目的：探讨酸性亮氨酸核磷酸蛋白32B （acidic leucine-rich nuclear phosphoprotein 32B, ANP32B）促进肿瘤发生、发展的分子机制,为其可能作为治疗靶点提供理论依据。方法：利用免疫沉淀联合质谱技术寻找和验证ANP32B相互作用蛋白;免疫荧光技术检测ANP32B 和核浆转运蛋白α6（karyopherin α6,KPNA6）在细胞内的定位;采用短发夹RNA（short hairpin RNA,shRNA）沉默KPNA6的表达;行GST-pulldown实验明确ANP32B与KPNA6直接结合及相互作用的区域。结果：通过免疫沉淀和质谱鉴定发现,ANP32B与KPNA6间存在相互作用,两者的结合不依赖ANP32B N端1~161氨基酸序列。体外GST-pulldown实验证实,KPNA6与ANP32B间存在直接的相互作用,且两者结合依赖ANP32B的核定位信号。利用shRNA抑制KPNA6的表达后,并不影响ANP32B的核内定位。结论：ANP32B通过核定位信号序列与KPNA6结合,两者在细胞核中有明显的共定位,而敲除KPNA6并不影响ANP32B的核内定位。
Objective: To investigate the molecular mechanism of ANP32B （acidic leucine-rich nuclear phosphoprotein 32B）in the promotion of tumors. Methods: ANP32B interaction proteins were searched and identified by immunoprecipitation(IP)-coupled LC-MS/MS technology. Immunofluorescence was used to identify the localization of ANP32B and karyopherin α6 (KPNA6). KPNA6 was knockdown by shRNA. GST-pulldown assay was used to identify the direct interaction of ANP32B and KPNA6 and domains of interaction. Results: Immunoprecipitation(IP)-coupled LC-MS/MS technology identified the interaction of KPNA6 with ANP32B, and the N-terminal 1-161 amino acid of ANP32B was not required for its interaction with KPNA6. In vitro GST-pulldown assay demonstrated the direct interaction between KPNA6 and ANP32B, and the nuclear localization signal (NLS) of ANP32B was essential for its interaction with KPNA6. Inhibition of KPNA6 expression by shRNA had no effect on the nuclear localization of ANP32B. Conclusions: ANP32B interactes with KPNA6 via its NLS domain, and ANP32B is co-localized with KPNA6 in the nucleus. Knockdown of KPNA6 does not influence the nuclear localization of ANP32B.
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