SHARP-2相互作用タンパク質の同定と特性

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The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is a basic helix-loop-helix transcriptional repressor. Many transcription factors interact with other proteins to form complex transcriptional regulation networks. To identify a SHARP-2-interacting protein(s), we screened a rat liver cDNA library using a yeast two-hybrid system. Approximately 3.5×107 independent clones were screened and 29 positive clones were obtained. Of these, multiple clones contained AT motif-binding factor-1 (ATBF1) and Sex-determining region Y-box 6 (Sox6) which both were transcription factors. We then focused on and analyzed physical protein-protein interaction of SHARP-2 with ATBF1 and Sox6. First, minimal interaction domain of ATBF1 and Sox6 with the SHARP-2 was determined. Amino acid residues between 3,271 and 3,379 of the ATBF1 and those between 201 and 362 of Sox6 were required for an interaction with SHARP-2, respectively. We then examined which domain of SHARP-2 is required for interactions with ATBF1 and Sox6. Amino acid residues between 301 and 355 of the SHARP-2 interacted with the ATBF1 and those between 384 and 397 of the SHARP-2 interacted with the Sox6. Thus, we concluded that both the ATBF1 and Sox6 were identified as the SHARP-2-interacting protein and that the SHARP-2 interacted with them using different domains.

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