Mechanism underlying IκB kinase activation mediated by the linear ubiquitin chain assembly complex.
The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.
Cell Biology and Metabolism Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
Hiroaki
H
Fujita
Simin
S
Rahighi
Mariko
M
Akita
Ryuichi
R
Kato
Yoshiteru
Y
Sasaki
Soichi
S
Wakatsuki
Kazuhiro
K
Iwai
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D051550:I-kappa B Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D025821:Polyubiquitin / Q000096:biosynthesis*; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; C514981:NEMO protein, mouse; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D025821:Polyubiquitin; C492590:Rnf31 protein, mouse; D044767:Ubiquitin-Protein Ligases; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse