SHARPIN Negatively Associates with TRAF2-Mediated NFκB Activation

NF kappa B is an inducible transcriptional factor controlled by two principal signaling cascades and plays pivotal roles in diverse physiological processes including inflammation, apoptosis, oncogenesis, immunity, and development. Activation of NF kappa B signaling was detected in skin of SHAPRIN-deficient mice and can be diminished by an NF kappa B inhibitor. However, in vitro studies demonstrated that SHARPIN activates NF kappa B signaling by forming a linear ubiquitin chain assembly complex with RNF31 (HOIP) and RBCK1 (HOIL1). The inconsistency between in vivo and in vitro findings about SHARPIN's function on NF kappa B activation could be partially due to SHARPIN's potential interactions with downstream molecules of NF kappa B pathway. In this study, 17 anti-flag immunoprecipitated proteins, including TRAF2, were identified by mass spectrum analysis among Sharpin-Flag transfected mouse fibroblasts, B lymphocytes, and BALB/c LN stroma 12 cells suggesting their interaction with SHARPIN. Interaction between SHARPIN and TRAF2 confirmed previous yeast two hybridization reports that SHARPIN was one TRAF2's partners. Furthermore, luciferase-based NF kappa B reporter assays demonstrated that SHARPIN negatively associates with NF kappa B activation, which can be partly compensated by over-expression of TRAF2. These data suggested that other than activating NF kappa B signaling by forming ubiquitin ligase complex with RNF31 and RBCK1, SHARPIN may also negatively associate with NF kappa B activation via interactions with other NF kappa B members, such as TRAF2.