Bruton酪氨酸激酶泛素化调节的研究.docxVIP

Bruton酪氨酸激酶泛素化调节的研究 房宜嘉胡媛媛 凌兰兰 陈月 史玉叶张莉 王春玲 于亮 南京医科大学附属淮安第一医院血液科 摘要： 目的:探讨Bruton酪氨酸激酶(Brutons Tyrosine Kinase, Btk)的代谢调节 途径和可能的分子机制。方法:用蛋口酶体抑制剂和/或佛波酯(PMA)处理内源 性表达Btk的B细胞系A20、Ramos细胞及转染了外源性Btk的293T、COS-7细 胞，应用RT-PCR法检测上述细胞Btk mRNA表达水平‘Western blot法检测Btk 蛋白表达水平;通过抗Tg M抗体交联BCR刺激A20细胞，检测细胞Btk泛素化水 平;用Btk、泛素和Cbl共转染COS-7细胞，检测其泛素化Btk水平;用Btk和编 码野生型或突变型泛素(K29R、K48R、K63R)即单泛素的质粒共转染293T细胞, 经蛋白酶体抑制剂处理后用抗泛素抗体和抗Btk抗体检测泛素化Btk水平;稳定 转染并表达Btk-GFP的293T细胞经氯唾处理后，应用Western blot检测其Btk 蛋白表达水平。结果:蛋白酶体特异性抑制剂和/或佛波酯可减少Btk转录，从而 引起Btk蛋白表达下降。Btk通过泛素化进行翻译后修饰，泛素化修饰程度与Btk 的表达及活化水平有关。Cbl为Btk的E3泛素连接酶，它可导致Btk泛素化。 Btk受多聚和/或单泛素化调节，Btk可在溶酶体内发生泛素化和降解，溶酶体抑 制剂氯唾可上调Btk的表达。结论:Btk蛋白的表达水平受泛素化途径调节，该 调节与Btk的表达水平有关。 关键词： Bruton酪氨酸激酶;泛素化;蛋白酶体抑制剂;B细胞; 作者简介：于亮，主任医师，副教授，博士生导师.E-mail :yuliangha@163. com 收稿日期:2016-07-03 基金：国家自然科学基金资助项目 Differential Regulation of Brutons Tyrosine Kinase by the Ubiquitin Pathway FANG Yi-Jia HU Yuan—Yuan LING Lan—Lan CHEN Yue SHI Yu-Ye ZHANG Li WANG Chun-Ling YU Liang Department of Hematology, Huai an First Peoples Hospital, Nanjing Medical University; Abstract： Objectivc: To investigatc the regulation of Bruton， s tyrosine kinase (Btk) and to explore its possible mechanism. Methods: After treatment with the proteasome inhibitors and/or phorbol esters (PMA) , the mRNA and prot ein expression level of Btk was detec ted by RT-PCR and Wes tern blot, respectively. The ubiquitination level of Btk in B lymphoblastoid A20 cells was estimated after stimulation via the crosslinking of BCR with anti-lgM antibody. The cotransfection of COS-7 cell with Btk, ubiquitin and Cbl was performed, then the ubiquitination level of Btk was measured. The Btk ubiquitination level was detected after ectopic expression of ubiquitin transfected with the wild type or triple mutant of Ub (K29 R, K48 R, K63 R) . Mono-ubiquitination of Btk was detected with anti bodies preferentially against monovalent ubiquitin; in addition, the protein expression levels of chloroquine-treated stably transfected cells expressin