Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR
Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR
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DOI：
10.1096/fj.06-6652com
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发表时间：
2007-04-01
期刊：
影响因子：
4.8
通讯作者：
Gomez-Cambronero, Julian
中科院分区：
文献类型：
--
作者：
Lehman, Nicholas;Ledford, Bill;Gomez-Cambronero, Julian
磷脂酸（PA）是磷脂酶D （PLD）在细胞膜上酶促作用的产物，它调节与NADPH氧化酶活化有关的激酶，以及哺乳动物雷帕霉素（mTOR）激酶的靶标。然而，为了解释其他关键的pa介导的细胞功能，必须存在其他脂质第二信使的蛋白靶点。在这项研究中，发现PA特异性和饱和结合并激活重组和免疫沉淀的内源性核糖体S6激酶（S6K），其化学计量为94:1脂质/蛋白。多磷酸肌苷PI4- p、PI4、5P(2)和心磷脂也可以结合并激活S6K，尽管其动力学不同。相反，至少有一个酰基侧链饱和（10:0）的PA在结合或激活酶方面无效。用野生型myc-(pcDNA)- pld2构建体转染COS-7细胞后，PLD活性高，核糖体p70S6K酶活性增加，T-389和T-421/S-424磷酸化增加，S-235/S-236中p70S6K天然底物S6蛋白磷酸化增加。然而，脂肪酶失活突变体（K758R）的过表达未能诱导PLD和S6K活性或磷酸化的增加，这表明PLD2的酶活性（即PA的合成）必须存在才能影响S6K。无论是用雷帕霉素抑制mTOR激酶活性，还是沉默mTOR基因表达，都不能改变PLD2对p70S6K活性的增强作用。这一发现表明，即使在没有mTOR的情况下，PA也能结合并激活p70S6K。最后，用PLD2转染COS-7改变了亚细胞表达模式，并通过免疫荧光显微镜观察到S6K和PLD2的共定位。这些结果首次表明PLD直接（不依赖mtor）参与p70S6K通路，并暗示PA是将细胞磷脂酶和激酶聚集在一起的纽带。
The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamycin ( mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA-mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichiometry of 94:1 lipid/protein. Polyphosphoinositides PI4-P and PI4,5P(2) and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated ( 10: 0) was ineffective in binding or activating the enzyme. Transfection of COS-7 cells with a wild-type myc-(pcDNA)-PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T-389 and T-421/S-424 as well as phosphorylation of p70S6K's natural substrate S6 protein in S-235/S-236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS-7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR-independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.