Caspase-mediated cleavage of ATM during cisplatin-induced tubular cell apoptosis: inactivation of its kinase activity toward p53.
Cisplatin induces renal cell injury and death, resulting in nephrotoxicity that limits its use in cancer therapy. Using cell culture models, recent work has suggested the involvement of p53 in renal cell apoptosis during cisplatin treatment. However, the signals upstream of p53 remain elusive. ATM and ATR are critical regulators of p53 under various conditions of DNA damage. Here, we show that ATM, and not ATR, was proteolytically cleaved into specific fragments of approximately 210 and 150 kDa during cisplatin-induced tubular cell apoptosis. ATM cleavage was paralleled by the development of apoptosis. VAD, a broad-spectrum inhibitor of caspases, attenuated the cleavage of ATM, whereas the inhibitors of specific caspases were less effective. In caspase-3-deficient MCF-7 cells, ATM was cleaved, releasing the 210- but not the 150-kDa fragment. Recombinant caspase-3 was much more effective than caspase-7 in cleaving ATM that was immunoprecipitated from cell lysates. During cisplatin incubation, VAD protected ATM and enhanced p53 phosphorylation. In vitro assay of protein kinase activity further showed that ATM immunoprecipitated from cisplatin-treated cells had significantly lower kinase activity toward p53 than that from control cells. Importantly, the protein kinase activity was restored in ATM that was protected by VAD during cisplatin incubation. ATM deficiency sensitized the cells to cisplatin-induced apoptosis, suggesting a cytoprotective role of ATM in this experimental model. Thus proteolysis of ATM by caspases may inactivate this regulatory molecule to facilitate the progression of apoptosis.
Wang
Jinzhao
J
Department of Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, GA 30912, USA.
Pabla
Navjotsingh
N
Wang
Cong-Yi
CY
Wang
Weixin
W
Schoenlein
Patricia V
PV
Dong
Zheng
Z
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D064007:Ataxia Telangiectasia Mutated Proteins; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002945:Cisplatin / Q000633:toxicity*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; D020170:Caspase 1; D002945:Cisplatin