Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains.

Polyubiquitin (pub) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical 1KB kinase (IKK) complex. Here, we demonstrate that nearly all of the Mi-pub chains formed in response to interleukin-1, or the Toll-Like Receptors 1/2 agonist Pam3CSK4, are covalently attached to K63-pUb chains either directly as K63-pub/M1-pub hybrids or indirectly by attachment to the same protein. lnterleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pub chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pub chains. We show that the heme-oxidized IRPZ ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pub chains in response to interleukin-1, that the formation of K63-pub chains is a prerequisite for the formation of Mi-pub chains, and that HOIP interacts with K63-pUb but not M1-pub linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGFbeta-activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFKB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pub chains and M1-pub chains, respectively. The formation of K63/M1pub hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pub chain, facilitating the TAK1-catalyzed activation of IKKa and IKKII. Our study may help to resolve the debate about the relative importance of K63pub and M1-pub chains in activating the canonical IKK complex. [ABSTRACT FROM AUTHOR]