Dok1与TrkA之间相互作用的酵母双杂交研究

Interaction between downstream of kinases 1 and TrkA:a yeast two-hybrid approach

目的:研究downstream of kinases1(dok1)与TrkA之间的相互作用,寻找TrkA的可能底物或调控蛋白,以深入认识TrkA下游信号转导机制。方法:将TrkA胞内域与LexA蛋白融合作为DNA结合蛋白,分别将dok1、dok1PTB及dok1ΔPTB与B42AD蛋白融合作为激活域蛋白,共转化酵母菌后,通过β-半乳糖苷酶活性检测以及氨基酸营养缺陷生长实验分析它们之间的相互作用。结果:dok1及dok1PTB与TrkA之间在酵母中存在结合,而dok1ΔPTB不与TrkA结合。结论:证实TrkA与dok1之间具有相互作用,dok1的PTB结构域在介导此相互作用的过程中非常重要。

Objective：To study the interaction between TrkA and downstream of kinases 1（dok 1） and search for the possible intracellular substrates or regulatory proteins of TrkA, so as to better understand the mechanism of downstream signal transduction of TrkA. Methods： The TrkA^IC （intracellular domain of TrkA） was fused with LexA and the product was used as a DNA-binding domain protein; dok1, dok1PTB and dok1△PTB（dok1 without PTB） were separately fused with B42AD and their products were used as activation domain protein. Cotransformants were subjected to β-galactosidase activity analysis and leucine medium growth analysis for understanding of their interaction. Results： Dokl and dok1PTB interacted with TrkA in yeast, while dok1△PTB did not. Conclusion： It is confirmed that TrkA can interact with dok1 in yeast, and the interaction may be mediated by PTB domain of dok1.