Gryz E A, Meakin S O
Neurodegeneration Group, The John P. Robarts Research Institute, London, Ontario, Canada.
Oncogene. 2000 Jan 20;19(3):417-30. doi: 10.1038/sj.onc.1203330.
TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons. TrkA expression in neuronal tumors also supports NGF-dependent differentiation of neuroblastomas and apoptosis of medulloblastomas. Phosphorylation of the activation loop tyrosines in RTK's are essential to activation as well as allosteric changes that facilitate substrate interaction and phosphorylation. Acidic amino acid substitution of the activation loop tyrosines in TrkA, Tyr683Tyr684, was performed to mimic the negative charges normally induced by ligand activation and receptor phosphorylation. A total of eight independent mutants containing single or double substitutions were generated for comparison. Herein, we demonstrate that acidic substitution of the activation loop tyrosines is sufficient to induce allosteric changes required for constitutive TrkA kinase activity as well as phosphorylation of TrkA signaling proteins such as Shc, PLCgamma-1, FRS-2 and erk1/2. The strongest constitutively active TrkA mutants, GluAsp and AspGlu, support NGF-independent neuritogenesis and cell survival to levels approximately 65 and 80-100%, respectively, of NGF-activated wild type TrkA. Thus, constitutively active TrkA may provide a useful strategy in future therapeutic approaches to limit the development and progression of neuronal tumors.
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。TrkA在神经元肿瘤中的表达也支持神经母细胞瘤的NGF依赖分化和成神经管细胞瘤的凋亡。RTK激活环酪氨酸的磷酸化对于激活以及促进底物相互作用和磷酸化的变构变化至关重要。对TrkA激活环酪氨酸Tyr683Tyr684进行酸性氨基酸替换，以模拟通常由配体激活和受体磷酸化诱导的负电荷。总共产生了八个包含单取代或双取代的独立突变体用于比较。在此，我们证明激活环酪氨酸的酸性替换足以诱导组成型TrkA激酶活性所需的变构变化以及TrkA信号蛋白（如Shc、PLCγ-1、FRS-2和erk1/2）的磷酸化。最强的组成型活性TrkA突变体GluAsp和AspGlu分别支持NGF非依赖的神经突生长和细胞存活，达到NGF激活的野生型TrkA水平的约65%和80 - 100%。因此，组成型活性TrkA可能为未来限制神经元肿瘤发生和进展的治疗方法提供一种有用的策略。
