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，被发现与抗凋亡蛋白Bcl2结合。钙调神经磷酸酶通过其直接结合特异性地去磷酸化Bc l-2。钙调神经磷酸酶去磷酸化的Bc l-2具有抑制细胞凋亡的功能，而通过失活钙调神经磷酸酶(主要的负性钙调神经磷酸酶过表达)而磷酸化的Bc l-2则失去了抗细胞凋亡的功能。磷酸化分析表明，钙调神经磷酸酶去磷酸化的靶区位于BH4结构域和Loop区域，这表明BH4上的Ser24和Seron环似乎分别是候选区域。用丙氨酸(A)或天冬氨酸(D)替代这些丝氨酸后，表达突变的Bcl2的细胞表现出与Bcl2通过磷酸化调节改变的功能的遗传分析一致的生物学行为。已有研究表明，作为Bcl2调控细胞凋亡的机制之一，Bcl2的磷酸化状态影响其与死亡诱导的Bax的亲和力。也就是说，去磷酸化的Bcl2有效地异二聚化为Bax，而磷酸化的Bcl2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的释放而导致细胞凋亡。在人白血病/淋巴瘤细胞系的体内研究表明，携带t(14；18)染色体易位的细胞主要是内源性Bcl2的去磷酸化形式的高表达。抗癌药物处理的白血病细胞显示内源性的Bcl2从去磷酸化形式向磷酸化形式迁移。

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.