Bruton酪氨酸激酶泛素化调节的研究  

Differential Regulation of Bruton's Tyrosine Kinase by the Ubiquitin Pathway

作　　者：房宜嘉 胡媛媛[1] 凌兰兰[1] 陈月[1] 史玉叶[1] 张莉[1] 王春玲[1] 于亮[1] 

机构地区：[1]南京医科大学附属淮安第一医院血液科,江苏淮安223300

出　　处：《中国实验血液学杂志》2017年第5期1559-1565,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(81170489);江苏省自然科学基金资助项目(BK20141254);南京医科大学科技发展基金面上项目(2014NJMU027)

摘　　要：目的:探讨Bruton酪氨酸激酶(Bruton's Tyrosine Kinase,Btk)的代谢调节途径和可能的分子机制。方法:用蛋白酶体抑制剂和/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Ig 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细胞经氯喹处理后,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,溶酶体抑制剂氯喹可上调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。Objective： To investigate 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 protein expression level of Btk was detected by RT-PCR and Western blot,respectively. The ubiquitination level of Btk in B lymphoblastoid A20 cells was estimated after stimulation via the crosslinking of BCR with anti-IgM 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 antibodies preferentially against monovalent ubiquitin; in addition,the protein expression levels of chloroquine-treated stably transfected cells expressing Btk-GFP were detected by Western blot,and quantified with the strength of GFP fluorescence. Results： In the presence of proteasome-specific inhibitors and/or PMA,steady-state levels of Btk protein were reduced due to decrease of transcription. Posttranslational modification of Btk by ubiquitination was observed,which was related with the level of Btk expression and activation. The E3 ubiquitin ligase Cbl,which binds to Btk,was also found to ubiquitinate this kinase. Altogether,the data of this study strongly suggest that Btk is regulated by poly-and/or monoubiquitination events. Conclusion： The Btk protein is dictated by its expression level through the ubiquitination pathway.

关 键 词：Bruton酪氨酸激酶 泛素化 蛋白酶体抑制剂 B细胞 

分 类 号：Q55[生物学—生物化学] R733.72[医药卫生—肿瘤]