The Crohn's disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO.
BACKGROUND
Crohn's disease is an autoimmune inflammatory disorder of the gastrointestinal tract and is characterized clinically by dysregulation of both pro-inflammatory and anti-inflammatory cytokine signaling networks. The function of the Crohn's disease protein, NOD2, highlights the biphasic nature of the pathology of Crohn's disease. NOD2 can both strongly activate and negatively attenuate NF-kB signaling. The biochemical mechanism for this dual function of NOD2 is unknown.


RESULTS
We demonstrate that NOD2 activation leads to ubiquitinylation of NEMO, a key component of the NF-kB signaling complex. This ubiquitinylation is agonist dependant, and it does not regulate proteosomal destruction of NEMO. We show the NOD2-dependent ubiquitinylation of NEMO is dependent on the scaffolding protein kinase RIP2. Crohn's disease-associated polymorphisms of NOD2 show a decreased ability to bind RIP2, and this decreased ability to bind RIP2 correlates with a decreased ability to ubiquitinylate NEMO. We map the site of NEMO ubiquitinylation to a novel NEMO ubiquitinylation site (Lysine 285) and show that this ubiquityinylation occurs in vivo. Lastly, we show functionally that RIP2-induced ubiquitinylation of NEMO is at least in part responsible for RIP2-mediated NF-kB activation.


CONCLUSIONS
These data suggest that this novel mode of regulation of the NF-kB signaling pathway could be a factor underlying the pathogenesis of Crohn's disease.
Abbott
Derek W
DW
Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
Wilkins
Andrew
A
Asara
John M
JM
Cantley
Lewis C
LC
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003424:Crohn Disease / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D053473:Nod2 Signaling Adaptor Protein; D011110:Polymorphism, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism*
D002352:Carrier Proteins; C491731:IKBKG protein, human; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D025801:Ubiquitin; D017346:Protein Serine-Threonine Kinases; C506253:RIPK2 protein, human; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2; D051550:I-kappa B Kinase