Identification of a functional nuclear localization signal mediating nuclear import of the zinc finger transcription factor ZNF24.
ZNF24 is a member of the SCAN domain family of Krüppel-like zinc finger (ZF) transcription factors, which plays a critical role in cell proliferation and differentiation. However, how ZNF24 enters the nucleus in order to exert its function remains unclear since its nuclear localization signal(s) (NLS) has not been identified. Here, we generated a series of GFP-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to ZF1-2. Deletion of ZF1-2 caused cytoplasmic accumulation of ZNF24. Fusion of the ZF1-2 to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the ZF1-2 is both necessary and sufficient for nuclear localization. ZNF24 containing histidine to leucine mutations that disrupt the structure of ZF1 or/and ZF2 retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. K286A and R290A mutation led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that ZNF24 interacted with importin-β and this interaction required the ZF motifs. The β-Catenin (CTNNB1) luciferase assays showed that the ZNF24 mutants defective in nuclear localization could not promote CTNNB1 promoter activation as the wild-type ZNF24 did. Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.
Department of Biochemical Pharmacy, Second Military Medical University, Shanghai, China.
Jian-Zhong
JZ
Li
Xia
X
Chen
Xue-Lian
XL
Gong
Hong-Yuan
HY
Hu
Duo
D
Shi
Yi-Ming
YM
Lu
Lei
L
Qiu
Fa
F
Lu
Zhen-Lin
ZL
Hu
Jun-Ping
JP
Zhang
D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
D051741:Kruppel-Like Transcription Factors; D014157:Transcription Factors; C457300:ZNF24 protein, human; D028961:beta Karyopherins