Sagoo J K, Fruman D A, Wesselborg S, Walsh C T, Bierer B E
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Biochem J. 1996 Dec 15;320 ( Pt 3)(Pt 3):879-84. doi: 10.1042/bj3200879.
Calcineurin (protein phosphatase 2B), a calmodulin- and calcium-dependent serine/threonine phosphatase, appears to be regulated by a C-terminal autoinhibitory domain. A 25 amino acid peptide derived from this domain inhibits calcineurin phosphatase activity in vitro. Here we show that a 97 amino acid fragment of the calcineurin A alpha C-terminus is approx. 8-fold more potent than the shorter peptide in calcineurin inhibition experiments. Mutation of an evolutionarily conserved Asp to Asn, previously shown to disrupt calcium-dependent signalling and calcineurin regulation in T-lymphocytes, greatly reduced inhibition by the autoinhibitory domain in vitro. Kinetic analysis of wild-type and mutated autoinhibitory domains show that both are competitive inhibitors of calcineurin phosphatase activity with Ki values of 5.0 +/- 0.2 microM and 36.0 +/- 3.7 microM respectively. This suggests intrasteric regulation of calcineurin, with the autoinhibitory domains interacting at the active site of the enzyme. The competitive behaviour of the autoinhibitory domains contrasts with the mechanism of calcineurin inhibition by immunosuppressant-immunophilin complexes, which have been shown to bind to calcineurin at a region removed from the active site.
钙调神经磷酸酶（蛋白磷酸酶2B）是一种钙调蛋白和钙依赖性丝氨酸/苏氨酸磷酸酶，似乎受C末端自抑制结构域调控。源自该结构域的一段25个氨基酸的肽在体外可抑制钙调神经磷酸酶的磷酸酶活性。在此我们表明，钙调神经磷酸酶Aα C末端的一个97个氨基酸的片段在钙调神经磷酸酶抑制实验中的效力比该较短肽约强8倍。一个进化上保守的天冬氨酸突变为天冬酰胺（先前已表明其会破坏T淋巴细胞中钙依赖性信号传导和钙调神经磷酸酶调控），在体外极大地降低了自抑制结构域的抑制作用。对野生型和突变型自抑制结构域的动力学分析表明，二者均为钙调神经磷酸酶磷酸酶活性的竞争性抑制剂，其Ki值分别为5.0±0.2微摩尔和36.0±3.7微摩尔。这表明钙调神经磷酸酶存在分子内调控，自抑制结构域在该酶的活性位点相互作用。自抑制结构域的竞争性行为与免疫抑制剂 - 亲免素复合物对钙调神经磷酸酶的抑制机制形成对比，后者已被证明是在远离活性位点的区域与钙调神经磷酸酶结合。
