酸性亮氨酸核磷酸蛋白32B促肿瘤机制的体外研究

doi:10.16150/j.1671-2870.2018.02.013

诊断学理论与实践 ›› 2018, Vol. 17 ›› Issue (02): 186-190.doi: 10.16150/j.1671-2870.2018.02.013

酸性亮氨酸核磷酸蛋白32B促肿瘤机制的体外研究

朱晓娜, 杨烁, 何平, 张辉林, 冯茹, 余韵

上海交通大学医学院附属瑞金医院分子中心,上海 200025

收稿日期:2018-01-10 出版日期:2018-04-25 发布日期:2018-04-25
通讯作者: 余韵 E-mail: yy@shsmu.edu.cn
基金资助:
上海市自然科学基金（15ZR1426600）

An in vitro study on mechanism of ANP32B in the promotion of tumors

ZHU Xiaona, YANG Shuo, HE Ping, ZHANG Huilin, FENG Ru, YU Yun

Center of Molecular Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2018-01-10 Online:2018-04-25 Published:2018-04-25

摘要/Abstract

摘要： 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。

关键词: 酸性亮氨酸核磷酸蛋白32B, 核浆转运蛋白α6, 相互作用, BT549细胞株

Abstract: 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.

Key words: Acidic leucine-rich nuclear phosphoprotein 32B, Karyopherin α6, Interaction, BT594 cell line

中图分类号:

R730.1

朱晓娜, 杨烁, 何平, 张辉林, 冯茹, 余韵. 酸性亮氨酸核磷酸蛋白32B促肿瘤机制的体外研究[J]. 诊断学理论与实践, 2018, 17(02): 186-190.

ZHU Xiaona, YANG Shuo, HE Ping, ZHANG Huilin, FENG Ru, YU Yun. An in vitro study on mechanism of ANP32B in the promotion of tumors[J]. Journal of Diagnostics Concepts & Practice, 2018, 17(02): 186-190.

参考文献

[1] Matilla A, Radrizzani M.The Anp32 family of proteins containing leucine-rich repeats[J]. Cerebellum,2005,4(1):7-18.
[2] Reilly PT, Yu Y, Hamiche A, et al.Cracking the ANP32 whips: important functions, unequal requirement, and hints at disease implications[J]. Bioessays,2014,36(11):1062-1071.
[3] Seo SB, McNamara P, Heo S, et al. Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the set oncoprotein[J]. Cell,2001, 104(1):119-130.
[4] Obri A, Ouararhni K, Papin C, et al.ANP32E is a histone chaperone that removes H2A.Z from chromatin[J]. Nature,2014,505(7485):648-653.
[5] Tochio N, Umehara T, Munemasa Y, et al.Solution structure of histone chaperone ANP32B: interaction with core histones H3-H4 through its acidic concave domain[J]. J Mol Biol,2010,401(1):97-114.
[6] Katayose Y, Li M, Al-Murrani SW, et al.Protein phosphatase 2A inhibitors, I(1)(PP2A) and I(2)(PP2A), asso-ciate with and modify the substrate specificity of protein phosphatase 1[J]. J Biol Chem,2000,275(13):9209-9014.
[7] Habrukowich C, Han DK, Le A, et al.Sphingosine interac-tion with acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) regulates PP2A activity and cyclooxygenase (COX)-2 expression in human endothelial cells[J]. J Biol Chem,2010,285(35):26825-26831.
[8] Kim HE, Jiang X, Du F, et al.PHAPI, CAS, and Hsp70 promote apoptosome formation by preventing Apaf-1 aggregation and enhancing nucleotide exchange on Apaf-1[J]. Mol Cell,2008,30(2):239-247.
[9] Hoffarth S, Zitzer A, Wiewrodt R, et al.pp32/PHAPI determines the apoptosis response of non-small-cell lung cancer[J]. Cell Death Differ,2008,15(1):161-170.
[10] Moore MJ, Rosbash M.Cell biology. TAPping into mRNA export[J]. Science,2001,294(5548):1841-1842.
[11] Fries B, Heukeshoven J, Hauber I, et al.Analysis of nucleocytoplasmic trafficking of the HuR ligand APRIL and its influence on CD83 expression[J]. J Biol Chem,2007, 282(7):4504-4515.
[12] Pan W, da Graca LS, Shao Y, et al. PHAPI/pp32 suppresses tumorigenesis by stimulating apoptosis[J]. J Biol Chem,2009,284(11):6946-6954.
[13] Bai J, Brody JR, Kadkol SS, et al.Tumor suppression and potentiation by manipulation of pp32 expression[J]. Oncogene,2001,20(17):2153-2160.
[14] Kadkol SS, Brody JR, Pevsner J, et al.Modulation of oncogenic potential by alternative gene use in human prostate cancer[J]. Nat Med,1999,5(3):275-279.
[15] Yu Y, Shen SM, Zhang FF, et al.Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) contributes to retinoic acid-induced differentiation of leukemic cells[J]. Biochem Biophys Res Commun,2012, 423(4):721-725.
[16] Shen SM, Yu Y, Wu YL, et al.Downregulation of ANP32B, a novel substrate of caspase-3, enhances caspase-3 activation and apoptosis induction in myeloid leukemic cells[J]. Carcinogenesis,2010,31(3):419-426.
[17] Yang S, Zhou L, Reilly PT, et al.ANP32B deficiency impairs proliferation and suppresses tumor progression by regulating AKT phosphorylation[J]. Cell Death Dis,2016, 7:e2082.
[18] Gao P, Wang QP, Chen L, et al.Prediction of human genes' regulatory functions based on proteinprotein interac-tion network[J]. Protein Pept Lett,2012,19(9):910-916.
[19] Adegbola O, Pasternack GR.Phosphorylated retinoblastoma protein complexes with pp32 and inhibits pp32-mediated apoptosis[J]. J Biol Chem,2005,280(16):15497-15502.
[20] Munemasa Y, Suzuki T, Aizawa K, et al.Promoter region-specific histone incorporation by the novel histone chaperone ANP32B and DNA-binding factor KLF5[J]. Mol Cell Biol,2008,28(3):1171-1181.
[21] Sun Z, Wu T, Zhao F, et al.KPNA6 (Importin {alpha}7)-mediated nuclear import of Keap1 represses the Nrf2-dependent antioxidant response[J]. Mol Cell Biol,2011,31(9):1800-1811.
[22] Goldfarb DS, Corbett AH, Mason DA, et al.Importin alpha: a multipurpose nuclear-transport receptor[J]. Trends Cell Biol,2004,14(9):505-514.
[23] Stewart M.Molecular mechanism of the nuclear protein import cycle[J]. Nat Rev Mol Cell Biol,2007,8(3):195-208.

酸性亮氨酸核磷酸蛋白32B促肿瘤机制的体外研究

An in vitro study on mechanism of ANP32B in the promotion of tumors

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