title: Mechanism of endogenous regulation of the type I interferon response by suppressor of IκB kinase epsilon (SIKE), a novel substrate of TANK-binding kinase 1 (TBK1).
abstract: TANK-binding kinase 1 (TBK1) serves as a key convergence point in multiple innate immune signaling pathways. In response to receptor-mediated pathogen detection, TBK1 phosphorylation promotes production of pro-inflammatory cytokines and type I interferons. Increasingly, TBK1 dysregulation has been linked to autoimmune disorders and cancers, heightening the need to understand the regulatory controls of TBK1 activity. Here, we describe the mechanism by which suppressor of IKKε (SIKE) inhibits TBK1-mediated phosphorylation of interferon regulatory factor 3 (IRF3), which is essential to type I interferon production. Kinetic analyses showed that SIKE not only inhibits IRF3 phosphorylation but is also a high affinity TBK1 substrate. With respect to IRF3 phosphorylation, SIKE functioned as a mixed-type inhibitor (K(i, app) = 350 nM) rather than, given its status as a TBK1 substrate, as a competitive inhibitor. TBK1 phosphorylation of IRF3 and SIKE displayed negative cooperativity. Both substrates shared a similar Km value at low substrate concentrations (∼50 nM) but deviated >8-fold at higher substrate concentrations (IRF3 = 3.5 μM; SIKE = 0.4 μM). TBK1-SIKE interactions were modulated by SIKE phosphorylation, clustered in the C-terminal portion of SIKE (Ser-133, -185, -187, -188, -190, and -198). These sites exhibited striking homology to the phosphorylation motif of IRF3. Mutagenic probing revealed that phosphorylation of Ser-185 controlled TBK1-SIKE interactions. Taken together, our studies demonstrate for the first time that SIKE functions as a TBK1 substrate and inhibits TBK1-mediated IRF3 phosphorylation by forming a high affinity TBK1-SIKE complex. These findings provide key insights into the endogenous control of a critical catalytic hub that is achieved not by direct repression of activity but by redirection of catalysis through substrate affinity.
authors: Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
authors: James D
authors: JD
authors: Marion
authors: Charlotte F
authors: CF
authors: Roberts
authors: R Jason
authors: RJ
authors: Call
authors: Jonathan L
authors: JL
authors: Forbes
authors: Kristina T
authors: KT
authors: Nelson
authors: J Ellis
authors: JE
authors: Bell
authors: Jessica K
authors: JK
authors: Bell
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism*; D007370:Interferon Type I / Q000378:metabolism*; D016898:Interferon-alpha / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D050838:Interferon Regulatory Factor-3; D007370:Interferon Type I; D016898:Interferon-alpha; D047908:Intracellular Signaling Peptides and Proteins; C583271:SIKE1 protein, human; D012694:Serine; D016899:Interferon-beta; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human
keywords: Cooperativity; Enzyme Inhibitors; Enzyme Kinetics; Enzyme Mechanisms; Innate Immunity
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