Recombinant IκB Kinases α and β Are Direct Kinases of IκBα

Abstract

Activation of the transcription factor NF-B is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IB. A large multiprotein complex, the IB kinase (IKK), catalyzes the phosphorylation of IB. The two kinase components of the IKK complex, IKK␣ and IKK␤, were overexpressed in insect cells and purified to homogeneity. Both purified IKK␣ and IKK␤ specifically catalyzed the phosphorylation of the regulatory serine residues of IB␣. Hence, IKK␣ and IKK␤ were functional catalytic subunits of the IKK complex. Purified IKK␣ and IKK␤ also preferentially phosphorylated serine as opposed to threonine residues of IB␣, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKK␣ and IKK␤ revealed that the kinase activity of IKK␤ on IB␣ is 50 -60-fold higher than that of IKK␣. The primary difference between the two activities is the K m for IB␣. The kinetics of both IKK␣ and IKK␤ followed a sequential Bi Bi mechanism. No synergistic effects on IB␣ phosphorylation were detected between IKK␣ and IKK␤. Thus, in vitro, IKK␣ and IKK␤ are two independent kinases of IB␣.The transcription factor NF-B plays a critical role in immune and inflammatory responses. It is regulated by the signaling of receptors for inflammatory cytokines such as TNF-␣, 1 IL-1, or other external stimuli (1). In resting cells, NF-B is sequestered in the cytoplasm through its association with inhibitory proteins termed IB. Two IB proteins, IB␣ and IB␤, are rapidly phosphorylated at Ser residues in the Nterminal region upon stimulation by TNF-␣ and IL-1. The regulated phosphorylation is at Ser-32 and Ser-36 of IB␣ and, correspondingly, Ser-19 and Ser-23 of IB␤ (2, 3). The more recently cloned IB isoform, IB⑀, also contains the two conserved Ser residues at the N terminus for signal-induced degradation (4). Phosphorylated IB␣ and IB␤ are subsequently ubiquitinated and undergo ubiquitin-dependent degradation by the 26 S proteasome (3, 5). Degradation of IB results in the release of NF-B, which then translocates to the nucleus, where it up-regulates the transcription of its target genes (1).A 500 -900-kDa protein complex that contains the TNF-␣-induced IB kinase (IKK) has been purified and characterized independently by two groups (6, 7). The IKK complex phosphorylates IB␣ at the specific Ser residues that target the protein for ubiquitination and degradation. Two kinase subunits of the IKK complex, denoted IKK␣ and IKK␤, have been cloned (6 -10). IKK␣ or IKK␤ overexpressed in mammalian cells specifically phosphorylates IB␣ and IB␤ after immunoprecipitation, and their kinase activities can be induced by TNF-␣ or IL-1 (6 -10). In HeLa cells, expression of antisense IKK␣ inhibited NF-B activation by TNF-␣ or IL-1 (6). Furthermore, overexpression of dominant-negative mutants of either IKK␣ or IKK␤ blocked TNF-␣/IL-1-induced NF-B activation (8, 10). Thus, both IKK␣ and IKK␤ contribute to the activity of the IKK complex and are involved in NF-B activation. An additional protein kinase, NF-B-inducing kina...

Phosphorylation of Serine 337 of NF-κB p50 Is Critical for DNA Binding

“…In the case of Ser 342 , it is unclear why this residue is important for p50 DNA binding. Phosphorylation may not be involved, as substitution with threonine failed to retain p50 DNA binding, although it is known that some kinases, notably IKKs, strongly prefer serine over threonine (46). In addition, despite their proximity, Ser 342 is not involved in phosphorylation of Ser 337 , as phosphorylation of Ser 337 by PKA was not disrupted by mutation of Ser 342 .…”

“…Although we have shown that Ser 337 is a target for phosphorylation by PKA in vitro, making it highly probable that p50 is regulated by PKA, it should be noted that the sequence surrounding Ser 337 also fits the profile of several other kinases such as CaMII, casein kinase II, and protein kinase G. Nonetheless, the conformational change induced by the phosphorylation of p50 Ser 337 could be different from that induced by phosphorylation of p65 Ser 276 because p50 lacks a transactivation domain. The crystal structures of DNA-bound NF-B show that Ser 337 is proximal to the hinge region linking the N-terminal and C-terminal domains of p50 (46). Serine 337 resides on the outer surface of p50, making it easily accessible by kinases.…”

Gold compound auranofin inhibits IκB kinase (IKK) by modifying Cys-179 of IKKβ subunit

“…IKK complex is composed of two catalytic subunits, IKKα (or IKK1) and IKKβ (IKK2), and a regulatory subunit, IKKγ/NEMO/IKKAP1 (reviewed in May and Ghosh, 1998;Zandi and Karin, 1999). IKKα and IKKβ are Ser/Thr kinases of similar structure which can form homodimers and heterodimers in vitro, and purified recombinant form of each can directly phosphorylate IκBα and β at proper sites (Lee et al, 1998;Li et al, 1998;Zandi et al, 1998). The activity of IKKα and IKKβ was shown to depend on their phosphorylation at specific serine residues (Ser-176/180 of IKKα and Ser-177/181 of IKKβ) located in the T (activation) loop of the kinase domain, and substitution of these sites with glutamate, which mimic the effect of phosphoserine, generated constitutively activated form of IKKα and IKKβ (Mercurio et al, 1997;Ling et al, 1998).…”

BMS-345541 Is a Highly Selective Inhibitor of IκB Kinase That Binds at an Allosteric Site of the Enzyme and Blocks NF-κB-dependent Transcription in Mice

“…Two catalytic subunits (termed IKK-1 and IKK-2) of IKK have recently been identified, cloned, and shown to be widely expressed in human tissues (12)(13)(14)(15)(16)(17). The use of gene-targeting experiments have clearly shown that all known proinflammatory stimuli, including cytokines, viruses, and lipopolysaccharide (LPS) require the IKK-2 subunit for NF-B activation (for a review see Ref.…”