Molecular mechanism of inhibitor of kappa B kinase (IKK) complex activation by inducible phosphorylation and ubiquitination upon T cell receptor (TCR) stimulation
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IκB kinase (IKK) complex serves as the master regulator for the activation of NF-κB by various stimuli. It contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKα/β at its activation loop, and the K63-linked polyubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. This study provides the first genetic evidence establishing that upon antigen receptor (AgR) stimulation, IKK phosphorylation is independent of CARMA1 and BCL10. We provided evidence that NEMO is required for IKK phosphorylation. Our data thus suggests that IKK phosphorylation is mediated by a kinase that functions independently of the CARMA1-BCL10-MALT1 (CBM) complex and the catalytic activity of IKK depends on a CBM mediated signaling event. We have provided the first genetic evidence that CARMA1 is essential for NEMO polyubiquitination. CARMA1 induces K-63 linked polyubiquitination of NEMO and assists in the interaction of BCL10 and NEMO. The Lys 399 residue and the zinc finger domain of NEMO are essential for NEMO ubiquitination and NF-κB activation. Phosphorylation of the IKK is independent of NEMO ubiquitination. Further, the sub-cellular localization of CARMA1 is critical for NEMO polyubiquitination. We demonstrated that CARMA1 recruits NEMO to the lipid rafts, where NEMO ubiquitination may occur. This study provides evidence that suggest the involvement of TAK1 in AgR induced NF-κB activation. Using a TAK1 specific inhibitor or TAK1 deficient Chicken B cells we showed that TAK1 regulates IKK phosphorylation and NF-κB activation. We showed that TAK1 functions as an upstream kinase for IKK in a PKC dependent, but CARMA1 independent manner. TAK1 is recruited to the immunological synapse in a CARMA1 and BCL10 independent manner. Thus, we have evaluated the roles and requirements of IKK phosphorylation and polyubiquitination. We have demonstrated that these events are independently regulated and are essential for IKK activation. This study proposes a working model for AgR mediated NF-κB activation, which depicts that the CARMA1-BCL10-MALT1 complex regulates NEMO polyubiquitination, whereas PKC-TAK1 regulates IKK phosphorylation. Thus, phosphorylation and ubiquitination of the IKK complex are controlled by two distinct signaling pathways.