Analysis of a novel mechanism of programmed cell death regulated by calcineurin.

丝氨酸/苏氨酸蛋白磷酸酶2B，钙调神经磷酸酶，被揭示为结合到抗凋亡蛋白，Bcl-2。钙调神经磷酸酶通过其直接结合特异性地使Bcl-2去磷酸化。激活的钙调磷酸酶使Bcl-2去磷酸化，而失活的钙调磷酸酶使Bcl-2磷酸化，磷酸化分析显示，钙调磷酸酶去磷酸化的靶区域位于BH 4结构域和Bcl-2内的Loop，这表明BH 4上的Ser 24和loop上的Ser 70似乎分别是候选者。表达突变Bcl-2的细胞用丙氨酸（A）或天冬氨酸（D）取代那些丝氨酸，对通过磷酸化调节改变Bcl-2功能的遗传分析显示出一致的生物学行为。已经阐明，作为Bcl-2调节细胞凋亡的机制，Bcl-2的磷酸化状态影响其对死亡诱导的Bax的亲和力。也就是说，去磷酸化的Bcl-2有效地异源二聚化为Bax，而磷酸化的Bcl-2失去了与Bax的结合，因此过量的Bax积累在线粒体上，通过促进细胞色素C的释放而引起细胞凋亡。体内研究发现，t（14 ; 18）易位的白血病细胞主要过表达脱磷酸化的Bcl-2，经抗癌药物处理后，凋亡的白血病细胞中Bcl-2的迁移率由脱磷酸化向磷酸化转变。

The serine/threonine protein phosphatase 2B, calcineurin, was revealed to be bound to anti-apoptotic protein, Bcl-2. Calcineurin specifically dephosphorylated Bcl-2 through its direct association. Dephosphorylated Bcl-2 by active calcineurin functioned as an apoptosis-suppressor, whereas phosphorylated Bcl-2 by inactivation of calcineurin (by overexpression of dominant negative type of calcineurin) lost the anti-apoptotic function.Phosphorylation assay revealed that target regions for dephosphorylation by calcineurin were located both a BH4 domain and Loop within Bcl-2, which suggested Ser24 on BH4 and Ser70 on loop seemed to be the candidates, respectively. Cells expressing mutative Bcl-2 substituted those Serines by Alanine(A) or Aspartate(D) showed consistent biological behaviors to genetic analyses of altered function of Bcl-2 by phorylation regulation. It was clarified, as a mechanism of apoptosis regulation by Bcl-2, that the phosphorylated status of Bcl-2 affected its affinity to death-induced, Bax. Namely, dephosphorylated Bcl-2 efficiently heterodimerized to Bax, whereas phosphorylated Bcl-2 lost its association to Bax, thus excess amount of Bax accumulated at mitochondria caused apoptosis by facilitating a relase of cytochrome C. In vivo study using human leukemia/lymphoma cell lines revealed that cells carrying t(14 ; 18) chromosomal translocation predominantly overexpressed dephosphorylated form of endogenous Bcl-2.It also revealed that apoptotic leukemia cells treated by anti-cancer drug showed mobility shift of endogenous Bcl-2 from dephosphorylated form to phosphorylated form.