The Scaffold Protein GIT2 Inhibits TRAF6 Ubiquitination To Repress The NF-κB Signaling Pathway The Ubiquitin Ligase TRAF6 Activates The JAK-STAT Signaling Pathway By Binding To STAT3 And Mediating Its Ubiquitination

Transcription factors NF-κB have important and evolutionarily conserved roles to regulate the expression of many genes involved in innate and adaptive immune response, cell survival, differentiation and proliferation. TNF receptor-associated factor 6 (TRAF6) is an ubiquitin ligase that regulates innate immune responses in interleukin-1 receptors (IL-1Rs)/Toll-like receptors (TLRs) signaling. The tumor suppressor cylindromatosis (CYLD) is a deubiquitylating enzyme that negatively regulates NF-κB pathway by removing lysine 63-linked polyubiquitin chains, including TRAF2, TRAF6 and NEMO. But the molecular mechanisms of CYLD in deubiquiting TRAF6 remaims to be further investigated.The GIT family proteins are ubiquitous multidomain proteins involved in diverse cellular functions, which include receptor endocytosis and cell motility. GITs regulate membrane trafficking between the plasma membrane and endosomes through their ARF GTPase-activating protein (ARF-GAP) activity. Furthermore, the SHD domain of GIT proteins binds to many molecules, such as MEK1 and PIX, to mediate ERK1/2 activation. Gene deletion of GIT2 leads to an immunodeficient state. But, whether GIT2 directly regulates NF-κB pathway and mechanmisms remain elusive.Additionly, we found the interaction of TRAF6 and STAT3. STAT3 is a key transcription factor that controls various cellular processes, such as cell growth, apoptosis, immune response and cancer. However, the molecular mechanisms of STAT3 activation by many cytokines, such as IL6, remain poorly understood.Here we report that G protein-coupled receptor kinase-interactor 2 (GIT2) interacted with TRAF6 and CYLD. Overexpression of GIT2 in HEK293 cells enhanced the association of CYLD and TRAF6. Thus, GIT2 appears to function as an adaptor that recruiting CYLD to TRAF6. Furthermore, GIT2 enhanced the deubquitination of CYLD to TRAF6 and blocked the NF-κB signaling pathway. As shown in GIT2-/- cells and mice, GIT2 inhibits the production of cytokins, includes IL-6 and TNFα.On the other hand, we identified TRAF6 as a new STAT3 interactor. TRAF6 augmented the ubiquitination of STAT3 and enhanced its transcriptional activity. Moreover, TRAF6 coordinated with Src kinase and IL-6 to enhance the activity of STAT3. Both the RING domain and the TRAF-type zinc finger domain of TRAF6 were indispensable for STAT3 activation. Furthermore, TRAF6 up-regulated the expression of several known STAT3 target genes, MCL1, and Bcl2.