20 research outputs found
Characterization of sequences in human TWIST required for nuclear localization
<p>Abstract</p> <p>Background</p> <p>Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) <sup>37</sup>RKRR<sup>40 </sup>and <sup>73</sup>KRGKK<sup>77 </sup>in the human TWIST (H-TWIST) protein.</p> <p>Results</p> <p>Using site-specific mutagenesis and immunofluorescences, we observed that altered TWIST<sup>NLS1 </sup>K38R, TWIST<sup>NLS2 </sup>K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWIST<sup>NLS2 </sup>K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWIST<sup>NLS1 </sup>with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays.</p> <p>Conclusion</p> <p>Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4.</p