Caspase cleavage of phospholipase D1 in vitro alters its regulation and reveals a novel property of the "loop" region.
Caspase cleavage of phospholipase D1 in vitro alters its regulation and reveals a novel property of the "loop" region.
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DOI：
10.1016/j.bbalip.2008.05.007
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发表时间：
2008-08
期刊：
影响因子：
--
通讯作者：
C. Riebeling;S. Bourgoin;D. Shields
中科院分区：
文献类型：
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作者：
C. Riebeling;S. Bourgoin;D. Shields
磷脂酶D（PLD）参与介导囊泡转运、有丝分裂、分化和凋亡。磷脂酶D活性的产物磷脂酸（PA）具有促有丝分裂的作用，在许多肿瘤细胞系中检测到PLD表达升高。一些报道已经证明，不同的PLD结构域调节其活性，并且PLD的截短形式保留酶活性。我们推测，在凋亡过程中，半胱天冬酶裂解的PLD可能会导致其活动的修改。为了测试这一想法，我们已经使用PLD 1和PLD 2的体外翻译，其产生的活性酶表现出模仿内源性蛋白质的特性。在这里，我们证明了PLD 1在体外被半胱天冬酶-8、-3和-7快速切割。相比之下，PLD 2裂解延迟，其活性不受与胱天蛋白酶-3孵育的影响。值得注意的是，caspase裂解后，PLD 1对调节刺激的反应发生了改变;它不再被PKC激活，而是对小GTP酶的反应活性增加。值得注意的是，这种增强的活性是由于“环”结构域中PLD 1的切割，该结构域是先前与负调控功能相关的区域。因此，我们的数据已经确定了一个新的调控域PLD 1。
Phospholipase D (PLD) has been implicated in mediating vesicular transport, mitosis, differentiation and apoptosis. The product of PLD activity, phosphatidic acid (PA) has mitogenic potential and elevated PLD expression has been detected in many tumor cell lines. Several reports have demonstrated that distinct PLD domains regulate its activity and that truncated forms of PLD retain enzymatic activity. We hypothesized that during apoptosis caspase cleavage of PLDs could result in modification of their activities. To test this idea, we have used in vitro translation of PLD1 and PLD2 which generated active enzymes exhibiting properties mimicking those of the endogenous proteins. Here we demonstrate that PLD1 was rapidly cleaved in vitro by caspases-8, -3 and -7. In contrast, PLD2 cleavage was delayed and its activity was unaffected by incubation with caspase-3. Significantly, following caspase cleavage the response of PLD1 to regulatory stimuli was altered; it was no longer activated by PKC and instead exhibited an increased activity in response to small GTPases. Notably, this enhanced activity was due to cleavage of PLD1 in the “loop” domain, a region previously associated with negative regulatory function. Thus our data have identified a novel regulatory domain in PLD1.