Gryz Ela A, Meakin Susan O
Laboratory of Neural Signalling, Cell Biology Group, The Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada N6A 5K8.
Oncogene. 2003 Nov 27;22(54):8774-85. doi: 10.1038/sj.onc.1206890.
TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons and also differentiation of neuroblastomas and apoptosis of medulloblastomas. We have previously shown that aspartic acid and glutamic acid substitution (AspGlu and GluAsp) of the activation loop tyrosines in TrkA (Tyr(683) and Tyr(684)) supports NGF-independent neuritogenesis and cell survival in PC12 cell-derived nnr5 cells. In this study, the AspGlu and GluAsp mutant Trks have been analysed for their ability to support NGF-independent and NGF-dependent neuritogenesis, proliferation and cell signalling in the human neuroblastoma cell line, SY5Y. We find that the AspGlu and GluAsp mutant Trks support NGF-dependent, but not NGF-independent, autophosphorylation, neuritogenic responses and/or inhibit cell cycle progression. The NGF-dependent neuritogenic responses are lower for the mutant Trks (approximately 30-60% for AspGlu and 50-60% for GluAsp), relative to wild-type TrkA. While both the AspGlu and GluAsp mutant Trks support NGF-dependent transient phosphorylation of Shc, PLCgamma-1, AKT, FRS2, SH2B as well as prolonged MAP kinase activation, the GluAsp mutant induces stronger NGF-dependent tyrosine phosphorylation of FRS2 and SH2B, as well as a stronger reduction in bromodeoxyuridine (BrdU) incorporation. Collectively, these data suggest that neither absolute levels of receptor autophosphorylation, high levels of TrkA expression nor the activation of a specific signalling pathway is dominant and absolutely essential for neuritogenesis and cell cycle arrest of SY5Y cells.
TrkA是神经生长因子（NGF）的受体酪氨酸激酶（RTK），可刺激原代神经元中NGF依赖的细胞存活和分化，也能促进神经母细胞瘤的分化以及髓母细胞瘤的凋亡。我们之前已经表明，TrkA激活环酪氨酸（Tyr(683)和Tyr(684)）的天冬氨酸和谷氨酸替换（AspGlu和GluAsp）可支持PC12细胞衍生的nnr5细胞中不依赖NGF的神经突生长和细胞存活。在本研究中，我们分析了AspGlu和GluAsp突变型Trk在人神经母细胞瘤细胞系SY5Y中支持不依赖NGF和依赖NGF的神经突生长、增殖及细胞信号传导的能力。我们发现，AspGlu和GluAsp突变型Trk支持依赖NGF的自磷酸化、神经突生成反应和/或抑制细胞周期进程，但不支持不依赖NGF的情况。相对于野生型TrkA，突变型Trk的依赖NGF的神经突生成反应较低（AspGlu约为30 - 60%，GluAsp约为50 - 60%）。虽然AspGlu和GluAsp突变型Trk都支持依赖NGF的Shc、PLCγ-1、AKT、FRS2、SH2B的瞬时磷酸化以及延长的MAP激酶激活，但GluAsp突变型诱导更强的依赖NGF的FRS2和SH2B酪氨酸磷酸化，以及更强的溴脱氧尿苷（BrdU）掺入减少。总体而言，这些数据表明，受体自磷酸化的绝对水平、TrkA的高表达水平以及特定信号通路的激活，对于SY5Y细胞的神经突生长和细胞周期停滞都不是主导且绝对必要的。
