Oxidative stress-induced ubiquitination of RCAN1 mediated by SCFβ-TrCP ubiquitin ligase

Abstract

A change in the protein level of RCAN1 (DSCR1/MCIP/Adapt78/CSP1) has been implicated in oxidative stress-induced cell death in neurons and in the pathogenesis of Alzheimer's disease. The pathogenic processes in neuro-degenerative diseases are closely related to oxidative stress and the ubiquitin proteasome system (UPS). Therefore, we investigated whether oxidative stress induces a change in the protein level of RCAN1 through the UPS. H2O2 induced ubiquitination of RCAN1 at the same concentrations as those causing a decrease in RCAN1 in HEK293T cells. β-TrCP, the F-box protein component of SCF ubiquitin ligase, interacted with RCAN1 in response to H2O2 stimulation. Although FBW4, another F-box protein, interacted with RCAN1, its interaction was independent of H2O2 stimulation. In vitro ubiquitination assay showed that SCFβ-TrCP but not SCFFBW4 increased ubiquitination of RCAN1, dependent on H2O2 stimulation. In addition, knockdown of β-TrCP by siRNA abolished the H2O2-induced decrease in RCAN1 in HEK293T cells. We further examined whether RCAN1 undergoes ubiquitination by H2O2 in primary neurons, similarly to that in HEK293T cells. An H2O2-induced decrease in RCAN1 was exhibited also in hippocampal and cortical neurons. Ubiquitination of RCAN1 was induced by 500 μM H2O2, the concentration at which H2O2 induced a decrease in RCAN1 in primary neurons. These results suggest that H2O2 induces SCFβ-TrCP-mediated ubiquitination of RCAN1, leading to a decrease in the protein level of RCAN1.