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Molecular and Cellular Biology, August 2001, p. 4856-4867, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.4856-4867.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Transcription Factor AP-2 Is Preferentially Cleaved by Caspase 6 and Degraded by Proteasome during Tumor Necrosis Factor Alpha-Induced Apoptosis in Breast Cancer Cells

Okot Nyormoi, Zhi Wang, Dao Doan, Maribelis Ruiz, David McConkey, and Menashe Bar-Eli^*

Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Received 24 January 2001/Returned for modification 7 March 2001/Accepted 9 May 2001

Several reports have linked activating protein 2 (AP-2) to apoptosis, leading us to hypothesize that AP-2 is a substrate for caspases. We tested this hypothesis by examining the effects of tumor necrosis factor alpha (TNF-) on the expression of AP-2 in breast cancer cells. Here, we provide evidence that TNF- downregulates AP-2 and AP-2 expression posttranscriptionally during TNF--induced apoptosis. Both a general caspase antagonist (zVADfmk) and a caspase 6-preferred antagonist (zVEIDfmk) inhibited TNF--induced apoptosis and AP-2 downregulation. In vivo tests showed that AP-2 was cleaved by caspases ahead of the DNA fragmentation phase of apoptosis. Recombinant caspase 6 cleaved AP-2 preferentially, although caspases 1 and 3 also cleaved it, albeit at 50-fold or higher concentrations. Activated caspase 6 was detected in TNF--treated cells, thus confirming its involvement in AP-2 cleavage. All three caspases cleaved AP-2 at asp¹⁹ of the sequence asp-arg-his-asp (DRHD¹⁹). Mutating D¹⁹ to A¹⁹ abrogated AP-2 cleavage by all three caspases. TNF--induced cleavage of AP-2 in vivo led to AP-2 degradation and loss of DNA-binding activity, both of which were prevented by pretreatment with zVEIDfmk. AP-2 degradation but not cleavage was inhibited in vivo by PS-431 (a proteasome antagonist), suggesting that AP-2 is degraded subsequent to cleavage by caspase 6 or caspase 6-like enzymes. Cells transfected with green fluorescent protein-tagged mutant AP-2 are resistant to TNF--induced apoptosis, further demonstrating the link between caspase-mediated cleavage of AP-2 and apoptosis. This is the first report to demonstrate that degradation of AP-2 is a critical event in TNF--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.

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^* Corresponding author. Mailing address: Department of Cancer Biology, Box 173, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 794-4004. Fax: (713) 792-8747. E-mail: mbareli{at}notes.mdacc.tmc.edu.

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Molecular and Cellular Biology, August 2001, p. 4856-4867, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.4856-4867.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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