before induction of apoptosis. (b) The percentage of detached MDCK cells 12 and 16 h after UV irradiation and HaCaT cells after 12 h of 500 nM and 1 μM STS treatment was determined as described in Materials and methods section. zVAD-fmk was added 2 h before induction of apoptosis. (c and d) Western blot analysis of total cell lysates from MDCK and HaCaT cells treated with UV light or STS in presence or absence of z-VAD-fmk. Cells were harvested and lysed 12 or 16 h after the treatment (same conditions as in b and c). The total lysates were subjected to western blotting with antibodies indicated in the figure

Figure 2

Loss of ZO-1 from the membrane during UV irradiation-induced apoptosis. (a) MDCK cells were subjected to UV irradiation in the presence or absence of z-VAD-fmk. Cells were fixed 6 h later and ZO-1 localization was analyzed by immunofluorescence (green). DAPI was used to stain the nuclei. White arrows (middle panel) point to loss of ZO-1 from the membrane and red arrows (middle panel) to possible subnuclear or nuclear localization of cleavage fragment(s) of ZO-1. Forty-fold magnification was used. (b and c) MDCK cells were subjected to UV irradiation and fixed at indicated times. After staining with ZO-1 antibody and DAPI, cells were visualized under fluorescent microscope (b) and the percentage of condensed and fragmented nuclei was determined (c). (d) At indicated times after UV irradiation, detached and attached cells were collected separately, counted and the percentage of detached cells was calculated

Figure 3

Time-dependent cleavage of ZO-1 during apoptosis in HaCaT cells. (a) HaCaT cells were treated with 1 μM STS in the presence or absence of zVAD-fmk and collected at indicated times. DEVDase activity was quantified by fluorogenic caspase substrate DEVD-afc. (b) Time-dependent cleavage of ZO-1, PARP and E-cadherin during STS-induced apoptosis in HaCaT cells was analyzed by western blot assays using specific antibodies. E-cadherin densiometry is indicated. (c) HaCaT cells were UV irradiated in the presence or absence of zVAD-fmk and detached and attached cells were collected separately 12 and 24 h later and the percentage of detached cells was determined. (d) HaCaT cells were UV irradiated in the presence or absence of zVAD-fmk. After 12 h, cells were collected either all together or detached and attached separately and stained with annexin V-PE and PI for flow cytometry analysis. In control experiment treatment was omitted. Each plot represents a typical experiment of 10 000 cells. Almost all detached HaCaT cells were annexin V-positive, while in the attached cells the percentage of the annexin V-positive cells was low. (e and f) Detached and attached HaCaT cells were collected separately at indicated times after UV-induced apoptosis. DEVD-ase activity of attached and detached cells was determined with fluorogenic caspase substrate Ac-DEVD-afc (e). In parallel, total cell lysates were subjected to western blotting with anti-ZO-1 and E-cadherin (f). Owing to the low number of detached cells remaining after 12 h in the presence of zVAD-fmk, we followed the cleavage of ZO-1 and E-cadherin 24 h after UV irradiation, when the number of detached cells is slightly lower compared with the 12-h time point without the inhibitor. zVAD-fmk almost completely abolished the cleavage of ZO-1 in both detached and attached cells, but not the cleavage of E-cadherin

Figure 4

In vitro cleavage of MAGUK proteins with different proteases involved in apoptosis. (a) In vitro translated MAGUK proteins were incubated with recombinant executioner caspases-3, -6 and -7 for 1 h at 37 °C. Samples were resolved on 10 or 12.5% SDS-PAGE and visualized by autoradiography. (b) In vitro translated MAGUK proteins were incubated with recombinant cathepsins as indicated in the figure. Cathepsins were activated at pH 6 and added to the translated proteins and buffer with pH 7 for 45 min at 37 °C. (c) Radiolabelled MAGUK proteins were incubated with recombinant granzyme B (GrB) or inactive form of recombinant granzyme B (NA) for 4 h at 37 °C. (d) In vitro translated wt, D761A and DD543AA mutants of MAGI-1 were incubated with granzyme B (GrB) or inactive form of granzyme B (NA) for 4 h at 37 °C. (e) In vitro translated MAGUK proteins and mutants of MAGI-1 (D761A and DD543AA) were incubated with recombinant caspase-8 for 1 h at 37 °C. All samples were resolved on 10 or 12.5% SDS-PAGE gels and visualized by autoradiography. In control experiments proteases were omitted

Figure 5

Cleavage of Dlg1, ZO-1 and ZO-3 in HaCaT and CaCo-2 cells after induction of apoptosis with LeuLeuOMe. (a) HaCaT and CaCo-2 cells were incubated with LeuLeuOMe for 20 h in the presence or absence of cysteine cathepsin inhibitor E64d or pan-caspase inhibitor zVAD-fmk. Morphology of cells was visualized under light microscopy. (b) HaCaT and CaCo-2 cells were treated with LeuLeuOMe in presence or absence of E64d or zVAD-fmk. Detached and attached cells were collected separately and percentage of detached cells was determined. (c and d) HaCaT (c) and CaCo-2 (d) cells were harvested 20 h after induction of apoptosis with LeuLeuOMe in presence or absence of inhibitors. Total cell lysates were subjected to western blotting with anti-Dlg1, anti-ZO-1, anti-ZO-3 and anti-PARP. (e) CaCo-2 cells were treated with LeuLeuOMe in the presence or absence of E64d and zVAD-fmk, and analyzed 12 h after induction of apoptosis by immunofluorescence. DAPI was used as a counterstain to visualize the nuclei. Forty-fold magnification was used

Figure 6

Model of cell–cell detachment during apoptosis. (a) Schematic representation of adherens junction complex. Adhesion molecule E-cadherin is connected to actin cytoskeleton via β- and α-catenin. MAGUK proteins strengthen the complex and also bind other adaptor and signalling molecules (not shown). (b) After caspase activation, MAGUK proteins are the first proteins cleaved in this complex thus allowing access to other components of cell junction closer to the membrane, such as β-catenin and E-cadherin. On LMP cathepsins translocate to the cytosol and can cleave MAGUKs. MMPs probably in later stages of apoptosis shed the extracellular part of E-cadherin, thus completing the disruption of cell–cell contact