A Comprehensive Model That Explains the Regulation of Phospholipase D2 Activity by Phosphorylation-Dephosphorylation
A Comprehensive Model That Explains the Regulation of Phospholipase D2 Activity by Phosphorylation-Dephosphorylation
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DOI：
10.1128/mcb.01239-09
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发表时间：
2010-05-01
影响因子：
5.3
通讯作者：
Gomez-Cambronero, Julian
中科院分区：
文献类型：
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作者：
Henkels, Karen M.;Peng, Hong-Juan;Gomez-Cambronero, Julian
我们在这里报告的磷脂酶D2（PLD 2）的酶活性调节磷酸化-去磷酸化。磷酸酶处理的PLD 2过表达细胞表现出双相性质的活动，表明存在的“激活”和“抑制”的网站的变化。我们确定了三个激酶能够磷酸化PLD 2在体外表皮生长因子受体（EGFR），JAK 3和Src（与JAK 3首次在本研究中报告）-磷酸化抑制，激活剂，和矛盾（一个可以产生任何效果）网站，分别。质谱分析表明，这些激酶中的每一种的靶点为EGFR的Y-296、JAK 3的Y-415和Src的Y-511。每个位点被激活或抑制的程度取决于所考虑的细胞类型。在COS-7细胞中，表现出最高水平的PLD 2活性，Y-415是一个突出的位点，JAK 3补偿了EGFR对Y-296的负调节。在MCF-7中，显示出最低水平的PLD 2活性的细胞，情况匡威，Y-296不能补偿Y-415的正调节。MTLn 3具有中等至低水平的脂肪酶活性，显示出中间的调节模式，但更接近MCF-7而不是COS-7细胞。EGFR对两种癌细胞系MTLn 3和MCF-7的负面作用通过RNA沉默实验进一步证明，所述RNA沉默实验产生显示出较低PLD 2活性的COS-7，以及显示出升高活性的MTLn 3和MCF-7细胞。MCF-7是源自具有相对低水平PLD活性的低侵袭性/侵袭性形式的乳腺癌的癌细胞系。我们提出，PLD 2活性在乳腺癌细胞系MCF-7中是低的，因为它被EGFR激酶对Y-296的酪氨酰磷酸化下调。因此，PLD 2-Y-296的磷酸化可能是降低具有低侵袭能力的转化细胞中PLD 2活性水平的信号。
We report here that the enzymatic activity of phospholipase D2 (PLD2) is regulated by phosphorylation-dephosphorylation. Phosphatase treatment of PLD2-overexpressing cells showed a biphasic nature of changes in activity that indicated the existence of "activator" and "inhibitory" sites. We identified three kinases capable of phosphorylating PLD2 in vitro-epidermal growth factor receptor (EGFR), JAK3, and Src (with JAK3 reported for the first time in this study)-that phosphorylate an inhibitory, an activator, and an ambivalent (one that can yield either effect) site, respectively. Mass spectrometry analyses indicated the target of each of these kinases as Y-296 for EGFR, Y-415 for JAK3, and Y-511 for Src. The extent to which each site is activated or inhibited depends on the cell type considered. In COS-7, cells that show the highest level of PLD2 activity, the Y-415 is a prominent site, and JAK3 compensates the negative modulation by EGFR on Y-296. In MCF-7, cells that show the lowest level of PLD2 activity, the converse is the case, with Y-296 unable to compensate the positive modulation by Y-415. MTLn3, with medium to low levels of lipase activity, show an intermediate pattern of regulation but closer to MCF-7 than to COS-7 cells. The negative effect of EGFR on the two cancer cell lines MTLn3 and MCF-7 is further proven by RNA silencing experiments that yield COS-7 showing lower PLD2 activity, and MTLn3 and MCF-7 cells showing an elevated activity. MCF-7 is a cancer cell line derived from a low-aggressive/invasive form of breast cancer that has relatively low levels of PLD activity. We propose that PLD2 activity is low in the breast cancer cell line MCF-7 because it is kept downregulated by tyrosyl phosphorylation of Y-296 by EGFR kinase. Thus, phosphorylation of PLD2-Y-296 could be the signal for lowering the level of PLD2 activity in transformed cells with low invasive capabilities.