2). These data suggest that STUB1 is required for T-cell activation, and it plays a role in the upstream of TAK1 and the IKK signalsome in TCR-NF-κB signaling pathway. In order to determine the target of STUB1, we examined the association between STUB1 and main components involved in TCR signaling by Co-IP. The results showed that overexpressed STUB1 interacted with MALT1, TRAF6, TAK1, and IKK-α, but not with BCL10, IKK-β, or IKK-γ (Fig. 2A and Supporting Information Fig. 3). Interestingly, altering the expression level of STUB1 by RNAi-mediated knockdown did not affect

the total expression levels of Temozolomide solubility dmso all these signal proteins markedly, indicating that STUB1 catalyzes the target protein by a nonlytic ubiquitin modification (Supporting Information Fig. 4). The STUB1-associated molecules were then cotransfected with ubiquitin to check whether their ubiquitination status was affected by STUB1 or not. As shown in Fig. 2B, cotransfection

with STUB1 caused a mass of ubiquitination of CARMA1, and moderate ubiquitination of MALT1. In contrast, ubiquitination of other STUB1-associated proteins, such as TRAF6, TAK1, and IKK-α, were not markedly altered by STUB1 (Fig. 2C), suggesting that STUB1 specifically catalyzes the ubiquitination of CARMA1 and MALT1. We next challenged stable Jurkat E6 cells expressing STUB1-RNAi or controls Protein Tyrosine Kinase inhibitor with P/I, and performed Co-IP and immunoblot Chorioepithelioma to determine the effects of STUB1 on endogenous ubiquitination of CARMA1 and MALT1 upon stimulation. The results showed that the ubiquitination of CARMA1 in control cells was induced at the early phase by P/I stimulation, and it was significantly impaired in STUB1-knockdown cell (Fig. 2D), suggesting that STUB1 is essential for P/I-induced CARMA1 ubiquitination. In contrast, the ubiquitination of MALT1 by P/I stimulation was not markedly affected by STUB1-knockdown, and the basic level of MALT1 ubiquitination in STUB1-RNAi-transfected cells was higher than that in control cells (Fig. 2E). The above results suggest that STUB1 facilitates

TCR signaling by specifically catalyzing the ubiquitination of CARMA1. To examine how STUB1 affected the ubiquitination of CARMA1 in detail, we first determined the minimal regions of CARMA1 responsible for its interaction with STUB1 by Co-IP. A series of truncation mutation expression plasmids of CARMA1 was constructed and used. The results showed that the PDZ (aa 661-742) and SH3 (aa 766-834) domains of CARMA1 were responsible for the interaction with STUB1 (Fig. 3A). MAGUK region of CARMA1, containing PDZ, SH3, and GUK domains, not only functions in localizing and clustering multiprotein signaling complexes on the cell membrane, but also controls the ubiquitination of CARMA1 [6, 18]. We then mutated each of all seven lysine residues at the PDZ and SH3 domains, and performed ubiquitination assays.