A nucleus-localization-deficient mutant serves as a dominant-negative inhibitor of gut-enriched Krüppel-like factor function.
Cancer cells differ from normal cells in many aspects, including loss of differentiation and uninhibited cell proliferation. Recent studies have suggested that gut-enriched Krüppel-like factor (GKLF) played an important role in the regulation of cell growth in the colon. Studies from this laboratory have shown that GKLF protein predominantly expressed in the cytoplasm but not the nucleus of colon cancer cells, suggesting that impaired nuclear translocation of GKLF might contribute to cancer formation. In this report, a region containing putative nuclear localization signal (NLS) of GKLF (PKRGRR; amino acids 385-390) was investigated. Mutation of KR to WT had no effect on the inhibitory properties of GKLF on cyclin D1 promoter activity and [(3)H]thymidine uptake in HT-29 cells, whereas mutation of RR to GL abolished GKLF function completely. Additional mutation analyses demonstrated that Arg(390) is the most critical moiety within this region that mediated GKLF function and its nucleus localization. Cotransfection of Arg(390) mutant (RR/RS) completely inhibited wild-type GKLF function, and GFP-RR/RS GKLF fusion proteins failed to translocate to the nucleus. The results from this study demonstrate that Arg(390) confers the NLS of GKLF and that the nucleus-localization-deficient mutant serves as dominant-negative inhibitor of GKLF function.
Shie
J L
JL
Department of Biomedical Sciences, Chang-Gung Institute of Nursing, Taiwan.
Tseng
C C
CC
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D019938:Cyclin D1 / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D005799:Genes, Dominant / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D019073:HT29 Cells; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013936:Thymidine / Q000378:metabolism / Q000493:pharmacokinetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D004268:DNA-Binding Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019938:Cyclin D1; D049452:Green Fluorescent Proteins; D004247:DNA; D013936:Thymidine