基于FRET成像探究棕榈酰化修饰调节Fyn激酶活性  

Palmitoylation in Regulating Fyn Kinase Activity Based on FRET Imaging

作　　者：张鑫[1,2] 郭佳 姚辉 邓林红[1] 欧阳明星 ZHANG Xin;GUO Jia;YAO Hui;DENG Linhong;OUYANG Mingxing(Institute of Biomedical Engineering and Health Sciences,School of Medical and Health Engineering;School of Pharmacy&School of Biological and Food Engineering,Changzhou University,Changzhou 213164,Jiangsu,China)

机构地区：[1]常州大学医学与健康工程学院,生物医学工程与健康科学研究院,江苏常州213164 [2]常州大学药学院生物与食品工程学院,江苏常州213164

出　　处：《医用生物力学》2023年第2期228-235,共8页Journal of Medical Biomechanics

基　　金：江苏省科技厅自然科学基金项目(BK20181464);国家自然科学基金项目(11872129,11532003)。

摘　　要：目的探讨棕榈酰化修饰调节非受体酪氨酸激酶Fyn活性的分子机制。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时检测细胞中的Fyn活性,并结合棕榈酰化位点缺失和共转染蛋白质酪氨酸激酶(C-terminal Src kinase,CSK)表达质粒研究其分子机制。结果实验发现,(C3,C6)任一位点的棕榈酰化缺失能引起Fyn的高活性表达,且C6位点影响更显著。已知CSK激活后发生膜转移,FRET检测证实其对细胞中的Fyn活性有下调作用,但不能有效调控(C3,C6)棕榈酰化位点缺失的Fyn(GSS)活性。结论本文结果初步支持了Fyn活性受细胞内的物理空间定位分布的一种调控机制假设,即棕榈酰化缺失的Fyn(GSS)受细胞膜上CSK抑制性的调节作用被减弱,从而促进了组成性的高活性表达。Objective To investigate the molecular mechanism of palmitoylation modification in regulating the activity of non-receptor tyrosine kinase Fyn.Methods The intracellular Fyn activity was detected by applying fluorescence resonance energy transfer(FRET)technology,and the mechanism was investigated by combining with Fyn palmitoylation deficiency and C-terminal Src kinase(CSK)plasmid co-expression.Results Experimental data showed that single loss of either of(C3,C6)palmitoylation sites resulted in higher Fyn activity,and C6 seemed more significant.It is known that CSK membrane translocation occurred after activation.FRET assay confirmed that CSK could down-regulate the activity of Fyn in cells,but could not effectively regulate the activity of Fyn(GSS)with the loss of palmitoylation sites.Conclusions The results in this study support the hypothesis on Fyn regulation by spatial localization,namely,non-palmitoylated Fyn(GSS)is less effective in the inhibitory regulation by CSK on cell membrane,thus promoting constitutive high activity expression.

关 键 词：Fyn激酶 棕榈酰化修饰 荧光共振能量转移 CSK激酶 

分 类 号：R318.01[医药卫生—生物医学工程]