Communication between the regulatory and the catalytic region of the cAMP-responsive guanine nucleotide exchange factor Epac
Communication between the regulatory and the catalytic region of the cAMP-responsive guanine nucleotide exchange factor Epac
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DOI：
10.1074/jbc.m301680200
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发表时间：
2003-06-27
影响因子：
4.8
通讯作者：
Wittinghofer, A
中科院分区：
文献类型：
--
作者：
Rehmann, H;Rueppel, A;Wittinghofer, A
Epac 1是一种鸟嘌呤核苷酸交换因子（GEF），可直接被cAMP激活。该蛋白由具有cAMP结合结构域的调节区和介导GEF活性的催化区组成。在不存在cAMP的情况下，Epac被cAMP结合结构域和催化区域之间的分子内相互作用抑制。cAMP结合被认为诱导构象变化，其允许LID（cAMP结合位点的C末端的α-螺旋）覆盖cAMP结合位点（Rehmann，H.，Prakash，B.，沃尔夫，E.，Rueppel，A.，de Rooij，J.，博斯，J.L.，和Wittinghousehold，A.（2003）Nat.Struct.Biol.10，26-32）。在这里，我们表明，在LID区域的保守残基的突变影响cAMP的结合只有轻微的，但有一个cAMP诱导的GEF活性的剧烈影响。令人惊讶的是，与野生型相比，一些突变体具有增加的最大GEF活性。此外，在LID的C-末端处的保守VLVLE序列突变成五个丙氨酸残基使得Epac具有组成型活性。从这些结果中，我们得出结论，LID区域起着至关重要的作用，在Epac的调节和催化部分之间的通信。
Epac1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1 that is directly activated by cAMP. This protein consists of a regulatory region with a cAMP-binding domain and a catalytic region that mediates the GEF activity. Epac is inhibited by an intramolecular interaction between the cAMP-binding domain and the catalytic region in the absence of cAMP. cAMP binding is proposed to induce a conformational change, which allows a LID, an alpha-helix at the C-terminal end of the cAMP-binding site, to cover the cAMP-binding site (Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., de Rooij, J., Bos, J. L., and Wittinghofer, A. ( 2003) Nat. Struct. Biol. 10, 26-32). Here we show that mutations of conserved residues in the LID region affect cAMP binding only marginally but have a drastic effect on cAMP-induced GEF activity. Surprisingly, some of the mutants have an increased maximal GEF activity compared with wild type. Furthermore, mutation of the conserved VLVLE sequence at the C-terminal end of the LID into five alanine residues makes Epac constitutively active. From these results we conclude that the LID region plays a pivotal role in the communication between the regulatory and catalytic part of Epac.