Di Fulvio Mauricio, Frondorf Kathleen, Henkels Karen M, Lehman Nicholas, Gomez-Cambronero Julian
Cell Biology and Physiology, Wright State University, School of Medicine, Dayton, OH 45435, USA.
J Mol Biol. 2007 Mar 30;367(3):814-24. doi: 10.1016/j.jmb.2007.01.021. Epub 2007 Jan 12.
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-knockdown cells (COS7(shGrb2)), resulted in the nearly full rescue of PLD2 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-Y169/179F, 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 EGF. 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.
衔接蛋白Grb2与磷脂酶D2（PLD2）相关联，但这种相互作用在体内对于该脂肪酶的功能是否必要尚不清楚。我们证明，基于稳定短发夹RNA（shRNA）对Grb2进行沉默，Grb2是表皮生长因子受体（EGFR）的关键信号转导子以及与Ras/Erk途径的连接蛋白，这导致COS7细胞中PLD2活性降低。将对shGrb2沉默具有抗性的Grb2构建体（XGrb2(SiL)）转染到Grb2敲低细胞（COS7(shGrb2)）中，导致PLD2活性几乎完全恢复。然而，Grb2-R86K是Grb2的一种SH2缺陷型突变体，无法与PLD2结合，它未能在COS7(shGrb2)细胞中增强受损的PLD2活性。Grb2和PLD2直接相关，并且无论EGFR是否激活，抗myc抗体都能沉淀Grb2。免疫荧光显微镜显示，在表皮生长因子（EGF）刺激后，共转染的PLD2和Grb2重新定位于类高尔基体结构。由于在与不与Grb2结合的脂肪酶突变体PLD2-Y169/179F进行共转染实验中未观察到这种情况，我们推断Grb2通过其SH2结构域将PLD2劫持到核周高尔基体区域。支持这一观点的是，原代细胞系人脐静脉内皮细胞（HUVEC）在细胞质和核周高尔基体区域中弥漫性表达PLD2，PLD2和Grb2在该区域共定位。在用EGF刺激后，原代细胞中的这种共定位增加。这些结果首次证明，Grb2的存在及其与局部细胞内结构的相互作用对于体内PLD2活性和信号传导至关重要。
