C-mip displays E3 ligase activity and targets WT1 to proteasome dégradation

Abstract

Introduction We show here, overproduction of C-mip was consistenly associated with a downregulation of WT1. C-mip interacts physically with WT1, and exhibits an E3 ligase activity, which targets WT1 to proteasome degradation. Description Overexpression of C-mip in M15 cell line induced a downregulation of WT1, which was prevented by lactacystin. We showed that C-mip exhibits an E3 ligase activity, which targets WT1 to proteasome degradation. Intravenous injection of c-mip-siRNA specifically prevented the repression of WT1 in LPS-induced proteinuria in mice. Methods (1) Transfected C-mip plasmid to M15 cell line, which expresses a high level of WT1, to see whether C-mip down-regulates the expression of WT1. (2) Cotransfected WT1 and C-mip expression plasmids into HEK cells, then the cells were incubated with lactacystin. (3) E3 ubiquitin ligase kit was used to determine whether C-mip could have an E3 ligase activity that drives ubiquitylation of WT1. (4) Knockdown of c-mip in vivo by RNA interference in LPS-treated mice. Results (1) WT1 abundance fell by 50% at 12 hours after transfection of C-mip plasmid to M15 cell line, shows C-mip destabilizes the WT1. (2) The abundance of WT1 protein was significantly reduced in the presence of C-mip, but coincubation with lactacystin inhibited WT1 decay, suggesting that C-mip targets WT1 to proteasome mediated degradation. (3) E3 ligase activity assay showed, C-mip stimulated the formation of polyubiquitin chains in a dose dependent manner, suggesting that C-mip exhibits an intrinsic ubiquitin E3 ligase activity. (4) In vivo c-mip siRNA treatment indicated knockdown of c-mip restores the expression of WT1 in LPS-treated mice. Conclusion (i) In vivo, in vitro overproduction of C-mip is associated with a downregulation of WT1; (ii) C-mip interacts directly with WT1 and displays E3 ligase activity, which promotes WT1 to proteasome-mediated degradation; (iii) Silencing endogenous c-mip expression with RNAi prevents the downregulation of WT1 in LPS-treated mice.