NOD2 Pathway Activation by MDP or Mycobacterium tuberculosis Infection Involves the Stable Polyubiquitination

The Rip2 kinase contains a caspase recruitment domain and has been implicated in the activation of the transcriptional fac-tor NF-!B downstream of Toll-like receptors, Nod-like recep-tors, and the T cell receptor. Although Rip2 has been linked to Nod signaling, how Nod-Rip2 proteins mediate NF-!B activa-tion has remained unclear. We find Rip2 required for Nod2-mediated NF-!B activation and to a lesser extent mitogen-acti-vated protein kinase activation. We demonstrate that Rip2 and I!B kinase- " become stably polyubiquitinated upon treatment of cells with the NOD2 ligand, muramyl dipeptide. We also demonstrate a requirement for the E2-conjugating enzyme Ubc13, the E3 ubiquitin ligase Traf6, and the ubiquitin-acti-vated kinase Tak1 in Nod2-mediated NF-!B activation. Rip2 polyubiquitination is also stimulated when macrophages are infected with live Mycobacterium tuberculosis but not whe