Abstract
The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, gamma-radiation, tumor necrosis factor-alpha, C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237 downward arrow sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.
Highlights
Apoptosis is characterized by organized dismantling of the cell structure and involves the action of various classes of proteases [1]
The main players in the proteolytic cascade activated during apoptosis are caspases, a group of cysteine proteases related to the cell death protein CED-3 in Caenorhabditis elegans
Enzyme poly(ADP-ribose) polymerase (PARP)1 was one of the first identified cellular substrates cleaved in apoptosis [6]
Summary
Apoptosis is characterized by organized dismantling of the cell structure and involves the action of various classes of proteases [1]. Both antibodies detected identical Nedd4 cleavage products during Fas-mediated apoptosis in Jurkat cells, data with N4ab2 only are shown (Fig. 1). Cleavage of Nedd4 protein, similar to that seen when Jurkat cells were treated with anti-Fas antibody was evident, the kinetics of cleavage were slower reflecting slower apoptotic induction (Fig. 2).
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