Allosteric Regulation of the Ubiquitin:nik and Traf3 E3 Ligases by the Lymphotoxin- Receptor*

The Lymphotoxinreceptor (LT R) activates the NFB2 transcription factors, p100 and RelB, by regulating the NFB-inducing kinase (NIK). Constitutive proteosomal degradation of NIK limits NFB activation in unstimulated cells by the ubiquitin:NIK E3 ligase comprised of subunits TNFR-associated factors (TRAF)3, TRAF2 and cellular inhibitor of apoptosis (cIAP). However, the mechanism releasing NIK from constitutive degradation remains unclear. We found that insertion of a chargerepulsion mutation in the receptor-binding crevice of TRAF3 ablated binding of both LT R and NIK suggesting a common recognition site. A homologous mutation in TRAF2 inhibited cIAP interaction and blocked NIK degradation. Furthermore, the recruitment of TRAF3 and TRAF2 to the ligated LT R competitively displaced NIK from TRAF3. Ligated LT R complexed with TRAF3 and TRAF2 redirected the specificity of the ubiquitin ligase reaction to polyubiquitinate TRAF3 and TRAF2, leading to their proteosomal degradation. Stimulus-dependent degradation of TRAF3 required the RING domain of TRAF2, but not of TRAF3, implicating TRAF2 as a key E3 ligase in TRAF turnover. The combined action of competitive displacement of NIK and TRAF degradation halted NIK turnover, and promoted its association with IKK and signal transmission. These results indicate the LT R modifies the ubiquitin:NIK E3 ligase, and also acts as an allosteric regulator of the ubiquitin:TRAF E3 ligase. INTRODUCTION The lymphotoxinreceptor (LT R), a member of the TNF receptor superfamily (1), is a key regulator of lymphoid organogenesis and homeostasis of the immune system(2,3). Ligation of the LT R activates a serine kinase cascade involving the NFB inducing kinase (NIK; MAPKKK14) and the inhibitor of B kinase(IKK ; IKK1). Activation of kinase cascade culminates in the proteosome-dependent processing of p100 (NFB2), degrading the ankyrin inhibitory domain and releasing a 52 kDa fragment (p52) (4-6). The p52 fragment forms a heterodimer with RelB that controls transcription of numerous effector genes, such as tissue organizing chemokines (CCL21, CXCL13) and integrins (intercellular adhesion molecule, ICAM1) that recruit lymphoid cells into the developing lymphoid organ.