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Caspase-mediated cleavage of the ubiquitin-protein ligase Nedd4 during apoptosis
K F Harvey1, N L Harvey, J M Michael
1Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia.
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.
Insights
Nedd4, a ubiquitin ligase, is cleaved during apoptosis by caspases. This study identifies Nedd4 as the first ubiquitin pathway enzyme targeted by caspases during programmed cell death.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Apoptosis involves the activation of caspases, a family of cysteine proteases.
- Caspases cleave specific target proteins at aspartate residues during programmed cell death.
- Previous studies identified various caspase substrates during apoptosis.
Purpose of the Study:
- To investigate whether Nedd4, a ubiquitin-protein ligase, is cleaved during apoptosis.
- To identify the caspases responsible for Nedd4 cleavage.
- To map the specific cleavage site(s) of Nedd4 by caspases.
Main Methods:
- Induction of apoptosis using various stimuli (Fas-ligation, gamma-radiation, TNF-alpha, etc.).
- Analysis of protein cleavage patterns in apoptotic cell extracts.
- In vitro cleavage assays using purified caspases and Nedd4.
- Site-directed mutagenesis to map caspase cleavage sites.
Main Results:
- Nedd4 was found to be cleaved during apoptosis induced by multiple stimuli.
- Cleavage of Nedd4 in apoptotic cells was inhibited by caspase-3-like protease inhibitors.
- In vitro, Nedd4 was cleaved by caspases-1, -3, -6, and -7.
- A conserved caspase cleavage site (DQPD237) in mouse Nedd4 was identified.
Conclusions:
- Nedd4 is a novel substrate for caspases during apoptosis.
- This study demonstrates the cleavage of a ubiquitin pathway enzyme by caspases.
- The findings provide new insights into the regulation of the ubiquitin system during programmed cell death.