Cleavage of MAGI-1, a tight junction PDZ protein, by caspases is an important step for cell-cell detachment in apoptosis

Abstract

MAGI-1, a member of the MAGUK family of proteins, is shown to be rapidly cleaved during Fas-induced apoptosis in mouse 3T3 A31 cells, and in UV irradiation- and staurosporine-induced apoptosis in HaCaT cells. This generates a 97 kDa N-terminal fragment that dissociates from the cell membrane; a process that is largely prevented in the presence of the caspase inhibitor Z-VAD-fmk. In addition, we show that in vitro translated radiolabelled MAGI-1 is efficiently cleaved into 97 kDa and 68 kDa fragments by caspases-3 and -7 at physiological concentrations and mutating the MAGI-1 Asp(761) to Ala completely abolished the caspase-induced cleavage. Moreover, in HaCaT cells overexpressing the MAGI-1 Asp(761)Ala mutant the disruption of cell-cell contacts was delayed during apoptosis, whereas other caspase-dependent processes such as nuclear condensation were not affected, suggesting that cell detachment is parallel to them. Thus, MAGI-1 cleavage appears to be an important step in the disassembly of cell-cell contacts during apoptosis.

Fig. 1. Fas-induced apoptosis in 3T3 A31 cells: time course of MAGI-1 and β-catenin cleavage and apoptosis quantification

(A) Experiments were performed in the absence (−) or presence (+) of 50 μM Z-VAD-fmk. Cells were harvested at indicated time points post induction of apoptosis by anti-Fas antibody. Following SDS polyacrylamide gel electrophoresis, samples were analyzed by Western blot using an antibody raised against the WW domains of MAGI-1 or an antibody against β-catenin (Santa Cruz). The full length MAGI-1c isoform is arrowed together with the 97 kDa cleavage product. Actin was used as a loading control. (B) Quantification of apoptosis by FACS analysis 1 and 2 h post induction of apoptosis. Experimental details were as described under Materials and methods

Fig. 2. Immunolocalisation of MAGI-1 and β-catenin during Fas induced apoptosis in 3T3 A31 cells

Experimental conditions were as described under Materials and methods. The panels show MAGI-1 (upper panel) at 0 h (a), 1 h (b) and 2 h (c) post addition of anti Fas antibody and β-catenin (lower panel) at 0 h (d), 1 h (e) and 2 h (f) post addition of anti Fas antibody

Fig. 3. MAGI-1 and E-cadherin are cleaved during UV irradiation-induced apoptosis in HaCaT cells

(A) Cells were harvested at indicated time points and Western blot analysis of MAGI-1 protein was performed as described in Material and methods. Experiments were performed in the absence (left panel) or presence (right panel) of 50 μM caspase inhibitor Z-VAD-fmk. The position of MAGI-1 and the 97 kDa cleavage product are indicated by the arrows. (B) Quantification of apoptosis by FACS analysis based on Annexin V staining. Analysis was done using the CellQuest software. Samples were taken at times indicated. (C) Western blot analysis of E-cadherin cleavage during UV irradiation-induced apoptosis. Cells were harvested at times indicated. The position of E-cadherin and its cleavage products are indicated by arrows

Fig. 4. MAGI-1 cleavage during staurosporine-induced apoptosis in HaCat cells

(A) Cells were harvested at indicated time points and Western blot analysis of MAGI-1 protein was performed as described in Fig. 1. Experiments were performed in the absence (−) or presence(+) of 50 μM concentration of Z-VAD-fmk. The position of MAGI-1c and the 97 kDa cleavage product are indicated by the arrows. (B) Quantification of apoptosis by FACS analysis 18 h post induction of apoptosis. Analysis was done using the CellQuest software

Fig. 5. Cleavage of MAGI-1 by caspases-3, -6 and -7 in vitro

(A) Radiolabelled full-length MAGI-1 protein was incubated for 1 h with 1μM caspase-3 (lane 2), caspase-6 (lane 3) or caspase-7 (lane 4). Lane 1, control experiment in the absence of caspases. (B) Radiola-belled MAGI-1 was incubated for 1 h with decreasing concentrations of caspase-3 (right panel) or caspase-7 (left panel) and protein bands were visualized by autoradiography. In control experiments caspase was omitted. (C) Radiolabelled WT and MAGI-1 Asp₇₆₁Ala protein were incubated for 1 h with (+) or without (−) 1 μM caspase-3 and protein bands visualized by autoradiography

Fig. 6. Fluorescence imaging of HaCaT cells overexpressing WT and MAGI-1 Asp7₆1Ala/EGFP fusion proteins

(A) HaCaT cells were transfected with C-terminally GFP tagged WT or D761A mutant MAGI-1 and examined by fluorescent microscopy for their localisation. Green fluorescence is visible at the sites of cell-cell contacts. (B) HaCaT cells were transfected with C-terminally GFP tagged WT or D761A mutant MAGI-1 and seeded on glass coverslips. Apoptosis was induced 24 h post transfection with UV irradiation. Pictures were taken four hours after apoptosis induction. (C) HaCaT cells were transfected with C-terminally GFP tagged WT or D761A mutant MAGI-1 and seeded on glass coverslips. Apoptosis was induced with 500 nM staurosporine and pictures were taken at times indicated

Fig. 7. Quantification of apoptotic cells after apoptosis induction by UV

PS exposure was measured after apoptosis induction at times indicated in nontransfected and in WT or D761A MAGI-1 transfected HaCaT cells (open, striped and closed bars, respectively). All other experimental details are described in Materials and methods

Fig. 8. Schematic representation of MAGI-1 and caspase cleavage site(s)

Diagram of MAGI-1 (grey bar) showing the positions of the guanylate kinase domain, two WW domains and 6 PDZ domains, and the positions and sequences of caspase cleavage sites

Fig. 9. Sequence of events during cell-cell detachment in apoptosis

(A) Schematic representation of cell-cell adhesion complex. E-cadherin is connected to actin cytoskeleton via α- and β-catenin. MAGI-1 strengthens the complex. (B) After caspase activation, MAGI-1 is the first protein cleaved in this complex thus allowing access to other constituents, such as β-catenin and E-cadherin