FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2060154087> ?p ?o ?g. }

• W2060154087 endingPage "121" @default.
• W2060154087 startingPage "111" @default.
• W2060154087 abstract "Besides activating NFκB by phosphorylating IκBs, IKKα/IKKβ kinases are also involved in regulating metabolic insulin signaling, the mTOR pathway, Wnt signaling, and autophagy. How IKKβ enzymatic activity is targeted to stimulus-specific substrates has remained unclear. We show here that NEMO, known to be essential for IKKβ activation by inflammatory stimuli, is also a specificity factor that directs IKKβ activity toward IκBα. Physical interaction and functional competition studies with mutant NEMO and IκB proteins indicate that NEMO functions as a scaffold to recruit IκBα to IKKβ. Interestingly, expression of NEMO mutants that allow for IKKβ activation by the cytokine IL-1, but fail to recruit IκBs, results in hyperphosphorylation of alternative IKKβ substrates. Furthermore IKK's function in autophagy, which is independent of NFκB, is significantly enhanced without NEMO as IκB scaffold. Our work establishes a role for scaffolds such as NEMO in determining stimulus-specific signal transduction via the pleiotropic signaling hub IKK. Besides activating NFκB by phosphorylating IκBs, IKKα/IKKβ kinases are also involved in regulating metabolic insulin signaling, the mTOR pathway, Wnt signaling, and autophagy. How IKKβ enzymatic activity is targeted to stimulus-specific substrates has remained unclear. We show here that NEMO, known to be essential for IKKβ activation by inflammatory stimuli, is also a specificity factor that directs IKKβ activity toward IκBα. Physical interaction and functional competition studies with mutant NEMO and IκB proteins indicate that NEMO functions as a scaffold to recruit IκBα to IKKβ. Interestingly, expression of NEMO mutants that allow for IKKβ activation by the cytokine IL-1, but fail to recruit IκBs, results in hyperphosphorylation of alternative IKKβ substrates. Furthermore IKK's function in autophagy, which is independent of NFκB, is significantly enhanced without NEMO as IκB scaffold. Our work establishes a role for scaffolds such as NEMO in determining stimulus-specific signal transduction via the pleiotropic signaling hub IKK. Constitutively active IKKβ is unable to activate NFκB in the absence of NEMO NEMO directly interacts with IκBα via its zinc finger NEMO functions as a scaffold to recruit IκBα to IKKβ Without NEMO, active IKKβ hyperactivates alternative substrates and autophagy Reliable cellular signal transduction depends on the specificity of kinases. Unlike some metabolic enzymes, in which the catalytic site may show great specificity for particular small molecule substrates, protein kinases require specificity domains. When such specificity domains are encoded by distinct specificity factors, the kinase activity can in principle be directed to alternate specific substrates, allowing for functional pleiotropy (Bhattacharyya et al., 2006Bhattacharyya R.P. Reményi A. Yeh B.J. Lim W.A. Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits.Annu. Rev. Biochem. 2006; 75: 655-680Crossref PubMed Scopus (369) Google Scholar, Ubersax and Ferrell, 2007Ubersax J.A. Ferrell Jr., J.E. Mechanisms of specificity in protein phosphorylation.Nat. Rev. Mol. Cell Biol. 2007; 8: 530-541Crossref PubMed Scopus (995) Google Scholar). One may distinguish between catalytic (or “allosteric”) specificity factors, that alter the intrinsic catalytic activity of the kinase toward a specific substrate, and scaffold (or “tethering”) specificity factors, that enhance otherwise weak interactions between substrate and kinase (Burack and Shaw, 2000Burack W.R. Shaw A.S. Signal transduction: hanging on a scaffold.Curr. Opin. Cell Biol. 2000; 12: 211-216Crossref Scopus (279) Google Scholar). Whereas the c-Jun N-terminal kinase (JNK)-interacting protein (JIP1), the JNK/SAPK-activating protein 1 (JSAP1), and the kinase suppressor of Ras (KSR) are thought to be in the former category, the axin scaffolding proteins target the pleiotropic kinases GSK3 and CK1 to the β-catenin pathway (Burack and Shaw, 2000Burack W.R. Shaw A.S. Signal transduction: hanging on a scaffold.Curr. Opin. Cell Biol. 2000; 12: 211-216Crossref Scopus (279) Google Scholar, Wu and Pan, 2010Wu D. Pan W. GSK3: a multifaceted kinase in Wnt signaling.Trends Biochem. Sci. 2010; 35: 161-168Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, Amit et al., 2002Amit S. Hatzubai A. Birman Y. Andersen J.S. Ben-Shushan E. Mann M. Ben-Neriah Y. Alkalay I. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway.Genes Dev. 2002; 16: 1066-1076Crossref PubMed Scopus (589) Google Scholar), and the Ste5 scaffold directs the specificity of yeast MAPK signaling (Schwartz and Madhani, 2004Schwartz M.A. Madhani H.D. Principles of MAP kinase signaling specificity in Saccharomyces cerevisiae.Annu. Rev. Genet. 2004; 38: 725-748Crossref PubMed Scopus (198) Google Scholar, van Drogen and Peter, 2002van Drogen F. Peter M. MAP kinase cascades: scaffolding signal specificity.Curr. Biol. 2002; 12: R53-R55Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Indeed, shuffling of binding pockets of Ste5 by site-directed mutagenesis enables redirection of signaling specificity (Dueber et al., 2003Dueber J.E. Yeh B.J. Chak K. Lim W.A. Reprogramming control of an allosteric signaling switch through modular recombination.Science. 2003; 301: 1904-1908Crossref PubMed Scopus (243) Google Scholar). Thus, multivalent scaffold proteins may not only direct kinase substrate specificity but also ensure signaling specificity of a pleiotropic kinase by coordinating upstream and downstream signaling axes. The IκB kinase (IKK) complex is ubiquitously expressed and functionally pleiotropic (Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Shared principles in NF-kappaB signaling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3526) Google Scholar, Scheidereit, 2006Scheidereit C. IkappaB kinase complexes: gateways to NF-kappaB activation and transcription.Oncogene. 2006; 25: 6685-6705Crossref PubMed Scopus (541) Google Scholar). Its major catalytic component, IKKβ (also known as IKK2), was purified as the IκB kinase that controls nuclear translocation of NFκB to initiate proinflammatory gene transcription by phosphorylating the canonical IκBs, IκBα, IκBβ, and IκBε (Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Shared principles in NF-kappaB signaling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3526) Google Scholar, Hoffmann and Baltimore, 2006Hoffmann A. Baltimore D. Circuitry of nuclear factor kappaB signaling.Immunol. Rev. 2006; 210: 171-186Crossref PubMed Scopus (747) Google Scholar, Scheidereit, 2006Scheidereit C. IkappaB kinase complexes: gateways to NF-kappaB activation and transcription.Oncogene. 2006; 25: 6685-6705Crossref PubMed Scopus (541) Google Scholar). However, recent literature suggests that IKKβ also plays critical roles in many other biological processes, including autophagy, insulin signaling, and DNA damage responses, by targeting diverse alternative cellular substrates for phosphorylation (Figure 1A ) (reviewed in Chariot, 2009Chariot A. The NF-kappaB-independent functions of IKK subunits in immunity and cancer.Trends Cell Biol. 2009; 19: 404-413Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar and Scheidereit, 2006Scheidereit C. IkappaB kinase complexes: gateways to NF-kappaB activation and transcription.Oncogene. 2006; 25: 6685-6705Crossref PubMed Scopus (541) Google Scholar). The large number of substrates, and the diverse biological functions regulated by their phosphorylation raise the question of whether IKKβ enzymatic activity may be targeted to distinct substrates. To ensure fidelity as a signal transducer of diverse signals, one would expect the activation of the kinase by upstream pathways to be coordinated with downstream substrate selection. In response to the inflammatory cytokine IL-1 IKKβ rapidly phosphorylates S32/36 of IκBα, thereby triggering its ubiquitin-dependent proteasomal degradation, while phosphorylation of the NFκB subunit p65 occurs with delayed kinetics (Figures 1B and S1A). However, using purified, constitutively active IKKβ (HA- IKKβEE) in an in vitro kinase assay, we find high phosphorylation of both IκBα and p65 proteins with no preference for IκBα (Figure 1C) suggesting that in vivo IKKβ's preference for IκB phosphorylation is regulated by additional factors. In vivo, IKKβ activation in response to inflammatory cytokine and pathogen-associated molecular patterns (PAMPs) stimulation requires NEMO, which connects the kinase complex to ubiquitin chains emanating from receptor-associated signaling complexes (Israël, 2010Israël A. The IKK complex, a central regulator of NF-kappaB activation.Cold Spring Harb Perspect Biol. 2010; 2: a000158Crossref Scopus (542) Google Scholar). To test whether NEMO may also be determining the target specificity of the catalytic enzyme, we used a constitutively active form of IKKβ generated through mutation of the critical activation loop serines 177 and 181 to phosphomimetic glutamates (IKKβEE). Retroviral transduction of wild-type fibroblasts with IKKβEE resulted in the expected increase in steady-state NFκB activity, but similar expression of IKKβEE in NEMO-deficient cells had only a modest effect (Figure 1D). However, reconstitution with retrovirally expressed NEMO restored the strong NFκB DNA binding activity. Strikingly, in vitro kinase assays with immunoprecipitated IKK using either FL-IκBα (Figure 1D) or GST-IκBα1-54 (Figure S1B) as substrate showed that NEMO did not alter the catalytic IKK activity in these cells, suggesting that it has a specific role in the NFκB activation pathway at a step downstream of its known function in IKK activation. Indeed, we found that IKKβEE caused only weak IκBα phosphorylation and degradation in unstimulated NEMO-deficient cells, unless NEMO was reconstituted (Figure 1E). Taken together these data indicate that intrinsic substrate specificity of IKKβ is not sufficient for efficient IκB phosphorylation, but that NEMO plays a role in targeting IKK activity toward the IκB-NFκB signaling module. NEMO's N-terminal region interacts with IKKβ, and the central CoZi-region allows for dimerization and binding to linear and K63 polyubiquitin chains, connecting IKK to inflammatory receptor pathways (Figure 2A ) (Israël, 2010Israël A. The IKK complex, a central regulator of NF-kappaB activation.Cold Spring Harb Perspect Biol. 2010; 2: a000158Crossref Scopus (542) Google Scholar). A frameshift mutation that completely removes NEMO's C-terminal Zinc finger (ZF) causes incontinentia pigmenti; its molecular function however remains unclear (Makris et al., 2002Makris C. Roberts J.L. Karin M. The carboxyl-terminal region of IkappaB kinase gamma (IKKgamma) is required for full IKK activation.Mol. Cell. Biol. 2002; 22: 6573-6581Crossref Scopus (77) Google Scholar). While it is critical for NFκB activation triggered by genotoxic stress (Huang et al., 2002Huang T.T. Feinberg S.L. Suryanarayanan S. Miyamoto S. The zinc finger domain of NEMO is selectively required for NF-kappa B activation by UV radiation and topoisomerase inhibitors.Mol. Cell. Biol. 2002; 22: 5813-5825Crossref Scopus (98) Google Scholar), its role in inflammation-induced NFκB activation is cell- and stimulus-specific. Disruption of the ZF was found to prevent TNF- but not IL-1β-induced NFκB activation in MEFs (Makris et al., 2002Makris C. Roberts J.L. Karin M. The carboxyl-terminal region of IkappaB kinase gamma (IKKgamma) is required for full IKK activation.Mol. Cell. Biol. 2002; 22: 6573-6581Crossref Scopus (77) Google Scholar). A complete defect in cytokine-induced NFκB activation was observed in human T cells expressing human C417R NEMO (corresponding to C410R in mouse NEMO) (Yang et al., 2004Yang F. Yamashita J. Tang E. Wang H.L. Guan K. Wang C.Y. The zinc finger mutation C417R of I-kappa B kinase gamma impairs lipopolysaccharide- and TNF-mediated NF-kappa B activation through inhibiting phosphorylation of the I-kappa B kinase beta activation loop.J. Immunol. 2004; 172: 2446-2452Google Scholar), while NFκB activation by LPS was normal in mouse B cells expressing this mutant (Huang et al., 2002Huang T.T. Feinberg S.L. Suryanarayanan S. Miyamoto S. The zinc finger domain of NEMO is selectively required for NF-kappa B activation by UV radiation and topoisomerase inhibitors.Mol. Cell. Biol. 2002; 22: 5813-5825Crossref Scopus (98) Google Scholar). More recently the ZF has been implicated in enhancing the affinity of NEMO for binding to K63 linked polyubiquitin chains, important for IKK activation by specific stimuli (Laplantine et al., 2009Laplantine E. Fontan E. Chiaravalli J. Lopez T. Lakisic G. Véron M. Agou F. Israël A. NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain.EMBO J. 2009; 28: 2885-2895Crossref PubMed Scopus (152) Google Scholar). We examined the possible role of the NEMO ZF on NFκB activation in the context of constitutively active IKKβ. We generated a truncation (ΔC25), a debilitating (C389/393S), and a neutral (M408S) mutant of the ZF domain. Reconstitution of NEMO-deficient cells with these constructs resulted in expression levels comparable to those in wild-type MEFs (Figure S2A). In cells expressing either WT or the M408S NEMO, which does not affect the overall structure of the ZF, strong NFκB activity was detected (Figure 2B), while only modest nuclear NFκB activity was observed in NEMO-deficient cells. Strikingly, truncating and debilitating the ZF abolished NEMO's ability to reduce IκBα levels (as well as IκBβ and IκBε, Figure S2B) and to enhance NFκB activity in the presence of IKKβEE (Figure 2B). These data suggest that the ZF of NEMO is critical for the active IKK complex to efficiently target IκBs for degradation in vivo, to allow for NFκB activation, even though ZF mutations had no effect on in vitro IKK kinase activity (Figure S2C). To examine NEMO's role in targeting IKK toward IκBs during inflammatory signaling, we had to identify conditions in which NEMO mutants, defective in IκB targeting, allowed for efficient IKK activation. NEMO-deficient cells reconstituted with NEMO mutants were exposed to TNF, IL-1β, and LPS. In cells expressing WT NEMO and M408S mutant NEMO, all stimuli induced in vitro kinase activity, although kinase activity induced by TNF was attenuated in M408S NEMO-expressing cells (Figures 2C, S2D, and S2E). IL-1β and LPS also induced strong, yet somewhat weaker kinase activity in cells expressing NEMO with a truncated or debilitated ZF (Figures 2C and S2D), though both mutants were defective in supporting TNFα-induced kinase activity (Figure S2E), consistent with data reported by Makris et al., 2002Makris C. Roberts J.L. Karin M. The carboxyl-terminal region of IkappaB kinase gamma (IKKgamma) is required for full IKK activation.Mol. Cell. Biol. 2002; 22: 6573-6581Crossref Scopus (77) Google Scholar. Thus, the ZF of NEMO appears to be largely dispensable for the activation of the IKK complex by IL-1β and LPS in fibroblasts, reflecting stimulus-specific requirements for IKK activation. If the ZF were necessary for recruiting the IKK complex to IκBs, a defect in IκBα phosphorylation and degradation and subsequent NFκB activation in cells expressing ZF-mutated NEMO would be anticipated. As expected, in NEMO−/−cells expressing WT NEMO or a neutral M408S mutant, phosphorylation and degradation of IκBα were induced by IL-1β (Figure 2D). However, in cells expressing NEMO with a truncated or debilitated ZF, IL-1β stimulation triggered neither IκBα phosphorylation nor degradation. Furthermore, NFκB DNA binding activity induced by IL-1β, LPS, and TNF was defective with ZF mutants while it was strongly activated in WT and M408S-expressing cells (Figures 2E, S2F, and S2G). The reduction in kinase activity measured in cells expressing truncated or C389/93S NEMO could not account for the observed defect in NFκB activation, as similarly low kinase activities in WT cells (elicited by lower stimulus concentrations) allowed for detection of strong NFκB DNA binding ability (Figure S2H). These data are consistent with results obtained with constitutively active IKKβ and further indicate that NEMO does indeed play a role in NFκB activation that is distinct from its function in activating the IKK complex, most likely by facilitating the recruitment of IκBs to the IKK complex. NEMO's apparent role as a specificity factor for IKK may be mediated by allostery altering the catalytic specificity of the enzyme or by functioning as a scaffold tethering IκB substrates toward the catalytic site. Given that in vitro kinase assays did not reveal differences in catalytic activity, we hypothesized that NEMO functions as a recruitment scaffold by enhancing the IKK-IκB interaction through an N-terminal interaction with IKKβ and a C-terminal interaction with IκBα (Figure 3A ). To test this hypothesis, IκBα was immunoprecipitated and its interaction with IKKβ and NEMO was analyzed by immunoblotting (Figure 3B). A strong interaction between IκB and IKK could be detected in the presence of WT NEMO, but not in its absence. Deletion of the ZF abolished binding. The interaction was dependent on IKKβ, as the interaction of IκB with WT NEMO was reduced in the absence of IKKβ or when an N-terminally truncated NEMO, which lacks the IKKβ binding site, was used. Comparable results were obtained when NEMO was immunoprecipitated (Figure S3). Together, these data indicate that NEMO interacts with IκBα, that the ZF is required for this interaction, and that the interaction is strengthened in the presence of IKKβ, suggesting the existence of a IκB:IKK:NEMO complex. In a functional assay, in which steady-state expression of IκBα was analyzed in the presence of small amounts of IKKβEE, reduced levels of IκBα could only be detected when WT NEMO was cotransfected, but not when N- and C-terminally truncated NEMO was expressed, further suggesting that NEMO indeed enhances IκBα turnover by means of complex formation with IκBα and IKKβ (Figure 3C). The N-terminal 54 amino acids of IκBα are known to be sufficient for phosphorylation by IKKβ (Figure 3D) (Wu and Ghosh, 2003Wu C. Ghosh S. Differential phosphorylation of the signal-responsive domain of I kappa B alpha and I kappa B beta by I kappa B kinases.J. Biol. Chem. 2003; 278: 31980-31987Crossref PubMed Scopus (39) Google Scholar). As NEMO appears to be important for targeting IKKβ to IκBα, IκBβ, and IκBε, we hypothesized that a region conserved in canonical IκBs would mediate the interaction with NEMO. Most mutations of conserved residues in IκBα showed no defects in phosphorylation and degradation upon stimulation with IL-1β (data not shown). However, mutation of negatively charged residues D27 and D28 (corresponding to D14 and E15 in IκBβ) to positively charged arginines resulted in a strong reduction of inducible phosphorylation and degradation upon stimulation with IL-1β (Figure 3E), indicating that these amino acids are critical for phosphorylation by IKKβ in cells. IKKβEE-induced phosphorylation of both the full-length and 1–54 IκB D27/28R mutants was comparable with WT IκBα in in vitro kinase assays (Figure 3F), indicating that the consensus phosphorylation site for IKKβ was not disrupted by the mutation. Instead, the in vivo defect in phosphorylation may be due to impaired recruitment by NEMO as the D27/28R IκBα mutation weakened IκBα's interaction with NEMO and IKK2 (Figure 3G). Together, these data indicate that in cells, NEMO forms a complex with IκBα and IKKβ and that amino acids D27 and D28 in the N terminus of IκBα are critical for complex formation. To test the interaction between NEMO and IκBα more directly, we produced highly purified recombinant, bacterially expressed NEMO and IκBα and performed gel filtration analysis. As shown in Figure 3H, free IκBα was detected in the low molecular weight fractions 26–35. In the presence of full-length NEMO IκBα was also present in NEMO-containing high molecular weight fractions (13–18). In contrast, no change in the elution profile of IκBα was detected in the presence of a C-terminal NEMO deletion mutant (lower panel). These data are indicative of a direct interaction between NEMO and IκBα that depends on NEMO's C terminus. A hallmark of scaffold proteins is a nonmonotonic dose response curve: increasing amounts of scaffold allow for increased complex formation, but when the scaffold concentration exceeds that of the kinase or substrate whose interaction it coordinates, then the excess will inhibit the formation of the full complex, by instead favoring the formation of many incomplete subcomplexes, an effect referred to as “combinatorial inhibition” (Ferrell, 2000Ferrell Jr., J.E. What do scaffold proteins really do?.Sci. STKE. 2000; 2000: pe1Crossref Scopus (71) Google Scholar, Levchenko et al., 2000Levchenko A. Bruck J. Sternberg P.W. Scaffold proteins may biphasically affect the levels of mitogen-activated protein kinase signaling and reduce its threshold properties.Proc. Natl. Acad. Sci. USA. 2000; 97: 5818-5823Crossref PubMed Scopus (387) Google Scholar). Accordingly, computational simulations of a mathematical model for the formation of the IκB-NEMO-IKK complex (Supplemental Experimental Procedures) showed increased complex formation when NEMO amounts are increased within a substoichiometric regime, but reduced functional complexes within a higher regime (Figure 4A ). To test this experimentally, we transfected 293T cells with increasing amounts of NEMO and limiting amounts of IKKβEE. Expression of small to intermediate amounts of NEMO led to a dose-dependent increase of IκB phosphorylation and reduced levels of total IκBα expression (Figure 4B and quantification Figure 4C) caused by intrinsic IKKβEE activity. Strikingly, high overexpression resulted in reduced phosphorylation of IκBα and increased IκBα levels. Expression of NEMO mutants that only interacted with either IKKβ (NEMO ΔC25) or IκBα (NEMO ΔN) had no effect. The kinase activity, assayed in vitro, remained unchanged throughout all NEMO expression levels (Figures 4D and S4). Thus, in the context of physical interactions delineated in Figures 3B–3H, the functional characteristics described in Figures 4A and 4B support the conclusion that NEMO functions as a specificity scaffold that recruits classical IκBs to IKK. Throughout these studies of NEMO's role as a specificity factor in vivo, we failed to observe such a function in vitro. We considered the explanation that the substrate availability/concentration in cells is lower than in in vitro kinase assay reaction conditions, in which substrate is supplied in excess to ensure sensitivity and linear dose responses. To test this hypothesis we attempted to mimic in vivo conditions by performing the kinase reaction in the presence of high concentrations of nonspecific competitor protein (BSA) and limiting concentrations of substrate. Using these conditions, IκBα phosphorylation was stronger and occurred with faster kinetics in the presence than in the absence of NEMO (Figure 4E). In contrast, ZF-deleted NEMO failed to enhance IκBα phosphorylation. Similarly NEMO had no effect on phosphorylation of IκBα DR, which is phosphorylation defective in vivo. These data further support the notion that NEMO acts as a scaffold for IKKβ likely by enhancing the local concentration of IκB to allow for its more efficient phosphorylation. IKKβ is a pleiotropic kinase that is known to phosphorylate numerous alternative substrates in vivo, in addition to IκBα. To analyze the potential effect of the scaffolding function of NEMO in this in vivo scenario we constructed an in silico model for IκB phosphorylation, in which IKKβ can bind to and phosphorylate IκBs or alternative substrates (Figure 5A ). Akin to our experimental results (Figure 4E), model simulations suggested that in the absence of alternative substrates (i.e., in vitro) the ZF-dependent NEMO scaffold function would have little effect on phosphorylation of IκB, while in their presence (i.e., in vivo), efficient phosphorylation of IκB may only occur in the presence of WT but not ZF mutant NEMO (Figure 5B, left panel). In contrast, NEMO's scaffolding function was predicted to restrict the phosphorylation of alternate substrates with the ZF mutant NEMO leading to the hyperphosphorylation of alternative IKKβ substrates (Figure 5B, right panel). To test this prediction experimentally, we analyzed steady-state phosphorylation of RelA/p65 Ser536, a well-established alternative target of IKKβ. Only low levels of p65 were phosphorylated in NEMO-deficient cells, but IKKβEE expression greatly enhanced phosphorylation levels (Figure 5C). Strikingly, expression of WT NEMO resulted in a strong reduction of phosphorylated p65, while ZF-mutated NEMO showed similar p-p65 levels to the parental NEMO-deficient IKKβEE cells, indicating that in contrast to IκBα, p65 is a NEMO-independent substrate. Indeed, in kinase assays performed in the presence of competitor protein and limiting amounts of substrate to reflect in vivo conditions, NEMO had no effect on the phosphorylation of p65 or p105, while IκBα phosphorylation was strongly enhanced by the addition of NEMO (Figures 5D and 5E). In contrast, without competitor, phosphorylation of IκBα, p65, and p105 was unaffected by NEMO (Figures 5E and S5C). These data not only demonstrate that NEMO does not affect IKKβ kinase activity per se, but that it specifically channels IKKβ kinase activity to IκBα in conditions of low substrate abundance. Next we asked whether we could observe similar substrate competition in the context of stimulated cells. As expected, IL-1β stimulation rapidly induced IκBα phosphorylation in NEMO-deficient cells reconstituted with WT NEMO, (Figure 6A ). No IκBα phosphorylation was detectable in the presence of ZF mutant NEMO but instead alternative IKKβ substrates, p65 and p105, were strongly phosphorylated. Similar results were obtained upon stimulation with LPS (Figure S6A). Phosphorylation of p65 and p105 was indeed caused by IKKβ, as treatment with an IKKβ-specific inhibitor abolished phosphorylation (Figure S6B). Despite hyperphosphorylation of p65 and p105 we did not detect measurable levels of NFκB activity (Figure 2E and data not shown). These data confirm that NEMO not only enhances phosphorylation of IκBs, but also restricts the phosphorylation of alternative substrates. We next addressed the implicit hypothesis that IκB and alternative substrates are effectively competing in vivo for limited enzyme activity. Using cells that lack the IκB substrates (iκbα−/−β−/−ε−/−), phosphorylation of both p65 and p105 was strongly induced as early as 5 min post-IL-1β stimulation, while it was barely induced in WT cells (Figure 6B). These data suggest that NEMO's role as a specificity factor is dependent on the availability of IκB substrates, further supporting a role of NEMO in acting as a scaffold/tethering factor to direct IKK's activity specifically to IκBs. Recent studies have shown that IκBα phosphorylation and degradation are not required for IKKβ-induced autophagy, suggesting the involvement of alternative IKKβ substrates (Comb et al., 2011Comb W.C. Cogswell P. Sitcheran R. Baldwin A.S. IKK-dependent, NF-κB-independent control of autophagic gene expression.Oncogene. 2011; 30: 1727-1732Crossref Scopus (89) Google Scholar, Criollo et al., 2010Criollo A. Senovilla L. Authier H. Maiuri M.C. Morselli E. Vitale I. Kepp O. Tasdemir E. Galluzzi L. Shen S. et al.The IKK complex contributes to the induction of autophagy.EMBO J. 2010; 29: 619-631Crossref PubMed Scopus (238) Google Scholar). Our model of NEMO as a specificity factor for IκBs that restricts the phosphorylation of alternative IKKβ substrates would predict that autophagy may be hyperactivated in cells that express ZF-debilitated NEMO. Indeed, when ZF-deleted NEMO was expressed along with IKKβEE, the number of cells that stained positive for the autophagy marker LC3 was significantly enhanced over that of WT NEMO controls (Figure 6C). Concomitantly, phosphorylation of p70S6K, a marker for mTOR activity, was strongly reduced, while the upstream AMPK was hyperphosphorylated in cells expressing the ZF mutant NEMO (Figure 6D), indicating that the signaling required for the induction of autophagy is hyperactivated by constitutively active IKKβ when NEMOs IκB targeting function is lost. Similarly, starvation increased the number of LC3 positive cells harboring a ZF-deleted NEMO (Figure 6E), despite comparable levels of NEMO-associated IKK kinase activity (Figure S6C). These data further show that autophagy is hyperactivated when IKKβ loses its ability to phosphorylate IκBs, but instead hyperphosphorylates alternative substrates. We have presented several lines of evidence that NEMO plays an essential role in targeting IKK to the IκB-NFκB signaling module. Previous studies focused on NEMO's essential role in the activation of IKK via its ability to bind ubiquitin chains, which are a hallmark of inflammatory signaling by receptors of the TNFR and TLR/IL1R superfamilies. Using a constitutively active IKK variant (Figure 1) and identifying a specific mutation in NEMO (Figure 2), we were able to distinguish between these two essential functions in activation and targeting. Physical interaction studies indicated that IKK-IκB interactions are enhanced by NEMO, which interacts not only with IKK through its N terminus (May et al., 2000May M.J. D'Acquisto F. Madge L.A. Glöckner J. Pober J.S. Ghosh S. Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex.Science. 2000; 289: 1550-1554Crossref PubMed Scopus (615) Google Scholar, Mercurio et al., 1999M" @default.
• W2060154087 created "2016-06-24" @default.
• W2060154087 creator A5034258593 @default.
• W2060154087 creator A5047177237 @default.
• W2060154087 creator A5058159306 @default.
• W2060154087 creator A5079762167 @default.
• W2060154087 creator A5089651139 @default.
• W2060154087 date "2012-07-01" @default.
• W2060154087 modified "2023-10-16" @default.
• W2060154087 title "NEMO Ensures Signaling Specificity of the Pleiotropic IKKβ by Directing Its Kinase Activity toward IκBα" @default.
• W2060154087 cites W1545189970 @default.
• W2060154087 cites W1694093395 @default.
• W2060154087 cites W1789358965 @default.
• W2060154087 cites W1881072801 @default.
• W2060154087 cites W1963904830 @default.
• W2060154087 cites W1966091813 @default.
• W2060154087 cites W1974251566 @default.
• W2060154087 cites W1997050095 @default.
• W2060154087 cites W2000387935 @default.
• W2060154087 cites W2007875429 @default.
• W2060154087 cites W2008472371 @default.
• W2060154087 cites W2011210301 @default.
• W2060154087 cites W2012045589 @default.
• W2060154087 cites W2018554367 @default.
• W2060154087 cites W2019478674 @default.
• W2060154087 cites W2021226225 @default.
• W2060154087 cites W2036931425 @default.
• W2060154087 cites W2043635727 @default.
• W2060154087 cites W2043794217 @default.
• W2060154087 cites W2059345773 @default.
• W2060154087 cites W2067117198 @default.
• W2060154087 cites W2074097004 @default.
• W2060154087 cites W2084252557 @default.
• W2060154087 cites W2085328161 @default.
• W2060154087 cites W2094863064 @default.
• W2060154087 cites W2096248361 @default.
• W2060154087 cites W2105089504 @default.
• W2060154087 cites W2109070574 @default.
• W2060154087 cites W2112805154 @default.
• W2060154087 cites W2124008135 @default.
• W2060154087 cites W2133275098 @default.
• W2060154087 cites W2134760231 @default.
• W2060154087 cites W2144842227 @default.
• W2060154087 cites W2145885182 @default.
• W2060154087 cites W2145933752 @default.
• W2060154087 cites W2152854378 @default.
• W2060154087 cites W2155246432 @default.
• W2060154087 cites W2155933617 @default.
• W2060154087 cites W2160444874 @default.
• W2060154087 cites W4251381517 @default.
• W2060154087 cites W4297063097 @default.
• W2060154087 doi "https://doi.org/10.1016/j.molcel.2012.04.020" @default.
• W2060154087 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3398199" @default.
• W2060154087 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/22633953" @default.
• W2060154087 hasPublicationYear "2012" @default.
• W2060154087 type Work @default.
• W2060154087 sameAs 2060154087 @default.
• W2060154087 citedByCount "78" @default.
• W2060154087 countsByYear W20601540872012 @default.
• W2060154087 countsByYear W20601540872013 @default.
• W2060154087 countsByYear W20601540872014 @default.
• W2060154087 countsByYear W20601540872015 @default.
• W2060154087 countsByYear W20601540872016 @default.
• W2060154087 countsByYear W20601540872017 @default.
• W2060154087 countsByYear W20601540872018 @default.
• W2060154087 countsByYear W20601540872019 @default.
• W2060154087 countsByYear W20601540872020 @default.
• W2060154087 countsByYear W20601540872021 @default.
• W2060154087 countsByYear W20601540872022 @default.
• W2060154087 countsByYear W20601540872023 @default.
• W2060154087 crossrefType "journal-article" @default.
• W2060154087 hasAuthorship W2060154087A5034258593 @default.
• W2060154087 hasAuthorship W2060154087A5047177237 @default.
• W2060154087 hasAuthorship W2060154087A5058159306 @default.
• W2060154087 hasAuthorship W2060154087A5079762167 @default.
• W2060154087 hasAuthorship W2060154087A5089651139 @default.
• W2060154087 hasBestOaLocation W20601540871 @default.
• W2060154087 hasConcept C100175707 @default.
• W2060154087 hasConcept C184235292 @default.
• W2060154087 hasConcept C2777730290 @default.
• W2060154087 hasConcept C502942594 @default.
• W2060154087 hasConcept C54355233 @default.
• W2060154087 hasConcept C62478195 @default.
• W2060154087 hasConcept C86803240 @default.
• W2060154087 hasConcept C95444343 @default.
• W2060154087 hasConceptScore W2060154087C100175707 @default.
• W2060154087 hasConceptScore W2060154087C184235292 @default.
• W2060154087 hasConceptScore W2060154087C2777730290 @default.
• W2060154087 hasConceptScore W2060154087C502942594 @default.
• W2060154087 hasConceptScore W2060154087C54355233 @default.
• W2060154087 hasConceptScore W2060154087C62478195 @default.
• W2060154087 hasConceptScore W2060154087C86803240 @default.
• W2060154087 hasConceptScore W2060154087C95444343 @default.
• W2060154087 hasIssue "1" @default.
• W2060154087 hasLocation W20601540871 @default.
• W2060154087 hasLocation W20601540872 @default.
• W2060154087 hasLocation W20601540873 @default.
• W2060154087 hasLocation W20601540874 @default.

• next