Publications (2)8.19 Total impact
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ABSTRACT: Transcription factor proteins function in the nucleus to regulate gene expression. Many transcription factors are critical regulators of tumor progression. Conversely, many oncogenic and tumor suppressor proteins are transcription factors or other types of nuclear proteins. Because of their critical physiological and pathological roles, these tumor regulators are tightly regulated not only in the protein expression but also in their subcellular localization. This chapter is focused on experimental strategies and method details for the identification and characterization of nuclear localization signal sequences for nuclear proteins using the Krüppel-like transcription factor 8 as an example.Methods in molecular biology (Clifton, N.J.) 01/2010; 647:171-86.
Article: A unique sequence in the N-terminal regulatory region controls the nuclear localization of KLF8 by cooperating with the C-terminal zinc-fingers.[show abstract] [hide abstract]
ABSTRACT: Krüppel-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 C(2)H(2) 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 C-terminus, 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-beta 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.Cell Research 06/2009; 19(9):1098-109. · 8.19 Impact Factor