The IkB Kinase Complex (IKK) Contains Two Kinase Subunits, IKKa and IKKb, Necessary for IkB Phosphorylation and NF-kB Activation

of the NF-kB dimer which then translocates to the nucleus to activate gene transcription. In the case of IkBa the inducible phosphorylation sites were mapped toserines (S) 32 and 36, whose substitution with alanines (A)or threonines (T) prevents phosphorylation, ubiquitination Ebrahim Zandi,† David M. Rothwarf,† Mireille Delhase, Makio Hayakawa, and Michael Karin* Laboratory of Gene Regulation and Signal Transduction Department of Pharmacology University of California at San Diego and degradation (Brockman et al., 1995; Brown et al., 9500 Gilman Drive 1995; Traenckner et al., 1995; DiDonato et al., 1996). As La Jolla, California 92093-0636 a result, nonphosphorylatable IkBa mutants, such as IkBa(A32/36), arepotent and specific inhibitors of NF-kB activation (Liu et al., 1996; Wang et al., 1996). Once it Summary was recognized that the key step in NF-kB activation is IkB phosphorylation, much of the effort to understand Recently we purified a 900 kDa cytokine-responsive the regulation of this signaling pathway has focused on IkB kinase complex (IKK) and molecularly cloned one identification of the protein kinase(s) responsible for this of its subunits, IKKa, a serine kinase. We now describe event. Recently we described the purification of a 900 the molecular cloning and characterization of IKKb, kDa protein kinase complex, the IkB kinase (IKK), that a second subunit of the IKK complex. IKKb is 50% phosphorylates IkBa and IkBb at the sites that mediate identical to IKKa and like it contains a kinase domain, their ubiquitination and degradation (DiDonato et al., a leucine zipper, and a helix-loop-helix. Although IKKa 1997). Another protein kinase complex that phosphoryand IKKb can undergo homotypic interaction, they lates IkB was described (Chen et al., 1996; Lee et al., also interact with each other and the functional IKK 1997), but its relationship to IKK is not clear at this point complex contains both subunits. The catalytic activias none of the components of that complex have been ties of both IKKa and IKKb make essential contribumolecularly defined. IKK activity is rapidly stimulated tions to IkB phosphorylation and NF-kB activation. by IL-1 or TNF and depends on its phosphorylation. While the interactions between IKKa and IKKb may Through peptide sequencing we identified and molecbe mediated through their leucine zipper motifs, their ularly cloned one component of the IKK complex, IKKa, helix-loop-helix motifs may be involved in interactions an 85 kDa polypeptide containing a protein kinase dowith essential regulatory subunits. main at its N-terminal half and protein interaction motifs (a leucine zipper [LZ] and a helix-loop-helix [HLH]) at its Introduction C-terminal half (DiDonato et al., 1997). Using antisense RNA, we demonstrated that expression of IKKa is essential for NF-kB activation by IL-1, TNF, TPA, or okadaic The dimeric NF-kB transcription factor, whose subunits acid (DiDonato et al., 1997). belong to the Rel family of DNA-binding proteins (Verma Interestingly, IKKa was also isolated by a two-hybrid et al., 1995; Baeuerle and Baltimore, 1996), plays a screen as a NIK interacting protein (Régnier et al., 1997). critical role in immune and inflammatory responses NIK is a member of theMEK kinase (MEKK) family whose (Baeuerle and Henkel, 1994; Baldwin, 1996; Barnes and overexpression results in NF-kB activation (Malinin et Karin, 1997). Recently NF-kB was found to protect cells al., 1997). NIK was originally isolated through interaction from apoptosis induced by tumor necrosis factor (TNF) with TRAF2 (Malinin et al., 1997), a component of the and various genotoxic agents (Beg and Baltimore, 1996; TNF receptor signaling complex, which plays a critical Liu et al., 1996; Van Antwerp et al., 1996; Wang et al., role in NF-kB activation (Hsu et al., 1996). It was sug1996). In most cell types, NF-kB dimers are kept in the gested that NIK directly interacts with IKKa, phosphorycytoplasm through interaction with the IkB inhibitory lating and activating it (Régnier et al., 1997). It was reproteins. The IkBs bind to the Rel homology domain ported that a catalytically inactive mutant of IKKa can (RHD), which contains the dimerization, nuclear transfer, completely inhibit NF-kB activation, suggesting its cataand DNA binding functions of the NF-kB/Rel proteins lytic activity is essential for IkB phosphorylation (Régnier (Beg and Baldwin, 1993; Gilmore and Morin, 1993; Baldet al., 1997). However, we found that catalytically inacwin, 1996). In response to cellstimulation with proinflamtive IKKa can still interact with other components of the matory cytokines (e.g., IL-1, TNF), bacterial lipopolysacIKK complex to generate a functional, cytokine-responcharide (LPS), and phorbol ester (TPA), at least two of sive IkB kinase, albeit of lower activity than the complex the IkBs (IkBa and IkBb) undergo rapid phosphorylation formed by wild-type IKKa. Therefore, we searched for at two conserved N-terminal residues, which targets other IKK components that interact with IKKa and would them for rapid polyubiquitination followed by degradaalso function as protein kinases. tion through the 26S proteasome (Alkalay et al., 1995; Here we describe the molecular characterization of Brockman et al., 1995; Brown et al., 1995; Chen et al., IKKb, a second component of the IKK complex. IKKb 1995; DiDonato et al., 1995, 1996; Traenckner et al., is closely related in structure to IKKa: it contains an 1995; Whiteside et al., 1995). This results in liberation N-terminal protein kinase domain and LZ and HLH motifs at its C-terminal half. IKKa has to interact with IKKb (and other subunits) to form a functional IkB kinase. We *To whom correspondence should be addressed. †These authors contributed equally to this work. demonstrate that the functional IKK complex contains brought to you by CORE View metadata, citation and similar papers at core.ac. uk