DNA sequence determinants of nuclear protein binding to the c-Ha-ras antioxidant/electrophile response element in vascular smooth muscle cells: Identification of Nrf2 and heat shock protein 90β as heterocomplex components

Kimberly P. Miller, Kenneth S. Ramos

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

The antioxidant/electrophile response element (ARE/EpRE) is a cis-acting element involved in redox regulation of c-Ha-ras gene. Protein binding to the ARE/EpRE may be credited to deoxyribonucleic acid sequence; therefore, studies were conducted to evaluate the influence of internal and flanking regions to the 10-bp human c-Ha-ras ARE/EpRE core (hHaras10) on nuclear protein binding in oxidant-treated vascular smooth muscle cells. A protein doublet bound to an extended oligonucleotide comprising the ARE/EpRE core in genomic context (hHaras27), whereas a single complex bound to hHaras10. Protein binding involved specific interactions of 25- and 23-kDa proteins with hHaras10, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO, and rGSTA2 confirmed the specificity of deoxyribonucleic acid-protein interactions and indicated preferred binding of p25 and p23 to the c-Ha-ras ARE/EpRE. "NNN" sequences within the core afforded unique protein-binding profiles to the c-Ha-ras ARE/EpRE. In addition, Nrf2 and heat shock protein 90β (p80) were identified as components of the c-Ha-ras ARE/EpRE heterocomplex. We conclude that both internal bases and flanking sequences regulate nuclear protein recruitment and complex assembly on the c-Ha-ras ARE/EpRE.

Original languageEnglish (US)
Pages (from-to)114-125
Number of pages12
JournalCell Stress and Chaperones
Volume10
Issue number2
DOIs
StatePublished - Jun 2005

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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