The elucidation of novel SH2 binding sites on PLD2
The elucidation of novel SH2 binding sites on PLD2
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DOI：
10.1038/sj.onc.1209340
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发表时间：
2006-05-18
期刊：
影响因子：
8
通讯作者：
Gomez-Cambronero, J.
中科院分区：
文献类型：
--
作者：
Di Fulvio, M.;Lehman, N.;Gomez-Cambronero, J.
我们实验室最近报道，磷脂酶 D2 (PLD2) 与 PTP1b 和生长因子受体结合蛋白 2 (Grb2) 形成三元复合物。在这里，我们建立了 PLD2/Grb2 关联的机制基础。我们已经确定 PLD2 蛋白中的残基 Y-169 和 Y-179 对于 Grb2 相互作用至关重要。我们提供的证据表明 Y-169 和 Y179 位于 PLD2 中介导 SH2 与 Grb2 相互作用的两个共有位点内。这已通过SH2缺陷型GSTGrb2 R86K突变体得到证实，该突变体未能在体外下拉PLD2。为了阐明两个相邻酪氨酸的功能，我们在 PLD2 中创建了一类新的缺失和点突变体。 Y-169 (PLD2 Y169F) 或 Y-179 (PLD2 Y179F) 的苯丙氨酸替代减少了 Grb2 结合，而同时突变则完全消除了它。发现 PLD2 上两个结合位点的作用在功能上并不等效：Y-169 用于调节酶的活性，而 Y-179 则调节蛋白质的总酪氨酸磷酸化。有趣的是，Grb2 与 PLD2 的结合与脂肪酶活性无关，因为 Grb2 与催化失活的 PLD2 突变体结合。最后，PLD2 残基 Y-169 和 Y-179 对于 Sos 的募集是必需的，但只有 PLD2 Y179F 突变体的过表达才会导致 Ras 活性增加、p44/42(Erk) 磷酸化和 DNA 合成增强。由于 Y-169 仍然能够调节酶活性，并且能够与 PLD2 Y179F 突变体中的 Grb2 结合，因此我们建议 Y-169 保持在 Y-179 的负调节之下。当其释放时，Y-169 通过 Ras/MAPK 途径介导细胞增殖。
Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association. We have identified residues Y-169 and Y-179 in the PLD2 protein as being essential for the Grb2 interaction. We present evidence indicating that Y-169 and Y179 are located within two consensus sites in PLD2 that mediate an SH2 interaction with Grb2. This was demonstrated with an SH2-deficient GSTGrb2 R86K mutant that failed to pull-down PLD2 in vitro. In order to elucidate the functions of the two neighboring tyrosines, we created a new class of deletion and point mutants in PLD2. Phenylalanine replacement of Y-169 (PLD2 Y169F)or Y-179 (PLD2 Y179F) reduced Grb2 binding while simultaneous mutation completely abolished it. The role of the two binding sites on PLD2 was found to be functionally nonequivalent: Y-169 serves to modulate the activity of the enzyme, whereas Y-179 regulates total tyrosine phosphorylation of the protein. Interestingly, binding of Grb2 to PLD2 occurs irrespectively of lipase activity, since Grb2 binds to catalytically inactive PLD2 mutants. Finally, PLD2 residues Y-169 and Y-179 are necessary for the recruitment of Sos, but only overexpression of the PLD2 Y179F mutant resulted in increased Ras activity, p44/42(Erk) phosphorylation and enhanced DNA synthesis. Since Y-169 remains able to modulate enzyme activity and is capable of binding to Grb2 in the PLD2 Y179F mutant, we propose that Y-169 is kept under negative regulation by Y-179. When this is released, Y-169 mediates cellular proliferation through the Ras/MAPK pathway.