BCR-ABL与泛素E3连接酶c-CBL的相互作用在慢性髓系白血病靶向治疗中的作用及相关机制研究

Role and Mechanism of the Interaction of BCR-ABL with E3 Ligase c-CBL in Targeting Therapy of Chronic Myelogenous Leukemia

目的:通过对BCR-ABL与泛素E3连接酶c-CBL的相互作用结构域的筛查,明确砷剂治疗慢性髓系白血病(CML)的结构基础。方法:根据BCR-ABL与c-CBL的结构信息,对二者的结合界面进行结构模拟与分析,基于此构建BCR-ABL的不同突变体[(△SH2(Src同源结构域2)、△TyrKC(酪氨酸激酶催化结构域)和△SH2/TyrKC(S/H))以及c-CBL的突变体△RF(环指结构域),转染293T及HeLa细胞,应用免疫共沉淀(Co-IP)]及免疫荧光(IF)共定位的方法筛查BCR-ABL与c-CBL的相互作用结构域。结果:Co-IP发现BCR-ABL的TyrKC结构域主要参与了与c-CBL的相互作用,SH2结构域也发挥一定的作用,但作用弱于TyrKC结构域;当两个结构域同时截短后,BCR-ABL与c-CBL几乎不发生相互作用;同时c-CBL的RF结构域在一定程度上影响了与BCR-ABL的相互作用。IF的结果与Co-IP相符,证明了结构模拟的准确性。结论:BCR-ABL的TyrKC和SH2结构域主要参与了与c-CBL的相互作用,而c-CBL的RF结构域参与了与BCR-ABL的相互作用。c-CBL与BCR-ABL的相互作用对BCR-ABL的稳定性发挥调控作用,对于深入理解砷剂靶向治疗CML分子机制提供了结构基础。

Objective：To explore the interaction domains between BCR-ABL and E3 liagase c-CBL,so as to reveal the structure-basis for the arsenic to treat chronic myelogenous leukemia（CML）.Methods：The interactional interface of BCR-ABL and c-CBL was simulated and analyzed according to the available structure model.Based on the structural information,the WT and mutant Migrl-BCR-ABL-GFP（ASH2,ATyrKC,ASH2/TyrKC（S/H）） and pFlag-c-CBL（ARF） were constructed and co-transfected into the 293 T and HeLa cells.The co-immunoprecipitation（Co-IP） was performed by using M2 beads（anti-Flag）,anti-GFP antibody and protein A beads,and the interaction was identified by using GFP and M2 antibody,respectively.Moreover,the colocalization of BCR-ABL and c-CBL was further evaluated by using immunofluorescent（IF） assay in transfected HeLa cells.Results：Co-IP demonstrated that the TyrKC domain of BCR-ABL was primarily involved in the interaction with c-CBL,while both the SH2 domain of BCR-ABL and the RF domain of c-CBL also participated in the interaction to a certain degree,which were consistent with the structure-based simulation.IF elucidated that the colocalization of BCR-ABL and c-CBL was almost entirely vanished when the deleted TyrKC domain of BCR-ABL was co-transfected with c-CBL,which were elegantly coincident with the results from CoIP.Conclusion：The TyrKC domain of BCR-ABL is sufficient and necessary to mediate the interaction between BCRABL and c-CBL,the SH2 domain of BCR-ABL and the RF domain of c-CBL are also involved in the association between the two proteins.It suggests that the association of BCR-ABL and c-CBL can modulate the stability and degradation of BCR-ABL,thus illustrating the molecular mechanisms of the targeting therapy for CML by arsenic.