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

分析钙调神经磷酸酶调节的程序性细胞死亡的新机制。

基本信息

批准号：
10670207
负责人：
KONDO Eisaku
金额：
$ 1.98万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670207/
关键词：
calcineurin Bcl-2 apoptosis dephosphorylation リン酸化制御神経細胞死 Bリンパ球 Bc1-2

项目摘要

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.

丝氨酸/苏氨酸蛋白磷酸酶2B钙调磷酸酶与抗凋亡蛋白Bcl-2结合。钙调磷酸酶通过其直接关联特异性地去磷酸化Bcl-2。活性钙调神经磷酸酶使Bcl-2去磷酸化具有细胞凋亡抑制作用，而钙调神经磷酸酶使Bcl-2失活（通过钙调神经磷酸酶显性阴性型的过度表达）使Bcl-2失去抗细胞凋亡功能。磷酸化分析显示，钙调磷酸酶的去磷酸化靶区位于Bcl-2的BH4结构域和Loop区域，这表明BH4上的Ser24和Loop上的Ser70可能分别是候选区域。表达突变Bcl-2的细胞用丙氨酸(A)或天冬氨酸(D)取代丝氨酸，在磷酸化调控下Bcl-2功能改变的遗传分析中显示出一致的生物学行为。Bcl-2的磷酸化状态影响其对死亡诱导的Bax的亲和力，这是Bcl-2调控细胞凋亡的一种机制。也就是说，去磷酸化的Bcl-2有效地异二聚化为Bax，而磷酸化的Bcl-2失去了与Bax的关联，因此过量的Bax积累在线粒体中，通过促进细胞色素c的释放而导致细胞凋亡。对人类白血病/淋巴瘤细胞系的体内研究显示，携带t（14; 18）染色体易位的细胞主要过度表达内源性去磷酸化形式的Bcl-2。结果还表明，经抗癌药物处理的凋亡白血病细胞内源性Bcl-2从去磷酸化形式向磷酸化形式迁移。