Lipopolysaccharide-mediated Interferon Regulatory Factor Activation Involves TBK1-IKKϵ-dependent Lys63-linked Polyubiquitination and Phosphorylation of TANK/I-TRAF

Abstract

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKK⑀-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/I-TRAF is required for the TBK1-and IKK⑀-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of doublestranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKK⑀ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide-and TBK1-IKK⑀-mediated Lys 63 -linked polyubiquitination, a mechanism that does not require TBK1-IKK⑀ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKK⑀ complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys 63 -linked polyubiquitination of their scaffold protein, TANK.

MDM2 restrains estrogen-mediated AKT activation by promoting TBK1-dependent HPIP degradation

“…Human primary fibroblasts, RAW 264.7 and HEK293 cells were maintained in culture as described, 27,39,40 whereas ZR-75, MCF7 and MDA-MB-231 cells were cultured in RPMI and DMEM, respectively, and supplemented with 10% fetal calf serum and antibiotics, as were p53-deficient MCF7 cells. For E2 treatments (10 nM), control or p53-deficient MCF7 cells were first cultured for 48 h with DMEM without phenol red supplemented with Charcoal/Dextran-treated FBS (DCC) (Hyclone/Fisher, Waltham, MA, USA) followed by 24 h without serum.…”

“…27 Both TBK1-DC70-and -DC150 -Myc expression plasmids were generated by PCR using TBK1-Myc as a template. The HPIP-coding sequence was subcloned into the pCMV-XL5 expression plasmid (Origen Technologies, Rockville, MD, USA).…”

“…Kinase assays in transfected cells were carried out as described. 27,42 IPs involving ectopically expressed or endogenous proteins were carried out as described. 40,43 For the detection of endogenous polyubiquitinated forms of HPIP (Figure 4h), MCF7 cells were pretreated with MG132.…”

“…Among 47 TBK1-interacting clones, four encoded TANK, which was previously reported as a TBK1-associated protein. 27 Two clones encoded a product lacking the first 205 amino acids of HPIP, whereas a third clone encoded the C-terminal part of HPIP (amino acids 275-731) ( Figure 1a). Co-immunoprecipitation (IP) experiments confirmed the interaction between exogenously expressed epitope-tagged TBK1 and HPIP in HEK293 cells (Figure 1b; Supplementary Figures S1A and S1B, see our Supplementary Data Section).…”

“…Immunoprecipitated FLAG-HPIP was phosphorylated in TBK1-overexpressing cells, similarly to FLAG-TANK, a known TBK1 substrate (Figure 3a). 27 Such phosphorylation of HPIP relies on TBK1 kinase activity, as a kinase-dead version of TBK1 failed to phosphorylate HPIP. An HPIP mutant lacking the first 60 N-terminal amino acids (HPIPDN60) was still phosphorylated by TBK1 (Supplementary Figure S4A), whereas deletion of the first 150 amino acids abolished both interaction and phosphorylation (Supplementary Figure S4A).…”