Inhibition of muscarinic receptor-linked phospholipase D activation by association with tubulin
Inhibition of muscarinic receptor-linked phospholipase D activation by association with tubulin
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DOI：
10.1074/jbc.m406987200
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发表时间：
2005-02-04
影响因子：
4.8
通讯作者：
Ryu, SH
中科院分区：
文献类型：
--
作者：
Chae, YC;Lee, HY;Ryu, SH
哺乳动物磷脂酶 D (PLD) 被认为是多种受体（包括毒蕈碱受体）传输信号的关键酶。 PLD激活是一个快速且短暂的过程，但尚未发现抑制信号依赖性PLD激活的负调节因子。在此，我们首次报道微管蛋白与 PLD2 的结合是毒蕈碱受体相关 PLD2 激活的抑制机制。通过肽质量指纹分析，在来自 COS-7 细胞的免疫沉淀 PLD2 复合物中鉴定出微管蛋白。发现 PLD2 和微管蛋白之间的直接相互作用是由 PLD2 的特定区域（氨基酸 476-612）介导的。体外微管蛋白结合可有效抑制 PLD2 (IC(50) < 10 nm)。在细胞中，微管破坏剂诺考达唑增强了 PLD2 与微管蛋白之间的相互作用，而微管稳定剂紫杉醇则减弱了 PLD2 和微管蛋白之间的相互作用。此外，发现 PLD2 活性与单体微管蛋白水平呈负相关。此外，我们发现单体微管蛋白与 PLD2 的相互作用以及对 PLD2 的抑制对于毒蕈碱受体相关的 PLD 信号通路非常重要。仅在卡巴胆碱刺激 1-2 分钟后，当卡巴胆碱刺激的 PLD2 活性降低时，PLD2 和微管蛋白之间的相互作用才会增加。 PLD2 的微管蛋白结合区的表达通过掩盖微管蛋白结合来阻止随后卡巴胆碱诱导的 PLD 活性的降低。总之，这些结果表明局部膜单体微管蛋白浓度的增加抑制PLD2活性，并提供了通过与微管蛋白相互作用抑制毒蕈碱受体诱导的PLD2激活的新机制。
Mammalian phospholipase D (PLD) is considered a key enzyme in the transmission signals from various receptors including muscarinic receptors. PLD activation is a rapid and transient process, but a negative regulator has not been found that inhibits signal-dependent PLD activation. Here, for the first time, we report that tubulin binding to PLD2 is an inhibition mechanism for muscarinic receptor-linked PLD2 activation. Tubulin was identified in an immunoprecipitated PLD2 complex from COS-7 cells by peptide mass fingerprinting. The direct interaction between PLD2 and tubulin was found to be mediated by a specific region of PLD2 (amino acids 476-612). PLD2 was potently inhibited (lC(50) < 10 nm) by tubulin binding in vitro. In cells, the interaction between PLD2 and tubulin was increased by the microtubule disrupting agent nocodazole and reduced by the microtubule stabilizing agent Taxol. Moreover, PLD2 activity was found to be inversely correlated with the level of monomeric tubulin. In addition, we found that interaction with and the inhibition of PLD2 by monomeric tubulin is important for the muscarinic receptor-linked PLD signaling pathway. Interaction between PLD2 and tubulin was increased only after 1-2 min of carbachol stimulation when carbachol-stimulated PLD2 activity was decreased. The expression of the tubulin binding region of PLD2 blocked the later decrease in carbachol-induced PLD activity by masking tubulin binding. Taken together, these results indicate that an increase in local membrane monomeric tubulin concentration inhibits PLD2 activity, and provides a novel mechanism for the inhibition of muscarinic receptor-induced PLD2 activation by interaction with tubulin.