Identification of two calcineurin B-binding proteins: tubulin and heat shock protein 60
Identification of two calcineurin B-binding proteins: tubulin and heat shock protein 60
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DOI：
10.1016/s1570-9639(02)00402-8
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发表时间：
2002-09-23
影响因子：
3.2
通讯作者：
Handschumacher, RE
中科院分区：
文献类型：
--
作者：
Li, W;Handschumacher, RE
钙调神经磷酸酶(CaN)是一种钙/钙调蛋白依赖性的蛋白磷酸酶，具有催化亚基(CANA)和调节亚基(CANB)两个亚基。CANB有四个钙离子结合位点，与钙调蛋白序列同源，被定义为CANA的调节亚单位。然而，我们已经证明CANB的线粒体表达远远超过CANA。为了研究这种过剩的CanB的作用，我们构建了谷胱甘肽-S-转移酶-CanB融合蛋白(GST-CANB)，并证明该融合蛋白主要与牛脑提取液中57 kDa的α-微管蛋白和牛肾提取液中的热休克蛋白60(HSP60)结合。免疫沉淀法证实了它们与CANB的钙依赖性相互作用。纯化的α/β微管蛋白能与Hsp60结合，但不能与细菌Hsp60类似物GroEL结合。CANB与HSP60的相互作用不受HSP10、ATP和Mg++的影响，提示CANB与HSP60不是错误折叠的底物，可能是一种调控蛋白。因此，CANB除了与CANA相互作用外，还可能在钙依赖事件中发挥其他调节作用，并可能在线粒体的钙依赖过程中发挥重要作用。(C)2002 Elsevier Science B.V.保留所有权利。
Calcineurin (CaN) is a Ca++/calmodulin-dependent protein phosphatase with two subunits: a catalytic subunit (CaNA) and a regulatory subunit (CaNB). With four Ca++-binding sites and a sequence homology to calmodulin, CaNB has been defined as the regulatory subunit for CaNA. However, we have shown that mitochondrial expression of CaNB far exceeds that of CaNA. To investigate the role of this excess CaNB, we have generated glutathione-S-transferase-CaNB (GST-CaNB) fusion protein and demonstrated that the fusion protein predominantly bound to alpha-tubulin, a 57 kDa protein in bovine brain extracts, and heat shock protein 60 (Hsp60) in bovine kidney extracts. Their Ca++-dependent interactions with CaNB were verified by immunoprecipitation. The binding of CaNB could be demonstrated with purified alpha/beta tubulins and Hsp60, but not GroEL, a bacterial Hsp60 analog. The interaction of CaNB and Hsp60 was not disrupted by the incubation with Hsp10, ATP and Mg++, suggesting that CaNB was not associated with Hsp60 as a misfolded substrate, and may serve as a regulatory protein. Thus, CaNB may play other regulatory roles in Ca++-dependent events in addition to its interaction with CaNA, and may be important for Ca++-dependent processes in mitochondria. (C) 2002 Elsevier Science B.V. All rights reserved.