Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis.
Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137*L, LSSD203*F and ALAD404*S), while only a single site (ALDD137*L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.
Department of Neuroscience Therapeutics, Division of Warner-Lambert Company, Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, USA. kevin.wang@wl.com
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Wang
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Posmantur
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Nadimpalli
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Nath
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Mohan
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Keegan
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Herzog
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D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001665:Binding Sites / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism* / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D019169:Jurkat Cells; D008970:Molecular Weight; D009447:Neuroblastoma; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; C033668:calpastatin; C505407:CASP3 protein, human; D002154:Calpain; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1