Identification of the Cytolinker Plectin as a Major Early In Vivo Substrate for Caspase 8 during CD95- and Tumor Necrosis Factor Receptor-Mediated Apoptosis

Abstract

Caspase 8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase 8, we used a panel of subunit-specific anti-caspase 8 monoclonal antibodies in confocal immunofluorescence microscopy. In the human breast carcinoma cell line MCF7, caspase 8 predominantly colocalized with and bound to mitochondria. After induction of apoptosis through CD95 or tumor necrosis factor receptor I, active caspase 8 translocated to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase 8 prodomain remained bound to mitochondria. Plectin was quantitatively cleaved by caspase 8 at Asp 2395 in the center of the molecule in all cells tested. Cleavage of plectin clearly preceded that of other caspase substrates such as poly(ADP-ribose) polymerase, gelsolin, cytokeratins, or lamin B. In primary fibroblasts from plectin-deficient mice, apoptosis-induced reorganization of the actin cytoskeleton, as seen in wild-type cells, was severely impaired, suggesting that during apoptosis, plectin is required for the reorganization of the microfilament system.

ACKNOWLEDGMENTS

We thank U. Matiba and D. Süss for excellent technical assistance, H. Heid for microsequencing fragments of recombinant plectin, and Branislav Nikolic for generating plectin cDNA expression plasmids and isolation of recombinant proteins. The MCF7-Fas and MCF7-Fas-Bcl-x_L cells and the MCF7-caspase 3 cells were generous gifts from M. Jäättelä and A. Porter, respectively. We are grateful to G. Salvesen for providing active recombinant caspases, to H. Söling for providing the anti-PDI antibody, and to P. Lichter for critically reading the manuscript.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Li 406/3-1 and PE 653/1-2), the Bundesministerium für Forschung und Technologie, the Tumor Center Heidelberg/Mannheim, the Deutsche Leukämieforschungshilfe, and the Austrian Science Foundation (PI2389 and SFB006/661). A.H.S. was supported by a stipend from the Boehringer Ingelheim Fonds.