YO-04 - Full basal transactivation activity of FOXO1 depends on its Cys612

Abstract

FOXO transcription factors regulate several aspects of the cellular stress response and antioxidant defence. It has been known for some time that FOXO cysteine residues are essential for oxidative stress-induced interactions with coregulators such as CBP/p300, which reversibly interacts with FOXO4 upon exposure to H2O2. Here, we investigated whether cysteine residues are essential for FOXO1 activity under basal conditions. To this end, we generated epitope-tagged cysteine-deficient mutants of human FOXO1 (all 7 cysteines were replaced by serines). Cysteine deficiency did not impair FOXO1 DNA binding, as determined using EMSAs, nor was nucleocytoplasmic distribution and shuttling affected in HEK293 or HepG2 cells overexpressing WT or Cys-deficient FOXO1. In contrast, stimulation of FOXO-responsive promoter constructs by overexpression of FOXO1 was far less prominent in cells overexpressing Cys-deficient FOXO1 as compared to those transfected with WT-FOXO1. Similarly, FOXO1 overexpression-induced increases in FOXO target gene mRNA levels was impaired in Cys-deficient FOXO1 relative to WT-FOXO1. A series of single-Cys mutation experiments led us to conclude that, of all 7 cysteines, only C612 is required for full FOXO1 transactivation activity under basal, non-stressed conditions. The exact role of C612 in controlling FOXO1 activity remains to be established.