Caspase-mediated cleavage of the ubiquitin-protein ligase Nedd4 during apoptosis.
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.
Harvey
K F
KF
Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia.
Harvey
N L
NL
Michael
J M
JM
Parasivam
G
G
Waterhouse
N
N
Alnemri
E S
ES
Watters
D
D
Kumar
S
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D008025:Ligases; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D010449:Peptide Mapping; D051381:Rats; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases; D019014:fas Receptor / Q000378:metabolism
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D019014:fas Receptor; D005047:Etoposide; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D008025:Ligases