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NF-jB essential modulator (NEMO) plays an essential role in the nuclear factor jB (NF-jB) pathway as a modulator of the two other subunits of the IjB kinase (IKK) complex, i.e. the protein kinases, IKKa and IKKb. Previous reports all envision the IKK complex to be a static entity. Using glycerol-gradient ultracentrifugation, we observed stimulus-dependent dynamic IKK complex assembly. | ễFEBS Journal NEMO oligomerization in the dynamic assembly of the IkB kinase core complex Elisabeth Fontan1 Francois Traincard1 Samuel G. Levy1 Shoji Yamaoka2 Michel Veron1 and Fabrice Agou1 1 Unite de Regulation Enzymatique des Activities Cellulaires CNRS URA 2185 Institut Pasteur Paris France 2 Tokyo Medicaland DentalUniversity Graduate Schoolof Medicine Japan Keywords fluorescence resonance energy transfer glycerol gradient ultracentrifugation IkB kinase complex NEMO Tax protein of human T-cellleukemia virus type 1 Correspondence F. Agou Unite de Regulation Enzymatique des Activites Cellulaires CNRS URA 2185 Institut Pasteur 25 28 rue du Dr Roux 75724 Paris Cedex 15 France Fax 33 1 45 68 83 99 Tel 33 1 44 38 95 69 E-mail fagou@pasteur.fr Received 15 December 2006 revised 13 March 2007 accepted 14 March 2007 doi 10.1111 j.1742-4658.2007.05788.x NF-kB essential modulator NEMO plays an essential role in the nuclear factor kB NF-kB pathway as a modulator of the two other subunits of the IkB kinase IKK complex i.e. the protein kinases IKKa and IKKp. Previous reports all envision the IKK complex to be a static entity. Using glycerol-gradient ultracentrifugation we observed stimulus-dependent dynamic IKK complex assembly. In wild-type fibroblasts the kinases and a portion of cellular nemo associate in a 350-kDa high-molecular-mass complex. In response to constitutive NF-kB stimulation by Tax we observed nemo recruitment and oligomerization to a shifted high-molecular-mass complex of 440 kDa which displayed increased IKK activity. This stimulus-dependent oligomerization of nemo was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin-1 p. In addition fully activated dimeric kinases not bound to nemo were detected in these Tax-activated fibroblasts. By glycerol gradient ultracentrifugation we also showed that a in fibroblasts deficient in IKKa and IKKp nemo predominantly exists as a monomer b in NEMO-deficient fibroblasts .
