Clathrin Complexes With The Inhibitor Kappa B Kinase Signalosome: Imaging The Interactome

Many receptors involved with innate immunity activate the inhibitor kappa B kinase signalosome (IKK). The active complex appears to be assembled from the two kinase units, IKK alpha and IKK beta with the regulatory protein NEMO. Because we previously found that RNA silencing of clathrin heavy chains (CHC), in transformed human lung pneumocytes (A549), decreased TNF alpha-induced signaling and phosphorylation of inhibitor kappa B (I kappa B), we hypothesized that CHC forms cytoplasmic complexes with members of the IKK signalosome. Widely available antibodies were used to immunoprecipitate IKK alpha and NEMO interactomes. Analysis of the affinity interactomes by mass spectrometry detected clathrin with both baits with high confidence. Using the same antibodies for indirect digital immunofluorescence microscopy and FRET, the CHC-IKK complexes were visualized together with NEMO or HSP90. The natural variability of protein amounts in unsynchronized A549 cells was used to obtain statistical correlation for several complexes, at natural levels and without invasive labeling. Analyses of voxel numbers indicated that: (i) CHC-IKK complexes are not part of the IKK signalosome itself but, likely, precursors of IKK-NEMO complexes. (ii) CHC-IKK beta complexes may arise from IKK beta-HSP90 complexes.