SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis

Abstract

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

Figure 1. SHARPIN is a novel component of the LUBAC complex

a, Schematic representation of the domain architecture of SHARPIN (A), HOIL-1L (B), and HOIP (C). b, Quantitative assessment of the binding of SHARPIN, HOIL-1L, and HOIP to a panel of ubiquitin species using isothermal titration calorimetry (ITC). The obtained equilibrium dissociation constants of individual measurements are listed. c, Formation of a trimeric complex between SHARPIN, HOIL-1L, and HOIP in HEK293T cells transfected with Myc- HOIP, HA-HOIL-1L, and GFP-SHARPIN or d, in MEFs. Lysates were immunoprecipitated and analyzed by Western blot.

Figure 2. SHARPIN and HOIP form a novel LUBAC complex with the ability to induce linear ubiquitylation and NF-κB activation

a, In vitro linear ubiquitin chain synthesis by purified SHARPIN and HOIP. b, Stimulation of linear ubiquitylation by SHARPIN and HOIP in vivo. Immobilised GST-ABIN-1-UBAN domain was used to detect linear ubiquitylation of proteins. c, MS/MS spectrum of the prototypic linear ubiquitin peptide present in vivo. d, e, SILAC experiments comparing relative levels of linear ubiquitin on immunoprecipitated NEMO induced by HOIP and SHARPIN (d) or HOIP-mut (RING domain)-SHARPIN-mut( TF_LV)(Lys8,Arg10) (e). f, g, Stimulation of NF-κB transcriptional activity by SHARPIN and HOIP in vivo. NF-κB-luciferase assays using the indicated combinations of SHARPIN, HOIL-1L, and HOIP were performed. Results are shown as means and s.e.m. (n=4). *P < 0.0001, determined by the Student's t-test

Figure 3. SHARPIN and HOIL-1L are essential for full activation of IKK and NF-κB

a, In vitro IKK-kinase activity in TNFα-treated Sharpin^cpdm/Sharpin^cpdm MEFs stably transduced with non-targeting (control) or HOIL-1L shRNA. b, c, Defect of TNFα-induced IκBα degradation and translocation of p65 in Sharpin^cpdm/Sharpin^cpdm MEFs stably transduced with HOIL-1L shRNA. (n = 19–23) d, e, Impaired NF-κB activation in primary B cells or macrophages of Sharpin^cpdm/Sharpin^cpdm stimulated with CD40L or LPS. e, f, g, Reduced capacity of Sharpin^cpdm/Sharpin^cpdm macrophages to secrete TNFα and MCP-1 in response to LPS. TNFα ELISA; n=5, MCP-1 ELISA; n=4. Results in c, f and g are shown as means and s.e.m. *P < 0.0001, determined by the Student's t-test.

Figure 4. Loss of SHARPIN sensitizes cells to FADD- and caspase-8-mediated apoptosis

a, Increased susceptibility to TNFα-induced apoptosis in SHARPIN deficient MEFs. Cell detachment and death were measured continuously in real-time using an impedance based real-time cell analyzer (RTCA). b, Increased caspase activation by TNFα and CHX in SHARPIN deficient MEFs. c, d, Cell death caused by loss of SHARPIN via FADD and caspase-8 dependent apoptotic signalling. MEFs were retrovirally transduced with indicated proteins and treated with TNFα. e, Increased apoptotic cell death in the skin of Sharpin^cpdm/Sharpin^cpdm mice. Normal epidermis (A) and Sharpin^cpdm/Sharpin^cpdm (B) skin samples were immunostained for caspase-8 (C), caspase-9 (D) and caspase-3 (E). None of these markers were present in control mouse epidermis (F). H&E; hematoxylin and eosin. Bar = 2 μm. Results in a and c are shown as means and s.e.m. (n=3).