Wang K K, Posmantur R, Nath R, McGinnis K, Whitton M, Talanian R V, Glantz S B, Morrow J S
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48106, USA.
J Biol Chem. 1998 Aug 28;273(35):22490-7. doi: 10.1074/jbc.273.35.22490.
The degradation of alphaII- and betaII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both alphaII- and betaII-subunits. Major C-terminal alphaII-spectrin BDPs were detected at approximately 150, 145, and 120 kDa (alphaII-BDP150, alphaII-BDP145, and alphaII-BDP120, respectively); major C-terminal betaII-spectrin BDPs were at approximately 110 and 85 kDa (betaII-BDP110 and betaII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that alphaII-BDP150 and alphaII-BDP120 were generated by cleavages at DETD1185S1186 and DSLD1478S1479, respectively. For betaII-spectrin, a major caspase site was detected at DEVD1457S1458, and both betaII-BDP110 and betaII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146S2147, was found to account for betaII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both alphaII- and betaII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.
研究了培养的人神经母细胞瘤SH-SY5Y细胞凋亡过程中αII-和βII-血影蛋白的降解情况。免疫荧光染色显示，在星形孢菌素刺激后，皮质血影蛋白细胞骨架的瓦解是一个早期事件。这种瓦解与源自αII-和βII-亚基的一系列显著的血影蛋白降解产物（BDPs）的产生相关。主要的C末端αII-血影蛋白BDPs分别在约150、145和120 kDa处检测到（分别为αII-BDP150、αII-BDP145和αII-BDP120）；主要的C末端βII-血影蛋白BDPs在约110和85 kDa处（分别为βII-BDP110和βII-BDP85）。对体外由半胱天冬酶3产生的主要片段进行N末端测序表明，αII-BDP150和αII-BDP120分别是通过在DETD1185S1186和DSLD1478S1479处切割产生的。对于βII-血影蛋白，在DEVD1457S1458处检测到一个主要的半胱天冬酶切割位点，βII-BDP110和βII-BDP85都共享一个以Ser1458开头的共同N末端序列。发现在C末端附近的另一个切割位点ETVD2146S2147导致了βII-BDP85的产生。使用特异性半胱天冬酶或钙蛋白酶抑制剂的研究表明，凋亡过程中血影蛋白的降解模式与非凋亡性细胞死亡过程中的不同。我们推测，半胱天冬酶与钙蛋白酶协同作用，迅速靶向αII-和βII-血影蛋白中的关键位点，从而在凋亡过程中引发血影蛋白-肌动蛋白皮质细胞骨架的快速溶解。
