Regulation Of TCR Mediated NF-κB Activation By STUB1

The response of lymphocyte to foreign antigen is the critical process for adaptive immune response. In T cell, TCR recognizes the antigen presented by APC and trigger NF-κB signaling, which is critical for T cell proliferation, cytokines production and initiating the adaptive immune response. Upon the TCR stimulation, CARMA1, which plays a pivotal role in TCR mediated NF-κB activation, assembles with BCL10-MALT1to form a signalosome to recruit downstream molecules and transduce the signal. Therefore, the regulation of CARMA1activity was extensively investigated in the past decade. It has demonstrated that post-translational modifications, such as phosphorylation, play essential roles in the regulation of CARMA1activity.By affinity purification and mass spectrometry analyze, we identified STUB1, a U-box containing E3ubiquitin ligase, as a specifically regulator of CARMA1by ubiquitination. Overexpressed STUB1interacted with CARMA1, and the PDZ and/or SH3domain of CARMA1were responsible for the interaction with STUB1, while the linker and U-box domain of STUB1were essential for its interaction with CARMA1. In the resting Jurkat T cell, STUB1constitutively associated with CARMA1, while after stimulation, their interaction increased in the early phage and then reduced, indicating this association was controlled by the stimulation. By RNAi mediated silencing of STUB1, PMA/Ionomycin (P/I) stimulation or CD3-CD28cross-linking induced NF-κB activation were markedly downregulated, accompany with reduced production of IL-2. However, TNFa triggered NF-κB activation was not affected. These results suggested that STUB1, as a binding protein of CARMA1, was specifically involved in TCR mediated NF-κB activation and IL-2production. Furthermore, overexpressed STUB1markedly enhanced the ubiquitination of CARMA1, but not other STUB1-associated proteins. Conversely, knockdown of STUB1blocked the ubiquitination of CARMA1induced by P/I stimulation. Subsequently, the ubiquitination of CARMA1catalyzed by STUB1was identified as a mainly Lys-27linked type, which was important for C ARM A1-mediated NF-κB activation. Taken together, these results suggested that STUB1specifically catalyze the Lys27-linked poly-ubiquitination of CARMA1to promote NF-κB activation upon TCR stimulation.In summary, we identified a U-box containing E3ubiquitin ligase STUB1as a binding partner of CARMA1. Upon TCR stimulation, STUB1catalyzes the Lys27-linked poly-ubiquitination of CARMA1, and promotes TCR induced canonical NF-κB activation and subsequent IL-2production in T cells. Different from previous study that STUB1catalyzed the substrate for ubiquitination-dependent degradation, our study evidenced that STUB1mediated ubiquitination of CARMA1could enhance its activity. Besides, MAGUK region of CARMA1was reported to control Lys48-linked proteolytical ubiquitination of CARMA1, whereas we found the ubiquitination on PDZ-SH3domain can enhance CARMA1activity. Our findings not only broaden our recognition of STUB1functions, but also provide new insight into the mechanism responsible for control of aberrant T-cell activation.