Activation of the heterodimeric IkappaB kinase alpha (IKKalpha)-IKKbeta complex is directional: IKKalpha regulates IKKbeta under both basal and stimulated conditions.
Signal-induced nuclear expression of the eukaryotic NF-kappaB transcription factor involves the stimulatory action of select mitogen-activated protein kinase kinase kinases on the IkappaB kinases (IKKalpha and IKKbeta) which reside in a macromolecular signaling complex termed the signalsome. While genetic studies indicate that IKKbeta is the principal kinase involved in proinflammatory cytokine-induced IkappaB phosphorylation, the function of the equivalently expressed IKKalpha is less clear. Here we demonstrate that assembly of IKKalpha with IKKbeta in the heterodimeric signalsome serves two important functions: (i) in unstimulated cells, IKKalpha inhibits the constitutive IkappaB kinase activity of IKKbeta; (ii) in activated cells, IKKalpha kinase activity is required for the induction of IKKbeta. The introduction of kinase-inactive IKKalpha, activation loop mutants of IKKalpha, or IKKalpha antisense RNA into 293 or HeLa cells blocks NIK (NF-kappaB-inducing kinase)-induced phosphorylation of the IKKbeta activation loop occurring in functional signalsomes. In contrast, catalytically inactive mutants of IKKbeta do not block NIK-mediated phosphorylation of IKKalpha in these macromolecular signaling complexes. This requirement for kinase-proficient IKKalpha to activate IKKbeta in heterodimeric IKK signalsomes is also observed with other NF-kappaB inducers, including tumor necrosis factor alpha, human T-cell leukemia virus type 1 Tax, Cot, and MEKK1. Conversely, the theta isoform of protein kinase C, which also induces NF-kappaB/Rel, directly targets IKKbeta for phosphorylation and activation, possibly acting through homodimeric IKKbeta complexes. Together, our findings indicate that activation of the heterodimeric IKK complex by a variety of different inducers proceeds in a directional manner and is dependent on the kinase activity of IKKalpha to activate IKKbeta.
O'Mahony
A
A
Gladstone Institute of Virology and Immunology, Microbiology and Immunology, University of California, San Francisco, California 94141, USA.
Lin
X
X
Geleziunas
R
R
Greene
W C
WC
D002460:Cell Line; D019281:Dimerization; D004789:Enzyme Activation; D016356:Gene Products, tax / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016356:Gene Products, tax; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; C482576:MAP3K1 protein, human; C069937:MAP3K8 protein, human