Transcription factor AP-2alpha is preferentially cleaved by caspase 6 and degraded by proteasome during tumor necrosis factor alpha-induced apoptosis in breast cancer cells.
Several reports have linked activating protein 2alpha (AP-2alpha) to apoptosis, leading us to hypothesize that AP-2alpha is a substrate for caspases. We tested this hypothesis by examining the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of AP-2 in breast cancer cells. Here, we provide evidence that TNF-alpha downregulates AP-2alpha and AP-2gamma expression posttranscriptionally during TNF-alpha-induced apoptosis. Both a general caspase antagonist (zVADfmk) and a caspase 6-preferred antagonist (zVEIDfmk) inhibited TNF-alpha-induced apoptosis and AP-2alpha downregulation. In vivo tests showed that AP-2alpha was cleaved by caspases ahead of the DNA fragmentation phase of apoptosis. Recombinant caspase 6 cleaved AP-2alpha preferentially, although caspases 1 and 3 also cleaved it, albeit at 50-fold or higher concentrations. Activated caspase 6 was detected in TNF-alpha-treated cells, thus confirming its involvement in AP-2alpha cleavage. All three caspases cleaved AP-2alpha at asp(19) of the sequence asp-arg-his-asp (DRHD(19)). Mutating D(19) to A(19) abrogated AP-2alpha cleavage by all three caspases. TNF-alpha-induced cleavage of AP-2alpha in vivo led to AP-2alpha degradation and loss of DNA-binding activity, both of which were prevented by pretreatment with zVEIDfmk. AP-2alpha degradation but not cleavage was inhibited in vivo by PS-431 (a proteasome antagonist), suggesting that AP-2alpha is degraded subsequent to cleavage by caspase 6 or caspase 6-like enzymes. Cells transfected with green fluorescent protein-tagged mutant AP-2alpha are resistant to TNF-alpha-induced apoptosis, further demonstrating the link between caspase-mediated cleavage of AP-2alpha and apoptosis. This is the first report to demonstrate that degradation of AP-2alpha is a critical event in TNF-alpha-induced apoptosis. Since the DRHD sequence in vertebrate AP-2 is widely conserved, its cleavage by caspases may represent an important mechanism for regulating cell survival, proliferation, differentiation, and apoptosis.
Nyormoi
O
O
Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Wang
Z
Z
Doan
D
D
Ruiz
M
M
McConkey
D
D
Bar-Eli
M
M
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D020287:In Situ Nick-End Labeling; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D012323:RNA Processing, Post-Transcriptional; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D011994:Recombinant Proteins; C493673:TFAP2A protein, human; C493679:TFAP2C protein, human; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D049452:Green Fluorescent Proteins; D004247:DNA; C505514:CASP6 protein, human; D053178:Caspase 6; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex