The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1.

The protein kinases IRAK [IL-1 (interleukin 1) receptor-associated kinase] 1 and 4 play key roles in a signalling pathway by which bacterial infection or IL-1 trigger the production of inflammatory mediators. In the present study, we demonstrate that IRAK1 and IRAK4 phosphorylate Pellino isoforms in vitro and that phosphorylation greatly enhances Pellino's E3 ubiquitin ligase activity. We show that, in vitro, Pellino 1 can combine with the E2 conjugating complex Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) to catalyse the formation of K63-pUb (Lys63-linked polyubiquitin) chains, with UbcH3 to catalyse the formation of K48-pUb chains and with UbcH4, UbcH5a or UbcH5b to catalyse the formation of pUb-chains linked mainly via Lys11 and Lys48 of ubiquitin. In IRAK1-/- cells, the co-transfection of DNA encoding wild-type IRAK1 and Pellino 2, but not inactive mutants of these proteins, induces the formation of K63-pUb-IRAK1 and its interaction with the NEMO [NF-kappaB (nuclear factor kappaB) essential modifier] regulatory subunit of the IKK (inhibitor of NF-kappaB kinase) complex, a K63-pUb-binding protein. These studies suggest that Pellino isoforms may be the E3 ubiquitin ligases that mediate the IL-1-stimulated formation of K63-pUb-IRAK1 in cells, which may contribute to the activation of IKKbeta and the transcription factor NF-kappaB, as well as other signalling pathways dependent on IRAK1/4.

[1]  Navin L Rao,et al.  IRAK1: a critical signaling mediator of innate immunity. , 2008, Cellular signalling.

[2]  A. Bowie,et al.  IRAK-2 Participates in Multiple Toll-like Receptor Signaling Pathways to NFκB via Activation of TRAF6 Ubiquitination* , 2007, Journal of Biological Chemistry.

[3]  P. Moynagh,et al.  Kinase-active Interleukin-1 Receptor-associated Kinases Promote Polyubiquitination and Degradation of the Pellino Family , 2007, Journal of Biological Chemistry.

[4]  S. Akira,et al.  Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor–mediated immune responses but not in TCR signaling , 2007, The Journal of experimental medicine.

[5]  H. Gram,et al.  IRAK-4 Kinase Activity Is Required for Interleukin-1 (IL-1) Receptor- and Toll-like Receptor 7-mediated Signaling and Gene Expression* , 2007, Journal of Biological Chemistry.

[6]  S. Janssens,et al.  Pellino Proteins: Novel Players in TLR and IL-1R Signalling , 2007, Journal of cellular and molecular medicine.

[7]  N. Walker,et al.  Crystal structures of IRAK-4 kinase in complex with inhibitors: a serine/threonine kinase with tyrosine as a gatekeeper. , 2006, Structure.

[8]  S. Park,et al.  Smad6 negatively regulates interleukin 1-receptor–Toll-like receptor signaling through direct interaction with the adaptor Pellino-1 , 2006, Nature Immunology.

[9]  S. Janssens,et al.  Pellino proteins are more than scaffold proteins in TLR/IL‐1R signalling: A role as novel RING E3–ubiquitin‐ligases , 2006, FEBS letters.

[10]  Gabriel Pineda,et al.  Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. , 2006, Molecular cell.

[11]  S. Srinivasula,et al.  Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-κB activation , 2006, Nature Cell Biology.

[12]  S. Srinivasula,et al.  Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected]. , 2006, Nature cell biology.

[13]  P. Cohen,et al.  Chaperoned ubiquitylation--crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex. , 2005, Molecular cell.

[14]  S. Akira,et al.  Essential function for the kinase TAK1 in innate and adaptive immune responses , 2005, Nature Immunology.

[15]  Zhijian J. Chen,et al.  TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. , 2004, Molecular cell.

[16]  D. Campbell,et al.  14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking. , 2004, The Biochemical journal.

[17]  P. Cao,et al.  Sequential Autophosphorylation Steps in the Interleukin-1 Receptor-associated Kinase-1 Regulate its Availability as an Adapter in Interleukin-1 Signaling* , 2004, Journal of Biological Chemistry.

[18]  A. Whitehead,et al.  Pellino3, a Novel Member of the Pellino Protein Family, Promotes Activation of c-Jun and Elk-1 and May Act as a Scaffolding Protein , 2003, The Journal of Immunology.

[19]  C. Kollewe,et al.  Characterization of Pellino2, a substrate of IRAK1 and IRAK4 , 2003, FEBS letters.

[20]  Zhengfan Jiang,et al.  Pellino 1 Is Required for Interleukin-1 (IL-1)-mediated Signaling through Its Interaction with the IL-1 Receptor-associated Kinase 4 (IRAK4)-IRAK-Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Complex* , 2003, The Journal of Biological Chemistry.

[21]  Zhengfan Jiang,et al.  Interleukin-1 (IL-1) Receptor-Associated Kinase-Dependent IL-1-Induced Signaling Complexes Phosphorylate TAK1 and TAB2 at the Plasma Membrane and Activate TAK1 in the Cytosol , 2002, Molecular and Cellular Biology.

[22]  T. Mak,et al.  Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4 , 2002, Nature.

[23]  Holger Wesche,et al.  IRAK-4: A novel member of the IRAK family with the properties of an IRAK-kinase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Y. Durocher,et al.  High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. , 2002, Nucleic acids research.

[25]  Zhijian J. Chen,et al.  TAK1 is a ubiquitin-dependent kinase of MKK and IKK , 2001, Nature.

[26]  G. Stark,et al.  IL-1-induced NFκB and c-Jun N-terminal kinase (JNK) activation diverge at IL-1 receptor-associated kinase (IRAK) , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[27]  H. Jockusch,et al.  Assignment1 of homologous genes, Peli1/PELI1 and Peli2/PELI2, for the Pelle adaptor protein Pellino to mouse chromosomes 11 and 14 and human chromosomes 2p13.3 and 14q21, respectively, by physical and radiation hybrid mapping , 2001, Cytogenetic and Genome Research.

[28]  H. Jockusch,et al.  Assignment of homologous genes, Peli1/PELI1 and Peli2/PELI2, for the Pelle adaptor protein Pellino to mouse chromosomes 11 and 14 and human chromosomes 2p13.3 and 14q21, respectively, by physical and radiation hybrid mapping. , 2001, Cytogenetics and cell genetics.

[29]  Jongdae Lee,et al.  TAK1 regulates multiple protein kinase cascades activated by bacterial lipopolysaccharide , 2000, Journal of leukocyte biology.

[30]  J. Trowsdale,et al.  Pellino-related sequences from Caenorhabditis elegans and Homo sapiens , 2000, Immunogenetics.

[31]  Zhijian J. Chen,et al.  Activation of the IκB Kinase Complex by TRAF6 Requires a Dimeric Ubiquitin-Conjugating Enzyme Complex and a Unique Polyubiquitin Chain , 2000, Cell.

[32]  S. Wasserman,et al.  Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase. , 1999, Journal of immunology.

[33]  C. Nüsslein-Volhard,et al.  Oligomerisation of Tube and Pelle leads to nuclear localisation of Dorsal , 1999, Mechanisms of Development.

[34]  Crafford A. Harris,et al.  Interleukin (IL)-1 Receptor–associated Kinase (IRAK) Requirement for Optimal Induction of Multiple IL-1 Signaling Pathways and IL-6 Production , 1998, The Journal of experimental medicine.

[35]  Z. Cao,et al.  MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. , 1997, Immunity.

[36]  P. Feng,et al.  IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. , 1997, Science.

[37]  Douglas K. Miller,et al.  The Interleukin-1 Receptor-associated Kinase Is Degraded by Proteasomes following Its Phosphorylation* , 1997, The Journal of Biological Chemistry.

[38]  Zhaodan Cao,et al.  TRAF6 is a signal transducer for interleukin-1 , 1996, Nature.

[39]  R. Brigelius-Flohé,et al.  Interleukin‐1‐induced activation of a protein kinase co‐precipitating with the type I interleukin‐1 receptor in T cells , 1994, European journal of immunology.