Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death

Cain/cabin1 is an endogenous inhibitor of calcineurin (Cn), a calcium-dependent serine/threonine phosphatase involved in various cellular functions including apoptosis. We show here that during apoptosis cain/cabin1 is cleaved by calpain at the carboxyl terminus to generate a cleavage product with a molecular mass of 32 kDa as a necessary step leading to Cn-mediated cell death. Mouse cain/cabin1 was identified from a thymus cDNA library by an in vitro substrate-screening assay with calpain. Exposure of Jurkat cells to the calcium ionophore, A23187, induced cain/cabin1 cleavage and cell death, accompanied by activation of calpain and Cn. The calpain inhibitors, calpeptin and zLLY, suppressed both A23187-induced cain/cabin1 cleavage and Cn activation, indicating that Cn activation and cain/cabin1 cleavage are calpain-dependent. Expression of cain/cabin1 or a catalytically inactive Cn mutant [CnAβ(2)(1–401/H160N)] and treatment with FK506 reduced A23187-induced cell death. In vitro calpain cleavage and immunoprecipitation assays with deletion mutants of cain/cabin1 showed that cleavage occurred in the Cn-binding domain of cain/cabin1, indicating that the cleavage at its C terminus by calpain prevented cain/cabin1 from binding to Cn. In addition, in vitro binding assays showed that cain/cabin1 bound to the Cn B-binding domain of Cn A. Taken together, these results indicate that calpain cleaves the calcineurin-binding domain of cain/cabin1 to activate Cn and elicit calcium-triggered cell death.

Cain/cabin1是钙调神经磷酸酶（calcineurin, Cn）的内源性抑制剂，而钙调神经磷酸酶是一种依赖钙离子的丝氨酸/苏氨酸磷酸酶，参与包括细胞凋亡在内的多种细胞功能活动。本研究证实，在细胞凋亡进程中，cain/cabin1会被钙蛋白酶（calpain）在羧基末端切割，产生分子量为32 kDa的切割产物，这是介导钙调神经磷酸酶依赖性细胞死亡的必要步骤。小鼠来源的cain/cabin1是通过以钙蛋白酶为探针的体外底物筛选实验，从胸腺cDNA文库中鉴定得到的。将Jurkat细胞暴露于钙离子载体A23187后，可诱导cain/cabin1切割与细胞死亡，同时伴随钙蛋白酶与钙调神经磷酸酶的激活。钙蛋白酶抑制剂calpeptin与zLLY可同时抑制A23187诱导的cain/cabin1切割与钙调神经磷酸酶激活，表明钙调神经磷酸酶激活与cain/cabin1切割均依赖钙蛋白酶通路。过表达cain/cabin1或催化失活型钙调神经磷酸酶突变体[CnAβ(2)(1–401/H160N)]，以及使用FK506进行处理，均可降低A23187诱导的细胞死亡比例。针对cain/cabin1缺失突变体开展的体外钙蛋白酶切割实验与免疫沉淀实验（immunoprecipitation assays）证实，切割位点位于cain/cabin1的钙调神经磷酸酶结合结构域内，表明钙蛋白酶在其羧基末端的切割可阻断cain/cabin1与钙调神经磷酸酶的结合。此外，体外结合实验显示，cain/cabin1可结合至钙调神经磷酸酶A的钙调神经磷酸酶B结合结构域。综合以上实验结果，本研究表明钙蛋白酶通过切割cain/cabin1的钙调神经磷酸酶结合结构域，激活钙调神经磷酸酶并引发钙离子触发的细胞死亡。