Chae Young Chan, Lee Sukmook, Lee Hye Young, Heo Kyun, Kim Jung Hwan, Kim Jong Hyun, Suh Pann-Ghill, Ryu Sung Ho
Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
J Biol Chem. 2005 Feb 4;280(5):3723-30. doi: 10.1074/jbc.M406987200. Epub 2004 Nov 16.
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 (IC50 <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.
哺乳动物磷脂酶D（PLD）被认为是包括毒蕈碱受体在内的各种受体信号转导中的关键酶。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激活的一种抑制机制。通过肽质量指纹图谱在COS-7细胞免疫沉淀的PLD2复合物中鉴定出了微管蛋白。发现PLD2与微管蛋白之间的直接相互作用是由PLD2的一个特定区域（氨基酸476 - 612）介导的。在体外，微管蛋白结合可有效抑制PLD2（IC50 <10 nM）。在细胞中，微管破坏剂诺考达唑可增加PLD2与微管蛋白之间的相互作用，而微管稳定剂紫杉醇则可降低这种相互作用。此外，发现PLD2活性与单体微管蛋白水平呈负相关。另外，我们发现单体微管蛋白与PLD2的相互作用及对其的抑制对于毒蕈碱受体相关的PLD信号通路很重要。只有在卡巴胆碱刺激1 - 2分钟后，当卡巴胆碱刺激的PLD2活性降低时，PLD2与微管蛋白之间的相互作用才会增加。PLD2微管蛋白结合区域的表达通过掩盖微管蛋白结合来阻止卡巴胆碱诱导的PLD活性随后的降低。综上所述，这些结果表明局部膜单体微管蛋白浓度的增加会抑制PLD2活性，并为通过与微管蛋白相互作用抑制毒蕈碱受体诱导的PLD2激活提供了一种新机制。
