Signal-induced Ubiquitination of IκB Kinase-β

Abstract

Initiation of the genetic programs for inflammation and immunity involves nuclear mobilization of transcription factor NF-B. This signal-dependent process is controlled in part by the ␤-catalytic subunit of IB kinase (IKK␤), which marks IB␣ and other cytoplasmic inhibitors of NF-B for proteolytic destruction. The catalytic activity of IKK␤ is stimulated by pathologic and physiologic inducers of NF-B, such as the Tax oncoprotein and proinflammatory cytokines. We now report evidence that these NF-B inducers target IKK␤ for conjugation to ubiquitin (Ub) in mammalian cells. The apparent molecular size of modified IKK␤ is compatible with monoubiquitination rather than attachment of a multimeric Ub chain. The modification is contingent upon signal-induced phosphorylation of the activation T loop in IKK␤ at Ser-177/Ser-181. The formation of IKK␤-Ub conjugates is disrupted in cells expressing YopJ, a Ub-like protein protease that interferes with the NF-B signaling pathway. These findings indicate an important mechanistic link between phosphorylation, ubiquitination, and the biologic action of IKK␤.The inducible transcription factor NF-B is biochemically coupled to cell-surface members of the tumor necrosis factor (TNF) 1 receptor, Toll-like receptor, and immunoglobulin superfamilies (1). NF-B is persistently activated in cells expressing the Tax protein of human T-cell leukemia virus type 1, which has potent oncogenic properties (2). This deregulated pattern of NF-B activity also underlies acute and chronic inflammatory diseases (1). Nuclear translocation of NF-B is controlled by an inducible multicomponent protein kinase, termed IKK, which targets IB␣ and other cytoplasmic inhibitors of NF-B for proteolytic destruction (3). IKK contains two catalytic subunits, designated IKK␣ and IKK␤, as well as a regulatory subunit called IKK␥ (3). IKK␤ and IKK␥ are essential for proteolytic inactivation of IB␣, whereas IKK␣ mediates NF-B subunit processing via an IKK␥-independent mechanism (4).The catalytic activity of IKK␤ is stimulated by signals that trigger its phosphorylation at Ser-177/Ser-181, such as TNF-␣ (5). These phosphoacceptors lie in a region of the catalytic domain that shares homology with regulatory "T loop" sequences found in mitogen-activated protein kinases and their upstream activators (5). In contrast to its transient pattern of phosphorylation and activation in TNF-stimulated cells, IKK␤ is chronically phosphorylated and activated in cells expressing the Tax oncoprotein (6). Tax-induced activation of IKK␤ is blocked by YopJ (6), a cysteine protease that removes ubiquitin (Ub)-related modifiers from target proteins in mammalian cells (7). This finding with YopJ raised the possibility that IKK␤ might be subject to signal-dependent ubiquitination. In keeping with this possibility, previous studies with partially purified kinase complexes suggested that IKK is conjugated to Ub in vitro (8). However, these pioneering in vitro experiments were conducted prior to molecular cloning of individual IKK subunits (3).We n...

Acetylation of MEK2 and IκB kinase (IKK) activation loop residues by YopJ inhibits signaling

“…These results are consistent with an earlier report (21) wherein it was demonstrated that phosphorylation of the activation loop of IKK␤ was inhibited in YopJ-expressing mammalian cells. Our results thus provide a mechanistic basis for the observed inhibition of the canonical NF-B proinf lammatory pathway by YopJ.…”

Yersinia Outer Protein P Suppresses TGF-β-Activated Kinase-1 Activity to Impair Innate Immune Signaling in Yersinia enterocolitica-Infected Cells

“…overexpressed YopP could potentially modify the ubiquitination of several signal transducers that are involved in NF-B activation. This idea is in line with the results of previous publications that have indicated that YopP/YopJ overexpression attenuates the conjugation of cellular proteins with ubiquitin-like SUMO-1 (6) and the ubiquitination of IKK␤ (46). This could give reason to speculations that YopP/YopJ may impair cellular signaling by antagonizing the ubiquitin modification of several critical signal transducers.…”

“…A similar effect of YopP/YopJ has been previously reported on the ubiquitination of IKK␤ (46) and on nonspecified cellular proteins modified with the ubiquitin-like molecule SUMO-1 (6). These results were ascribed to the putative cysteine protease activity of YopP/YopJ, which could mediate cleavage of ubiquitin-like molecules (6,46). However, we could not reveal a reliable effect of YopP on the ubiquitination of TRAF6, NEMO, or TAB2 in Yersinia-infected cells (data not shown).…”

Oxidative Stress Transiently Decreases the IKK Complex (IKKα, β, and γ), an Upstream Component of NF-κB Signaling, after Transient Focal Cerebral Ischemia in Mice

“…Nuclear factor-kB inducers target IKKb for conjugation to ubiquitin in mammalian cells. Carter et al (2003) proposed that T loop phosphorylation at Ser177/Ser181 generates a conditional ubiquitintargeting signal in IKKb by a post-translational mechanism. Increases in accumulation of multiubiquitin after 30 mins of tFCI may show the ubiquitination of the IKK complex.…”