ORIGINAL ARTICLE Ubiquitylated Tax targets and binds the IKK signalosome at the centrosome YKfoury 1 ,RNasr 1,2,7 ,AFavre-Bonvin 3,7 ,MEl-Sabban 4 ,NRenault 2 ,M-LGiron 2 ,NSetterblad 5 , HElHajj 1 , E Chiari 3 , AG Mikati 1 , O Hermine 6 ,ASaib 2 ,HdeThe´ 2 ,CPique 3 and A Bazarbachi 1 1 Department of Internal Medicine, American University of Beirut, Beirut, Lebanon; 2 UMR 7151 CNRS, Laboratoire Associe´au Comite´de Paris de la Ligue contre le Cancer, Paris, France; 3 UMR 7151 CNRS/Universite´Paris 7, INSERM U567, Universite´Paris 5, Faculte´de Me´decine Rene´Descartes, UMR3, Institut Cochin, De´partement de Biologie Cellulaire, Paris, France; 4 Department of Human Morphology, American University of Beirut, Beirut, Lebanon; 5 Imagery Department of the Institut Universitaire d’He´matologie IFR105 Paris, France and 6 CNRS UMR 8603 and Department of Hematology, Necker Hospital, Paris, France Constitutive activation of the NF-jB pathway by the Tax oncoprotein plays a crucial role in the proliferation and transformation of HTLV-I infected T lymphocytes. We have previously shown that Tax ubiquitylation on C-terminal lysines is critical for binding of Tax to IjB kinase (IKK) and its subsequent activation. Here, we report that ubiquitylated Tax is not associated with active cytosolic IKK subunits, but binds endogenous IKK-a,-b, -c, targeting them to the centrosome. K63-ubiquitylated Tax colocalizes at the centrosome with IKK-c, while K48-ubiquitylated Tax is stabilized upon proteasome inhibition. Altogether, these results support a model in which K63-ubiquitylated Tax activates IKK in a centro- some-associated signalosome, leading to the production of Tax-free active cytoplasmic IKK. These observations highlight an unsuspected link between Tax-induced IKK activation and the centrosome. Oncogene (2008) 27, 1665–1676; doi:10.1038/sj.onc.1210804; published online 24 September 2007 Keywords: NF-kB; IkB kinase; centrosome; HTLV-I; Tax; ubiquitin Introduction Activation of the NF-kB pathway plays a crucial role in the proliferation and transformation of HTLV-I infected T cells (Robek and Ratner, 1999). Tax acts at multiple levels to initiate and maintain a permanent NF-kB activation (reviewed in Sun et al., 2000). A critical step is the recruitment of Tax to the IkB kinase (IKK)-g regulatory component of the IKK complex. Tax/IKK-g association leads to activation of the IKK-a and -b kinases resulting in IkB phosphorylation, ubiquitylationandproteasomaldegradation.Theprecise subcellular localization where these events occur and their molecular requirements remain largely unknown. Tax displays a dual nuclear and cytoplasmic localiza- tion. In the nucleus, Tax is predominantly located in RelA-enriched nuclear bodies (Bex et al., 1998). In the cytoplasm,asubstantialfractionofTaxlaysinperinuclear speckles, adjacent to the centrosome or microtubule organizing center (MTOC), in close association with the cis-Golgi compartment (Nejmeddine et al., 2005). Tax targets the centrosomal protein Tax1BP2, causing centro- some amplification, genomic instability and aneuploidy (Ching etal.,2006;Nitta etal.,2006;Pumfery etal.,2006). The activity of several transcription factors is regu- lated through ubiquitin-mediated proteolysis. However, ubiquitylationofsometranscriptionfactors(reviewedin Conaway et al., 2002) increases their transcriptional activity in a degradation-independent manner. We and others have shown that Tax is ubiquitylated (Chiari et al., 2004; Peloponese et al., 2004). We also showed that Tax ubiquitylation on C-terminal lysines is critical for both Tax binding to IKK and for the nuclear translocationofRelA(Nasr etal.,2006).Inaddition,we and others showed that Tax is post-translationally modified by small ubiquitin-related modifier (SUMO) in the nucleus and that sumoylation is critical for the formation of RelA-enriched Tax nuclear bodies (Lamsoul et al., 2005; Nasr et al., 2006). In this study, we investigated the molecular determi- nants involved in Tax association to IKK. We demon- strate that Tax binds IKK in a centrosome-associated signalosome, while it was no more associated to active cytosolic IKK complexes, and that IKK targeting to the centrosome depends on Tax conjugation to K63-linked ubiquitin chains. Results TaxinducesperinucleardelocalizationofendogenousIKK subunits TolocatethecellularsitesofTax/IKKinteraction,HeLa cellsweretransfectedwithTax,andimmunostainedwith Received 20 June 2007; revised 7 August 2007; accepted 20 August 2007; published online 24 September 2007 Correspondence: Dr A Bazarbachi, Department of Internal Medicine, American University of Beirut, Medical center, Hamra Street, PO Box 113-6044, Beirut, Lebanon. E-mail: bazarbac@aub.edu.lb 7 These two authors contributed equally to this work. Oncogene (2008) 27, 1665–1676 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc