McGinnis K M, Whitton M M, Gnegy M E, Wang K K
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1998 Aug 7;273(32):19993-20000. doi: 10.1074/jbc.273.32.19993.
We have previously demonstrated cleavage of alpha-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Asp-fluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD176A and CG201A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.
我们之前已证明，在SH-SY5Y神经母细胞瘤细胞凋亡过程中，半胱天冬酶-3（caspase-3）和钙蛋白酶可切割α-血影蛋白（Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683 - 690）。我们在此证明，在凋亡过程中，钙/钙调蛋白依赖性蛋白激酶IV（CaMK IV）会被caspase-3和钙蛋白酶切割。我们用促凋亡剂毒胡萝卜素处理SH-SY5Y细胞。蛋白质免疫印迹分析显示，主要的CaMK IV降解产物为40、38和33 kDa。用纯化的caspase-3消化对照SH-SY5Y裂解物产生一个38 kDa的CaMK IV片段；用纯化的钙蛋白酶消化产生一个主要的40 kDa片段。用苄氧羰基-天冬氨酸-CH2OC(O)-2,6-二氯苯或Z-缬氨酸-丙氨酸-天冬氨酸-氟甲基酮预处理能够在原位和体外阻断caspase-3介导的38 kDa片段的产生。钙蛋白酶抑制剂II同样在原位和体外阻断了钙蛋白酶介导的40 kDa片段的形成。其他半胱天冬酶家族成员对重组CaMK IV的消化显示，只有caspase-3产生的片段化模式与原位观察到的一致。主要的caspase-3和钙蛋白酶切割位点分别被鉴定为PAPD176A和CG201A，均在CaMK IV催化域内。此外，在毒胡萝卜素处理的SH-SY5Y细胞中，钙调蛋白刺激的蛋白激酶活性在6小时内下降。活性丧失先于细胞死亡。
