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Nuclear localization of coactivator RAC3 is mediated by a bipartite NLS and importin alpha3

Percy Luk Yeung1, Aihua Zhang, J Don Chen

  • 1Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ, USA.

Biochemical and Biophysical Research Communications
|August 1, 2006
PubMed

Insights

Nuclear receptor coactivator RAC3 uses a bipartite nuclear localization signal (NLS) in its bHLH domain for nuclear import via importin alpha3. This nuclear translocation is essential for RAC3

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Nuclear receptor coactivators, such as RAC3 (also known as SRC-3/ACTR/AIB1/p/CIP/TRAM-1), are critical regulators of gene expression.
  • The p160 coactivator family plays roles in numerous physiological processes and diseases.
  • Understanding the mechanisms of coactivator localization is crucial for deciphering their function.

Purpose of the Study:

  • To investigate the mechanism of nuclear translocation of the nuclear receptor coactivator RAC3.
  • To identify the specific regions and interactions involved in RAC3's nuclear import.
  • To determine the functional significance of RAC3 nuclear localization.

Main Methods:

  • Site-directed mutagenesis of the RAC3 protein, specifically targeting the bHLH domain.
  • Nuclear localization signal (NLS) assays using enhanced green fluorescent protein (EGFP) fusions.
  • Co-immunoprecipitation to study protein-protein interactions between RAC3 and importin alpha3.
  • Functional assays measuring transcriptional enhancement activity of wild-type and mutant RAC3.

Main Results:

  • RAC3 nuclear translocation is mediated by a bipartite NLS located within its conserved bHLH domain.
  • Mutation of this bipartite NLS abolishes RAC3 nuclear localization.
  • The identified NLS is sufficient to direct EGFP to the nucleus, requiring specific basic amino acids.
  • RAC3 binds strongly to importin alpha3, dependent on these basic amino acids.
  • A cytoplasmic RAC3 mutant exhibits impaired transcriptional enhancement, highlighting the necessity of nuclear localization for function.

Conclusions:

  • A novel mechanism for nuclear translocation of p160 coactivators has been elucidated.
  • The conserved bHLH domain of RAC3 contains a critical bipartite NLS essential for nuclear import.
  • Nuclear import mediated by importin alpha3 is indispensable for the transcriptional coactivator function of RAC3.

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