Apoptotic cell death induces dramatic molecular changes in cells, becoming apparent on the structural level as membrane blebbing, condensation of the cytoplasm and nucleus, and loss of cell-cell contacts. The activation of caspases is one of the fundamental steps during programmed cell death. Here we report a detailed analysis of the fate of the Ca 2؉ -dependent cell adhesion molecule E-cadherin in apoptotic epithelial cells and show that during apoptosis fragments of Ecadherin with apparent molecular masses of 24, 29, and 84 kDa are generated by two distinct proteolytic activities. In addition to a caspase-3-mediated cleavage releasing the cytoplasmic domain of E-cadherin, a metalloproteinase sheds the extracellular domain from the cell surface during apoptosis. Immunofluorescence analysis confirmed that concomitant with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulates in the cytosol. In the presence of inhibitors of caspase-3 and/or metalloproteinases, cleavage of E-cadherin was almost completely blocked. The simultaneous cleavage of the intracellular and extracellular domains of E-cadherin may provide a highly efficient mechanism to disrupt cadherin-mediated cell-cell contacts in apoptotic cells, a prerequisite for cell rounding and exit from the epithelium.The crucial role of apoptosis during development and for tissue homeostasis of multicellular organisms is well established (1). Malfunctions of the death program and its control mechanisms often result in prenatal death during development and contribute to immune and neuronal diseases or cancer in the adult organism (2-4). The central mechanism of this cell death machinery is a proteolytic cascade mediated by the caspase family of cysteine proteinases (5, 6), which specifically cleave their substrates after aspartate residues. Caspases are synthesized as inactive proenzymes. After initiation of the apoptotic program these proenzymes are processed by two proteolytic events, generating a large subunit and a small subunit that form a heterodimer. The association of two heterodimers results in the formation of the active enzyme containing two catalytic sites (7,8). Depending on their position in the proteolytic cascade, caspase family members are divided into upstream initiator caspases and downstream effector caspases.The activation of this caspase cascade leads to the specific cleavage of substrate proteins and finally results in the morphological changes becoming apparent in apoptotic cells. The identification of an increasing number of caspase substrates has revealed different classes of cellular proteins that are cleaved during the effector phase of apoptosis. A set of substrate proteins is represented by proteins that protect living cells from apoptosis, e.g. ICAD/DFF45 (9, 10) and Bcl-2 (11, 12). Nuclear envelope proteins (e.g. lamin A and B (13-15) and LAP2 and Nup153 (16) (27)) and DNA repair (DNA-PK CS ) and of the splicing machinery (U1-70K) is assumed to support the disruption of structural...
-Catenin is a member of the Armadillo repeat protein family with a dual cellular function as a component of both the adherens junction complex and the Wnt/wingless signaling pathway. Here we show that -catenin is proteolytically cleaved during anoikis and staurosporine-induced apoptosis. Cleavage of -catenin was found to be caspase-dependent. Five cleavage products of -catenin were identified in vivo and after in vitro cleavage by caspase-3. Amino acid sequencing and mass spectrometry analysis indicated two caspase-3 cleavage sites at the C terminus and three further sites at the N terminus, whereas the central Armadillo repeat region remained unaffected. All -catenin cleavage products were still able to associate with E-cadherin and ␣-catenin and were found to be enriched in the cytoplasm. Functional analysis revealed that -catenin deletion constructs resembling the observed proteolytic fragments show a strongly reduced transcription activation potential when analyzed in gene reporter assays. We therefore conclude that an important role of the -catenin cleavage during apoptosis is the removal of its transcription activation domains to prevent its transcription activation potential.
We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.
Keratin 15 (K15) and keratin 17 (K17) are intermediate filament (IF) type I proteins that are responsible for the mechanical integrity of epithelial cells. By analyzing the human breast epithelial cell line H184A1 before and after induction of apoptosis by high-resolution two-dimensional gel electrophoresis (2-DE) we identified the caspase-mediated cleavage of keratins 15 and 17. After induction of apoptosis three fragments of both K15 and K17 could be observed by 2 -DE. K15 and K17 proteolysis was observed during staurosporineinduced apoptosis and anoikis (anchorage-dependent apoptosis) as well and was shown to be caspase-dependent. By using mass spectrometry we could determine the caspase cleavage sites, one in K15 and two in K17. The sequence VEMD/A at the cleavage site located in the conserved linker region was found in K15 and K17. A further cleavage site was identified in the tail region of K17 with the recognition motif EVQD/G. Cell Death and Differentiation (2001) 8, 308 ± 315.
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