NEMO is rapidly recruited to the TCR via polyubiquitin chain recognition and is dissociated via IKKβ kinase activity 2310337

Abstract

Abstract Introduction The IκB kinase (IKK) complex is best known for its roles in cell survival and in the activation of canonical NFκB isoforms. The complex contains two kinase subunits (IKKα/β) and a dimeric polyubiquitin-binding adaptor subunit known as the NFκB essential modulator protein (NEMO) [IKBKG]. While oversimplified models often place the IKK complex entirely downstream of the adaptor protein Carma1, T cell receptor (TCR)-induced IKKβ phosphorylation is Carma1-independent. Thus, IKK complexes may play unconventional roles downstream of the TCR, but upstream of Carma1. Methods Here, we examine how IKK complexes participate in TCR proximal signals via dynamic imaging analyses of fluorescent NEMO chimeras expressed in Jurkat T cells and primary human T cell blasts. Results We observe that NEMO enters TCR- and ZAP-70-containing microclusters within ∼70 seconds of their formation, via processes that rely on the activities of Lck and ZAP-70, and that are independent of downstream effectors implicated in canonical NFκB activation. The mutational inactivation of either the K63 or the linear polyubiquitin-binding sites in NEMO, the pharmacological inhibition the K63-chain forming E2 ligase Ubc13, or the inhibition of the linear polyubiquitin forming LUBAC complex all prevent the recruitment of NEMO to the TCR. If NEMO is co-expressed with wild-type IKKβ, the co-clustering of NEMO with the TCR is largely eliminated. However, kinase dead IKKβ co-clusters with NEMO at the TCR, and IKKβ inhibitors shift wild-type IKK complexes into TCR microclusters. Conclusion These data suggest that the IKK complex is rapidly recruited to the TCR via multipoint interactions with ubiquitin polymers assembled within ∼1 minute of ZAP-70 recruitment, whereas the activity of the IKK complex releases NEMO from the TCR. These findings define a window within which partially activated IKK complexes may influence TCR-proximal signaling events upstream of Carma1. Funding Source NIH R01 AI103022 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)