Krüppel-like zinc fingers bind to nuclear import proteins and are required for efficient nuclear localization of erythroid Krüppel-like factor.
Erythroid Krüppel-like Factor (EKLF/KLF-1) is an erythroid-specific transcription factor that contains three C(2)H(2) zinc fingers and is required for correct chromatin structure and expression of the beta-globin locus. However, regions within the EKLF protein that serve as signals for its nuclear localization and the proteins that may enable it to become localized are unknown. Two approaches were used to address these issues. First, green fluorescent protein or pyruvate kinase was fused to EKLF domains, and localization was monitored and quantitated by confocal microscopy. Two necessary and sufficient nuclear localization signals (NLSs) were identified: one (NLS1) adjacent to the zinc finger DNA binding domain within a highly basic stretch of amino acids (275-296), and another more efficient signal (NLS2) within the zinc finger domain itself (amino acids 293-376). Interestingly, each zinc finger contributes to the overall effectiveness of NLS2 and requires an intact finger structure. Second, each NLS was tested in vitro for binding to importin proteins. Surprisingly, both EKLF NLSs, but principally the zinc finger domain, bind importin alpha and importin beta. These findings demonstrate that two nuclear localization signals target EKLF to the nucleus and suggest this transport relies primarily on a novel zinc finger/importin protein interaction.
Quadrini
Karen J
KJ
Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Bieker
James J
JJ
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D016296:Mutagenesis; D010957:Plasmids; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013552:Swine; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D011994:Recombinant Proteins; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor