Limits...
Identification of a functional nuclear localization signal mediating nuclear import of the zinc finger transcription factor ZNF24.

Li JZ, Chen X, Gong XL, Hu HY, Shi D, Lu YM, Qiu L, Lu F, Hu ZL, Zhang JP - PLoS ONE (2013)

Bottom Line: Our results delimit the NLS to ZF1-2.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.Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacy, Second Military Medical University, Shanghai, China.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

The lysine 286(K286) and arginine 290(R290) residues within the first and second ZF region are critical for the nuclear localization of ZNF24.(A) Diagram of the ZNF24 mutants with point mutation of the lysine(K), argine(R), serines (S), threonines (T) or lysines (K) to alanines (A) within the first and second ZF region as indicated. (B) The protein localization was analyzed similarly as in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c). (D) Conservation of the first and second ZF region of ZNF24 among the species.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815127&req=5

pone-0079910-g005: The lysine 286(K286) and arginine 290(R290) residues within the first and second ZF region are critical for the nuclear localization of ZNF24.(A) Diagram of the ZNF24 mutants with point mutation of the lysine(K), argine(R), serines (S), threonines (T) or lysines (K) to alanines (A) within the first and second ZF region as indicated. (B) The protein localization was analyzed similarly as in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c). (D) Conservation of the first and second ZF region of ZNF24 among the species.

Mentions: The lysine and arginine residues are important for NLS activity[32]. The primary amino acid sequence of ZNF24 zinc fingers contains a total of 6 basic residues in the ZF1 and ZF2 region; two and four basic residues are present in the first and second zinc fingers, respectively (Figure 5A). We therefore sequentially replaced each lysine and arginine in ZF1 and ZF2 with alanine to investigate which amino acid residues are essential for the ZNF24 NLS. Figure 5A indicates the zinc finger amino acid sequences of these mutant proteins. The mutation of basic residues 286 or 290 to alanine (K286A or R290A) resulted in a partial mislocalization of the protein to the cytoplasm, whereas the nuclear localization of the other mutants did not significantly change (Figure 5B). Figure 5C quantifies the Fn/c ratios of these mutants and confirms the visual phenotypes seen in Figure 5B. Importantly, these residues are completely conserved across the species (Figure 5D). These results indicate that the basic residues 286 and 290 within the ZF1 and ZF2 region play an important role in nuclear localization.


Identification of a functional nuclear localization signal mediating nuclear import of the zinc finger transcription factor ZNF24.

Li JZ, Chen X, Gong XL, Hu HY, Shi D, Lu YM, Qiu L, Lu F, Hu ZL, Zhang JP - PLoS ONE (2013)

The lysine 286(K286) and arginine 290(R290) residues within the first and second ZF region are critical for the nuclear localization of ZNF24.(A) Diagram of the ZNF24 mutants with point mutation of the lysine(K), argine(R), serines (S), threonines (T) or lysines (K) to alanines (A) within the first and second ZF region as indicated. (B) The protein localization was analyzed similarly as in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c). (D) Conservation of the first and second ZF region of ZNF24 among the species.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815127&req=5

pone-0079910-g005: The lysine 286(K286) and arginine 290(R290) residues within the first and second ZF region are critical for the nuclear localization of ZNF24.(A) Diagram of the ZNF24 mutants with point mutation of the lysine(K), argine(R), serines (S), threonines (T) or lysines (K) to alanines (A) within the first and second ZF region as indicated. (B) The protein localization was analyzed similarly as in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c). (D) Conservation of the first and second ZF region of ZNF24 among the species.
Mentions: The lysine and arginine residues are important for NLS activity[32]. The primary amino acid sequence of ZNF24 zinc fingers contains a total of 6 basic residues in the ZF1 and ZF2 region; two and four basic residues are present in the first and second zinc fingers, respectively (Figure 5A). We therefore sequentially replaced each lysine and arginine in ZF1 and ZF2 with alanine to investigate which amino acid residues are essential for the ZNF24 NLS. Figure 5A indicates the zinc finger amino acid sequences of these mutant proteins. The mutation of basic residues 286 or 290 to alanine (K286A or R290A) resulted in a partial mislocalization of the protein to the cytoplasm, whereas the nuclear localization of the other mutants did not significantly change (Figure 5B). Figure 5C quantifies the Fn/c ratios of these mutants and confirms the visual phenotypes seen in Figure 5B. Importantly, these residues are completely conserved across the species (Figure 5D). These results indicate that the basic residues 286 and 290 within the ZF1 and ZF2 region play an important role in nuclear localization.

Bottom Line: Our results delimit the NLS to ZF1-2.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.Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacy, Second Military Medical University, Shanghai, China.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus