The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity
The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity
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DOI：
10.1074/jbc.m414234200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Kuno, T
中科院分区：
文献类型：
--
作者：
Sio, SO;Suehiro, T;Kuno, T
钙调磷酸酶是一种由催化和调节亚基组成的异源二聚体，是许多细胞类型中Ca2+信号转导所需的蛋白磷酸酶。裂变酵母基因组编码一组催化（Ppb1）和调节（Cnb1）亚基，为研究这些亚基在体内的功能提供了理想的模型系统。在这里，我们克隆了cnb1（+）基因，发现cnb1敲除（Delta cnb1）与Delta ppb1表现出相同的表型，ppb1的过表达未能抑制Delta cnb1的表型。有趣的是，过表达C端缺失的Ppb1 (Ppb1 Delta C)， Ppb1的组成活性形式，也未能抑制Delta cnb1的表型。FK506以依赖于fkbp12的方式引起MgCl2对野生型细胞的敏感性。Ppb1和Cnb1的共同过表达抑制了fk506诱导的MgCl2敏感性，但抑制只是部分的，这表明过量的Ppb1-Cnb1复合物不能竞争FKBP12-FK506复合物。虽然Ppb1 δ C单独过表达对细胞生长影响不大，但Ppb1 δ C和Cnb1共同过表达会引起明显的生长缺陷。使用减毒型nmt1启动子共表达Cnb1时，FK506抑制了生长缺陷，但使用野生型nmt1启动子共表达Cnb1时，FK506未能抑制生长缺陷。敲除编码钙调磷酸酶下游靶转录因子的prz1（+）基因，无论启动子的效力如何，都能抑制生长缺陷。这些结果表明Cnb1对钙调神经磷酸酶的激活至关重要，激活的钙调神经磷酸酶是FKBP12-FK506复合物在体内的药理学靶点。
Calcineurin, a protein phosphatase required for Ca2+ signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits. The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study the functions of these subunits in vivo. Here, we cloned the cnb1(+) gene and showed that the cnb1 knock-out (Delta cnb1) exhibits identical phenotypes with Delta ppb1 and that overexpression of Ppb1 failed to suppress the phenotypes of Delta cnb1. Interestingly, overexpression of the C-terminal-deleted Ppb1 (Ppb1 Delta C), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Delta cnb1. FK506 caused MgCl2 sensitivity to the wild-type cells in an FKBP12-dependent manner. Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl2 sensitivity, but the suppression was only partial, suggesting that an excess amount of the Ppb1-Cnb1 complex cannot compete out the FKBP12-FK506 complex. Although overexpression of Ppb1 Delta C alone had little effect on cell growth, co-overexpression of Ppb1 Delta C and Cnb1 caused a distinct growth defect. FK506 suppressed the growth defect when Cnb1 was co-expressed using the attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using the wild-type nmt1 promoter. Knock-out of the prz1(+) gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency. These results suggest that Cnb1 is essential for the activation of calcineurin and that the activated calcineurin is the pharmacological target of the FKBP12-FK506 complex in vivo.