Kruppel-like factor 8 （KLF8） transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signa...Kruppel-like factor 8 （KLF8） transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal（s） （NLS） has not been identified. KLF8 shares with other KLFs monopartite NLSs （mNLS） and C2H2 zinc fingers （ZFs）, both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn^2＋-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of 〉1.5 ZFs from Cterminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid （aa） 151-200 region almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear localization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.展开更多
本研究旨在探讨信号转导和转录活化蛋白4(signal transducer and activator of transcription 4,STAT4)受白介素-12(in-terleukin-12,IL-12)刺激后移位入细胞核的机制。对STATs家族成员进行同源性比对分析结果显示,STAT4的DNA结合域的39...本研究旨在探讨信号转导和转录活化蛋白4(signal transducer and activator of transcription 4,STAT4)受白介素-12(in-terleukin-12,IL-12)刺激后移位入细胞核的机制。对STATs家族成员进行同源性比对分析结果显示,STAT4的DNA结合域的395～416位氨基酸残基序列可能具有二聚体特异性核定位信号(dimer-specific nuclear localization signal,dsNLS)功能。有鉴于此,本研究以pEGFP-C1为表达载体,分别构建了pEGFP-STAT4质粒、缺失395～416位氨基酸残基序列的缺失型STAT4质粒(pEGFP-STAT4-Del)、将SV40大T抗原上经典的NLS核酸序列插入表达载体的阳性对照质粒(pEGFP-NLS)和将缺失型STAT4插入pEGFP-NLS的pEGFP-NLS-STAT4-Del质粒。将这些质粒瞬时转染宫颈癌腺癌细胞系Caski细胞,经过IL-12刺激,发现野生型STAT4移位入细胞核,而缺失型STAT4分布在细胞浆中;进一步用leptomycinB处理,IL-12再刺激后野生型STAT4被滞留于细胞核中,而缺失型STAT4仍然分布在胞浆中;将NLS插入缺失型STAT4,能恢复缺失型STAT4的核移位。以上结果说明野生型STAT4在IL-12刺激下能移位入细胞核,其入核机制是在其DNA结合域的395～416位氨基酸残基序列具有dsNLS功能,能介导活化的STAT4移位入细胞核。展开更多
SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptiona...SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Pall complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Pall complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degrada- tion, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.展开更多
文摘Kruppel-like factor 8 （KLF8） transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal（s） （NLS） has not been identified. KLF8 shares with other KLFs monopartite NLSs （mNLS） and C2H2 zinc fingers （ZFs）, both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn^2＋-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of 〉1.5 ZFs from Cterminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid （aa） 151-200 region almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear localization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.
基金supported by the National Natural Scientific Foundation of China (No. 30771122)National Undergraduate Innovation Training Project of Ministry of Education,China (No.AE11528)
文摘本研究旨在探讨信号转导和转录活化蛋白4(signal transducer and activator of transcription 4,STAT4)受白介素-12(in-terleukin-12,IL-12)刺激后移位入细胞核的机制。对STATs家族成员进行同源性比对分析结果显示,STAT4的DNA结合域的395～416位氨基酸残基序列可能具有二聚体特异性核定位信号(dimer-specific nuclear localization signal,dsNLS)功能。有鉴于此,本研究以pEGFP-C1为表达载体,分别构建了pEGFP-STAT4质粒、缺失395～416位氨基酸残基序列的缺失型STAT4质粒(pEGFP-STAT4-Del)、将SV40大T抗原上经典的NLS核酸序列插入表达载体的阳性对照质粒(pEGFP-NLS)和将缺失型STAT4插入pEGFP-NLS的pEGFP-NLS-STAT4-Del质粒。将这些质粒瞬时转染宫颈癌腺癌细胞系Caski细胞,经过IL-12刺激,发现野生型STAT4移位入细胞核,而缺失型STAT4分布在细胞浆中;进一步用leptomycinB处理,IL-12再刺激后野生型STAT4被滞留于细胞核中,而缺失型STAT4仍然分布在胞浆中;将NLS插入缺失型STAT4,能恢复缺失型STAT4的核移位。以上结果说明野生型STAT4在IL-12刺激下能移位入细胞核,其入核机制是在其DNA结合域的395～416位氨基酸残基序列具有dsNLS功能,能介导活化的STAT4移位入细胞核。
文摘SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Pall complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Pall complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degrada- tion, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.
