The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF
The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF
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DOI：
10.1016/j.jmb.2007.01.021
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发表时间：
2007-03-30
影响因子：
5.6
通讯作者：
Gomez-Cambronero, Julian
中科院分区：
文献类型：
--
作者：
Di Fulvio, Mauricio;Frondorf, Kathleen;Gomez-Cambronero, Julian
衔接蛋白Grb 2与磷脂酶D2（PLD 2）相关，但尚不清楚这种相互作用是否是体内脂肪酶功能所必需的。我们证明，稳定的短发夹RNA（shRNA）为基础的沉默Grb 2，表皮生长因子受体（EGFR）的关键信号转导子和连接器的Ras/Erk途径，导致减少PLD 2的活性在COS 7细胞。将对shGrb 2沉默难治的Grb 2构建体（XGrb 2（SiL））转染到Grb 2（shGrb 2）中导致几乎完全拯救PLD 2敲低细胞（C 0 S7活性。然而，Grb 2-R86 K，一种不能与PLD 2结合的Grb 2的SH 2缺陷突变体，未能诱导COS 7 shGrb 2细胞中受损的PLD 2活性的增强。Grb 2和PLD 2直接相关，并且Grb 2被抗myc抗体降低，而不管EGFR活化的存在与否。免疫荧光显微镜显示，共转染PLD 2和Grb 2重新定位到高尔基体样结构EGF刺激后。由于这在Grb 2和PLD 2-YI 69/179 F（一种不与Grb 2结合的脂肪酶突变体）的共转染实验中没有观察到，我们推断Grb 2通过其SH 2结构域将PLD 2劫持到核周高尔基体区域。支持这一点的发现是，原代细胞系HUVEC在细胞质和核周高尔基体区域中弥散表达PLD 2，其中PLD 2和Grb 2共定位。这些结果首次证明Grb 2的存在及其与局部细胞内结构的相互作用对于体内PLD 2活性和信号传导是必不可少的。(c)2007爱思唯尔有限公司保留所有权利。
The adaptor protein Grb2 associates with phospholipase D2 (PLD2), but it is not known if this interaction is necessary for the functionality of the lipase in vivo. We demonstrate that stable short hairpin RNA (shRNA)based silencing of Grb2, a critical signal transducer of the epidermal growth factor receptor (EGFR) and linker to the Ras/Erk pathway, resulted in the reduction of PLD2 activity in COS7 cells. Transfection of a Grb2 construct refractory to shGrb2 silencing (XGrb2(SiL)) into the Grb2(shGrb2)), resulted in the nearly full rescue of PLD2 knockdown cells (COS7 activity. However, Grb2-R86K, an SH2-deficient mutant of Grb2 that is incapable of binding to PLD2, failed to induce an enhancement of the impaired PLD2 activity in COS7 shGrb2 cells. Grb2 and PLD2 are directly associated and Grb2 is brought down with anti-myc antibodies irrespective of the presence or absence of EGFR activation. Immunofluorescence microscopy showed that co-transfected PLD2 and Grb2 re-localize to Golgi-like structures after EGF stimulation. Since this was not observed in cotransfection experiments with Grb2 and PLD2-YI69/179F, a lipase mutant that does not bind to Grb2, we inferred that Grb2 serves to hijack PLD2 to the perinuclear Golgi region through its SH2 domain. Supporting this is the finding that the primary cell line HUVEC expresses PLD2 diffusely in the cytoplasm and in the perinuclear Golgi region, where PLD2 and Grb2 colocalize. Such colocalization in primary cells increased after stimulation with EGE These results demonstrate for the first time that the presence of Grb2 and its interaction with localized intracellular structures is essential for PLD2 activity and signaling in vivo. (c) 2007 Elsevier Ltd. All rights reserved.