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A nucleus-localization-deficient mutant serves as a dominant-negative inhibitor of gut-enriched Krüppel-like factor
Abstract:
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.
Insights
Gut-enriched Krüppel-like factor (GKLF) nuclear localization is crucial for its function in colon cancer. Arg(390) acts as a nuclear localization signal (NLS), and its mutation impairs GKLF function, potentially inhibiting cancer growth.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Colon cancer is characterized by uncontrolled cell proliferation and loss of differentiation.
- Gut-enriched Krüppel-like factor (GKLF) regulates cell growth, but its function is impaired in colon cancer cells due to altered localization.
- GKLF protein is found in the cytoplasm, not the nucleus, of colon cancer cells, suggesting impaired nuclear translocation contributes to cancer formation.
Purpose of the Study:
- To investigate the role of a specific region (amino acids 385-390) containing a putative nuclear localization signal (NLS) in GKLF function.
- To identify the critical amino acid residue responsible for GKLF nuclear localization and inhibitory activity.
- To determine if a nucleus-localization-deficient GKLF mutant can act as a dominant-negative inhibitor.
Main Methods:
- Site-directed mutagenesis of the putative NLS region (PKRGRR) in GKLF.
- Assays to evaluate GKLF's inhibitory effects on cyclin D1 promoter activity and [(3)H]thymidine uptake in HT-29 cells.
- Cotransfection experiments with wild-type GKLF and mutant forms.
- Expression of GFP-fused GKLF mutants to assess nuclear translocation via fluorescence microscopy.
Main Results:
- Mutation of the RR motif to GL completely abolished GKLF function, while KR to WT mutation had no effect.
- Arg(390) was identified as the critical residue mediating GKLF function and nuclear localization.
- The Arg(390) mutant (RR/RS) inhibited wild-type GKLF activity and failed to translocate to the nucleus.
- GFP-tagged Arg(390) mutants did not enter the nucleus, confirming its role in nuclear import.
Conclusions:
- Arg(390) within the GKLF protein serves as the nuclear localization signal (NLS).
- Impaired nuclear translocation of GKLF, due to mutations affecting Arg(390), leads to loss of its growth-inhibitory function.
- The nucleus-localization-deficient GKLF mutant acts as a dominant-negative inhibitor, suggesting therapeutic potential by blocking GKLF function in cancer cells.