Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.
Mol Cell Biol. 2010 May;30(9):2251-63. doi: 10.1128/MCB.01239-09. Epub 2010 Feb 22.
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.
我们在此报告，磷脂酶 D2（PLD2）的酶活性受磷酸化-去磷酸化调节。磷酸酶处理过的 PLD2 过表达细胞显示出活性的两相变化，表明存在“激活剂”和“抑制剂”位点。我们鉴定了三种能够在体外磷酸化 PLD2 的激酶-表皮生长因子受体（EGFR）、JAK3 和Src（在本研究中首次报道 JAK3）-分别磷酸化一个抑制性、一个激活剂和一个两用（可产生两种效果）位点。质谱分析表明，这些激酶的每个靶点分别为 EGFR 的 Y（296）、JAK3 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，PLD2 活性最高，Y（415）是一个主要的位点，JAK3 补偿了 EGFR 对 Y（296）的负调节。在 MCF-7 细胞中，PLD2 活性最低，情况正好相反，Y（296）无法补偿 Y（415）的正调节。MTLn3 具有中低水平的脂肪酶活性，表现出一种中间调节模式，但更接近 MCF-7 细胞，而不是 COS-7 细胞。RNA 沉默实验进一步证明了 EGFR 对两种癌细胞系 MTLn3 和 MCF-7 的负效应，实验结果显示 COS-7 细胞的 PLD2 活性降低，而 MTLn3 和 MCF-7 细胞的活性升高。MCF-7 是一种源自低侵袭/浸润性乳腺癌的癌细胞系，PLD 活性相对较低。我们提出，PLD2 活性在乳腺癌细胞系 MCF-7 中较低，是因为它被 EGFR 激酶的酪氨酸磷酸化所下调。因此，PLD2-Y（296）的磷酸化可能是降低低侵袭能力转化细胞中 PLD2 活性的信号。
