title: Clathrin complexes with the inhibitor kappa B kinase signalosome: imaging the interactome.
abstract: 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, IKKα and IKKβ with the regulatory protein NEMO. Because we previously found that RNA silencing of clathrin heavy chains (CHC), in transformed human lung pneumocytes (A549), decreased TNFα-induced signaling and phosphorylation of inhibitor kappa B (IκB), we hypothesized that CHC forms cytoplasmic complexes with members of the IKK signalosome. Widely available antibodies were used to immunoprecipitate IKKα 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-IKKβ complexes may arise from IKKβ-HSP90 complexes.
authors.affiliation: Department of Surgery, University of Colorado Denver, Aurora, Colorado.
authors.forename: Fabia
authors.initials: F
authors.lastname: Gamboni
authors.affiliation: Department of Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Escobar
authors.affiliation: Department of Surgery, University of Colorado Denver, Aurora, Colorado Department of Surgery, Denver Health Medical Center, Denver, Colorado.
authors.forename: Ernest E
authors.initials: EE
authors.lastname: Moore
authors.affiliation: UC Denver Mass Spectrometry and Proteomics Facility, Aurora, Colorado.
authors.forename: Monika
authors.initials: M
authors.lastname: Dzieciatkowska
authors.affiliation: Department of Biochemistry and Molecular Genetics, UC Denver Mass Spectrometry and Proteomics Facility, Aurora, Colorado.
authors.forename: Kirk C
authors.initials: KC
authors.lastname: Hansen
authors.affiliation: Department of Surgery, University of Colorado Denver, Aurora, Colorado.
authors.forename: Sanchayita
authors.initials: S
authors.lastname: Mitra
authors.affiliation: Department of Surgery, University of Colorado Denver, Aurora, Colorado.
authors.forename: Trevor A
authors.initials: TA
authors.lastname: Nydam
authors.affiliation: Department of Surgery, University of Colorado Denver, Aurora, Colorado Belle Bonfils Blood Center, Denver, Colorado.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Silliman
authors.affiliation: Department of Surgery, University of Colorado Denver, Aurora, Colorado.
authors.forename: Anirban
authors.initials: A
authors.lastname: Banerjee
keywords: Affinity proteomics; Hsp90; NEMO; fluorescent resonance energy transfer