The inhibitor of κB kinase β (IKKβ) phosphorylates IκBα twice in a single binding event through a sequential mechanism

Abstract

Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre–steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s−1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1–54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event. Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre–steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s−1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1–54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event. The nuclear factor κB (NF-κB)2 family of transcription factors are evolutionarily conserved master regulators of cell proliferation, innate immunity, inflammation, cell differentiation, and apoptosis (1Hayden M.S. Ghosh S. Shared principles in NF-kappaB signaling.Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3638) Google Scholar, 2Vallabhapurapu S. Karin M. Regulation and function of NF-kappaB transcription factors in the immune system.Annu. Rev. Immunol. 2009; 27: 693-733Crossref PubMed Scopus (2112) Google Scholar). Dysregulation of NF-κB is associated with many disorders, including cancer, autoimmune disease, neurodegenerative diseases, arthritis, and diabetes (3Karin M. Nuclear factor-kappaB in cancer development and progression.Nature. 2006; 441: 431-436Crossref PubMed Scopus (3042) Google Scholar, 4Courtois G. Gilmore T.D. Mutations in the NF-kappaB signaling pathway: implications for human disease.Oncogene. 2006; 25: 6831-6843Crossref PubMed Scopus (420) Google Scholar, 5Baker R.G. Hayden M.S. Ghosh S. NF-kappaB, inflammation, and metabolic disease.Cell Metab. 2011; 13: 11-22Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar). Thus, investigation of the molecular mechanisms of NF-κB activation is important for our understanding of human disease. The mammalian NF-κB family consists of RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2), which form 15 separate homodimeric or heterodimeric complexes (6Oeckinghaus A. Ghosh S. The NF-kappaB family of transcription factors and its regulation.Cold Spring Harb. Perspect. Biol. 2009; 1: a000034Crossref PubMed Scopus (1833) Google Scholar). In resting cells, NF-κB dimers containing RelA, RelB, and/or c-Rel are sequestered in the cytoplasm through interactions with Inhibitor of κB (IκB) proteins IκBα, IκBβ, or IκBε. In contrast, NF-κB dimers containing p100 and p105 are localized to the cytoplasm through a C-terminal inhibitory domain containing multiple ankyrin repeat motifs. In response to various stimuli, NF-kB is activated through one of two separate pathways: the canonical or the noncanonical pathway. Within the canonical pathway, a Ser/Thr-specific IκB kinase (IKK) complex phosphorylates IκB proteins within a conserved DSGXXS destruction box motif, leading to IκB polyubiquitination, 26S proteasome-mediated degradation, and subsequent NF-κB release. Free NF-κB dimers then translocate to the nucleus to regulate transcription. In the noncanonical pathway, the IKK complex phosphorylates p100 to induce proteolytic processing of p100 to the activated NF-κB2 subunit p52, which also localizes to the nucleus. The IKK complex also catalyzes the phosphorylation of several protein substrates within alternative signaling pathways, and this activity is thought to coordinate the functions of the NF-κB pathways with other cellular pathways (reviewed in reference (7Hinz M. Scheidereit C. The IkappaB kinase complex in NF-kappaB regulation and beyond.EMBO Rep. 2014; 15: 46-61Crossref PubMed Scopus (361) Google Scholar)). The IKK complex consists of the nonenzymatic protein NEMO (NF-κB essential modulator, also called IKKγ) and a homodimer or heterodimer of the catalytic subunits IKKα and IKKβ (8Chen Z.J. Parent L. Maniatis T. Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity.Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar, 9DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB.Nature. 1997; 388: 548-554Crossref PubMed Scopus (1919) Google Scholar, 10Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation.Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1600) Google Scholar, 11Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. et al.Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation.Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar, 12Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. et al.IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.Mol. Cell Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar). Although IKKα and IKKβ share 54% amino acid sequence identity, these kinases possess distinct substrate specificities. For example, IKKα predominantly catalyzes phosphorylation of p100 within the noncanonical pathway (13Xiao G. Fong A. Sun S.C. Induction of p100 processing by NF-kappaB-inducing kinase involves docking IkappaB kinase alpha (IKKalpha) to p100 and IKKalpha-mediated phosphorylation.J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, G. G. G. et by IKKalpha of a signaling PubMed Scopus Google Scholar). In contrast, IKKβ is for the phosphorylation of IκBβ, within the canonical pathway E. Karin M. phosphorylation of IkappaB by IKKalpha and and 1998; PubMed Google Scholar, F. in the IkappaB kinase 1999; PubMed Scopus Google Scholar, Delhase M. T. M. et subunit of IkappaB kinase (IKK) is essential for nuclear factor activation and of 1999; PubMed Scopus Google Scholar). that IKKβ a of kinase domain a domain and a C-terminal domain S. A. R. et of a human IkappaB kinase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar, S. D.B. et for IkappaB kinase activation Biol. PubMed Scopus Google Scholar). The contains activation with the and in human IKKβ, activation is essential for the activity of IKKβ analysis that these residues to IKKβ whereas substitution of these residues with phosphomimetic residues the kinase constitutively F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. et al.IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.Mol. Cell Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar, F. Murray B.W. Shevchenko A. Bennett B.L. Li et and IkappaB kinases essential for NF-kappaB 1997; PubMed Scopus Google Scholar, M. Hayakawa M. Karin M. and regulation of IkappaB kinase activity through subunit 1999; PubMed Scopus Google Scholar). Although the phosphorylation of IκB proteins by IKKβ is a within the canonical pathway of NF-κB the molecular mechanism of IKKβ is we pre–steady-state kinetic analysis to investigate the molecular mechanism of IκBα phosphorylation catalyzed by constitutively IKKβ. Our data that IKKβ phosphorylates full-length IκBα twice within the conserved DSGXXS destruction box a single binding event. Our also that IKKβ phosphorylates IκBα at followed by Ser36. that these of the kinetic mechanism of IKKβ the IKK and The activity of human IKKβ is phosphorylation of activation residues and within the kinase domain F. Murray B.W. Shevchenko A. Bennett B.L. Li et and IkappaB kinases essential for NF-kappaB 1997; PubMed Scopus Google Scholar, M. Hayakawa M. Karin M. and regulation of IkappaB kinase activity through subunit 1999; PubMed Scopus Google Scholar). In to the catalytic of activated IKKβ, we to a constitutively IKKβ phosphomimetic amino acid substitution F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. et al.IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.Mol. Cell Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar, F. Murray B.W. Shevchenko A. Bennett B.L. Li et and IkappaB kinases essential for NF-kappaB 1997; PubMed Scopus Google for our Within the canonical pathway of NF-κB IKKβ phosphorylates IκBα at residues Ser32 and Ser36 1, and to induce the proteolytic degradation of IκBα and the subsequent of NF-κB IκBα is also at other in within the C-terminal domain phosphorylation of alpha by kinase S. A. PubMed Scopus Google Scholar, R. C. S. Phosphorylation of IkappaBalpha in the C-terminal domain by kinase protein Biol. 1996; PubMed Google Scholar). phosphorylation of residues within the destruction box motif, phosphorylation of residues within the domain IκBα for and 26S proteasome-mediated degradation R. C. S. Phosphorylation of IkappaBalpha in the C-terminal domain by kinase protein Biol. 1996; PubMed Google Scholar). kinetic analysis that IKKβ phosphorylates IκBα by using a mechanism Li IkappaB kinases alpha and a kinetic mechanism and are by and Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google that IKKβ IκBα and ATP in any to IKKβ we to the pre–steady-state kinetic of IKKβ-catalyzed phosphorylation of IκBα by first IKKβ with the IκBα protein substrate to the IKKβ•IκBα complex and the by the of To the molecular mechanism of IKKβ-catalyzed phosphorylation of full-length IκBα, the affinity of ATP (Kd) for the IKKβ•IκBα complex and the maximum observed rate (kp) of IKKβ-catalyzed phosphorylation were by using pre–steady-state kinetic To this we the ATP of the phosphorylation rate of IKKβ IKKβ was in a 4-fold to the IκBα substrate to that nearly of the IκBα substrate was by the IKKβ to IκBα with (Kd) of R. F. M. R. T. et of and phosphorylation of IkappaB by IkappaB kinase with cellular regulation in human 1999; PubMed Scopus Google and we that of the IκBα substrate was by IKKβ our A of full-length IκBα and IKKβ was with a containing of for various The were then by and phosphorylation of IκBα was by using A was a was IKKβ and IκBα by and a with IKKβ, IκBα, and ATP The of IκBα at was to the and a function of The that IKKβ IκBα with fast exponential phase rate followed by a slower linear phase rate and these data were to The of the observed in were in the that the exponential rate to phosphorylation of IκBα at residues Ser32 and Ser36 within the destruction box motif, and the slower linear rate either the phosphorylation of IκBα at of the destruction box motif, the domain or phosphorylation of IκBα substrate that was in a complex by IKKβ, or The exponential of IKKβ-catalyzed phosphorylation of IκBα were then a function of ATP and to a of 0.32 s−1 for the maximum observed rate of IKKβ-catalyzed phosphorylation of full-length IκBα and a of 12 for ATP binding to the IKKβ•IκBα phosphorylation of full-length IκBα and full-length IκBα amino acid substitution A of IKKβ and the full-length IκBα was with a containing a and for various Phosphorylation of the full-length IκBα substrates was then to the and phosphorylation a function of to or to The the to The exponential phase the exponential phase and the linear phase were WT IκBα and its various of IKKβ, IκB Kinase To investigate the molecular mechanism of IKKβ we to the of IKKβ to either Ser32 or Ser36 within the destruction box of IκBα 1, and To this we the phosphorylation of separate full-length IκBα amino acid substitution in which either Ser32 or Ser36 were to to the phosphorylation to the and of a or Cys to the and of the phosphorylation substitution mutant was also in which residues within the destruction box were The of IκBα was a function of and to data the are in and of the full-length IκBα substrates by IKKβ were to 1, A is the exponential phase is the exponential rate and is the linear rate of full-length IκBα phosphorylation by IκBα phosphorylation phase amplitude rate rate Phosphorylation of the full-length IκBα substrates by IKKβ were to 1, A is the exponential phase is the exponential rate and is the linear rate in a the kinetic of IKKβ-catalyzed phosphorylation of IκBα and the IκBα amino acid substitution mutants, the amplitude of the exponential phase by phosphorylation of of the IκBα single amino acid substitution was reduced approximately to the exponential phase amplitude of IκBα and the observed exponential phase amplitude by phosphorylation of the amino acid substitution mutant was reduced by to IκBα and These data that the exponential phase of IKKβ-catalyzed phosphorylation of IκBα to phosphorylation of IκBα at Ser32 and Ser36. Interestingly, the exponential phase of IκBα phosphorylation to a single rate that IKKβ IκBα at Ser32 and Ser36 a that IKKβ IκBα and of the IKKβ•IκBα Thus, we that IKKβ phosphorylates IκBα within a single binding event. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic substitution mutant was by approximately to the exponential rate of phosphorylation of IκBα and In contrast, the exponential rate of phosphorylation for of the other IκBα single amino acid substitution mutants, including the phosphomimetic amino acid substitution was reduced by approximately to IκBα phosphorylation and These data suggest that IκBα is at the rate at which IKKβ catalyzes the phosphorylation at Ser36 is phosphorylation of IκBα Ser36 the rate of phosphorylation at The linear rate of IKKβ-catalyzed phosphorylation of the IκBα single amino acid substitution was to the linear rate of IKKβ-catalyzed phosphorylation of IκBα and These are with the that the linear phase of phosphorylation of IκBα and the IκBα amino acid substitution phosphorylation of IκBα at of the destruction box 1, and by IKKβ. Interestingly, the linear rate of IKKβ-catalyzed phosphorylation of the amino acid substitution mutant was to 4-fold faster than for IκBα and the IκBα single amino acid substitution and suggesting that IKKβ-catalyzed phosphorylation of Ser32 and Ser36 the linear domain phosphorylation rate in these Although IKKβ phosphorylates IκBα within the destruction box 1, and we that IKKβ is also of IκBα at within the C-terminal within the domain To the that the observed of IKKβ-catalyzed phosphorylation of full-length IκBα were by phosphorylation of IκBα residues of the destruction box motif, we the phosphorylation of fusion proteins 1, and These GST-IκBα(1–54) protein substrates the C-terminal of IκBα. Thus, we that substrate phosphorylation by IKKβ reduced these protein the IKKβ-catalyzed phosphorylation of GST-IκBα(1–54) bearing the first 54 residues of IκBα, including the destruction box motif, and separate amino acid substitution of GST-IκBα(1–54) 1, and pre–steady-state The of IκBα substrate was a function of A and In to phosphorylation of the full-length IκBα substrates A and a single exponential phase of GST-IκBα(1–54) was observed A and these data were to data the are in and The of a linear phase was to either a in phosphorylation of the GST-IκBα(1–54) fusion protein at of the destruction box to phosphorylation of the full-length IκBα substrates and/or a in the of protein the rate of phosphorylation was to A and of of the substrates by IKKβ were to A is the exponential phase amplitude and is the exponential rate phosphorylation by phosphorylation phase amplitude rate that phosphorylation of by IKKβ was to Phosphorylation of the substrates by IKKβ were to A is the exponential phase amplitude and is the exponential rate A that phosphorylation of by IKKβ was to in a that the C-terminal of IκBα with the and of IKKβ G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar). to the destruction box of IκBα for phosphorylation by the of IKKβ and the molecular mechanism of IKKβ. Interestingly, in with our of IKKβ-catalyzed phosphorylation of the full-length IκBα the amplitude of the exponential phase of GST-IκBα(1–54) phosphorylation was approximately than the exponential phase amplitude of of the GST-IκBα(1–54) single amino acid substitution and Thus, we that interactions IKKβ and the C-terminal of IκBα are for IKKβ to IκBα twice within a single binding event. the full-length IκBα and the GST-IκBα(1–54) substrates were at exponential and and suggesting that interactions IKKβ and the C-terminal domain of IκBα the exponential rate of IKKβ-catalyzed in to the with the full-length IκBα amino acid substitution and the phosphomimetic amino acid substitution mutant was at a rate to the GST-IκBα(1–54) and the GST-IκBα(1–54) single amino acid substitution were at a rate that was reduced by approximately to GST-IκBα(1–54) and Thus, substitution of destruction box residues Ser32 or Ser36 of the GST-IκBα(1–54) substrates the exponential rate of phosphorylation of these substrates to a than substitution of Ser32 or Ser36 within the full-length IκBα that interactions IKKβ and the of IκBα the exponential rate of IKKβ-catalyzed these interactions the of phosphorylation of IκBα Ser32 and Ser36. The pathways of NF-κB regulation are of of the NF-kappaB pathway in the of and PubMed Scopus Google Scholar, E. G. The pathway: a for novel molecular in 2008; PubMed Scopus Google Scholar). Thus, understanding of the molecular mechanisms of the in the regulation of these pathways, IKKβ, for the development of novel for the of a of human diseases, The IKKβ-catalyzed phosphorylation of IκB protein substrates is essential within the canonical pathway of NF-κB Thus, the IKKβ pathway the of M.S. Ghosh S. NF-kappaB, the first and PubMed Scopus Google Scholar). of IKKβ S. A. R. et of a human IkappaB kinase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar, S. D.B. et for IkappaB kinase activation Biol. PubMed Scopus Google the molecular mechanism of IKKβ-catalyzed phosphorylation we a pre–steady-state kinetic to investigate the phosphorylation of IκBα by a constitutively form of human IKKβ. we to the phosphorylation of full-length IκBα and fusion protein substrates 1, and to investigate important interactions IKKβ and the C-terminal domain of IκBα. that IKKβ phosphorylates IκBα twice within a single binding and that phosphorylation of Ser32 of IκBα increases the rate of phosphorylation of Ser36. Thus, we a model IKKβ IκBα and phosphorylates IκBα first at followed by a phosphorylation at Ser36. experiments that the and of IKKβ to the C-terminal of IκBα G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar). a binding the of IκBα for phosphorylation at Ser32 and Ser36 by the kinase domain of IKKβ. of the domain of IKKβ in the of phosphorylation the Ser32 and Ser36 of IκBα to its C-terminal domain G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar). the model in IKKβ Ser36 followed by or either Ser32 or Ser36 Our kinetic data and that the model in to the two IKKβ to the IκBα phosphorylation of the two IκBα phosphorylation the IKKβ kinase domain IκBα at Ser32 first by of a for phosphorylation of the amino acid sequence phosphorylation of IκBα at the Ser32 possess a and acid Thus, phosphorylation of the amino acid sequence Ser36 also the amino acid sequence of the IκBα C-terminal by IKKβ important for this phosphorylation the exponential rate of phosphorylation of the full-length IκBα(S32D) phosphomimetic substitution mutant was by approximately with to the IκBα substrate and whereas this in the exponential rate of phosphorylation was observed with the fusion protein which the C-terminal of IκBα and this phosphorylation of Ser32 followed by Ser36 is a for IKKβ phosphorylation of Ser32 and Ser36 of one and is that IKKβ is to in the amino acid sequence of the IκBα destruction box by the that the exponential rate of phosphorylation of IκBα amino acid substitution for the phosphomimetic was slower than the exponential rate of phosphorylation of IκBα substrates bearing the WT destruction box and IKKβ IκBα at Ser32 and Ser36 to induce the and degradation of IκBα and the subsequent of NF-κB are to protein substrates at multiple through one of two separate mechanisms (reviewed in reference mechanism and 19: PubMed Scopus Google a in which a kinase phosphorylates a substrate binding or a in which a kinase phosphorylates a substrate two or binding event. IKKβ a mechanism to IκBα, then we that pre–steady-state analysis separate for the first and phosphorylation to the and our that the exponential phase of IKKβ-catalyzed phosphorylation of full-length IκBα to a single rate is with the phosphorylation of Ser32 and Ser36 a In of this the amplitude of the exponential phase by phosphorylation of of the single amino acid substitution in the IκBα destruction box was reduced approximately to the exponential phase amplitude of IκBα and to amino acid at Ser32 or Ser36 IκBα to and degradation and IκBα a of NF-κB in T. S. Phosphorylation of human and and activation in response to PubMed Scopus Google Scholar, F. et phosphorylation alpha to the PubMed Scopus Google Scholar, S. L. G. of by PubMed Scopus Google Scholar, J.A. et of a response domain in alpha to multiple pathways for Biol. 15: PubMed Google Scholar). Thus, a single phosphorylation catalyzed by IKKβ is to The of two phosphorylation within a single binding is mechanism by which IKKβ IKKβ is to two separate phosphorylation with multiple including IκBα, IκBβ, and (reviewed in reference M.S. Ghosh S. to 2004; PubMed Scopus Google suggesting that the of two phosphorylation a mechanism by which IKKβ phosphorylates protein IKKα is to at multiple residues to induce the proteolytic activation of this NF-κB subunit (13Xiao G. Fong A. Sun S.C. Induction of p100 processing by NF-kappaB-inducing kinase involves docking IkappaB kinase alpha (IKKalpha) to p100 and IKKalpha-mediated phosphorylation.J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google and the called and kinase 1, are to the factor proteins and at two or to these regulators of the innate response (reviewed in reference S. The innate to 2008; PubMed Scopus Google Scholar)). Thus, the of multiple phosphorylation a single binding a conserved mechanism of the IKK and were the ATP and A was by using the to human IKKβ bearing a C-terminal affinity The kinase was by of in in the were and IKKβ was G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar). cell IKKβ were in A and by and by at for The cell was then with and proteins were by with A. proteins were with a of to with and were then and to a The was with and proteins were using a linear of to with containing IKKβ were and to a to The containing IKKβ were then and IKKβ was to the of of human IκBα was a protein in by using G. Li G. et of of kinase 2011; PubMed Scopus Google Scholar). The fusion protein followed by a and residues to of IκBα. IκBα was in at for The were then in and by and by at for The cell was with and proteins were by with proteins were by using a linear of to 15 reduced with 15 reduced containing IκBα were then and the was full-length IκBα by with in analysis of the and of catalyzed by human 2011; PubMed Scopus Google Scholar). the protein were to a and with binding IκBα was then by using a linear of to with containing IκBα were then to a with containing full-length IκBα were and full-length IκBα amino acid substitution were and by using the IκBα and IκBα amino acid substitution were to the of of L. J.A. of a the 2009; PubMed Scopus Google Scholar, J.A. T. of the by PubMed Scopus Google Scholar). The GST-IκBα(1–54) fusion protein was in E. fusion protein affinity followed by a a and residues to 54 of IκBα. The GST-IκBα(1–54) fusion protein was in at for The were then in binding using a and by at for The cell was then with and proteins were by with binding proteins were by using a linear of to with containing GST-IκBα(1–54) were and to a with containing GST-IκBα(1–54) were then and for full-length IκBα The GST-IκBα(1–54) fusion protein substrate and GST-IκBα(1–54) amino acid substitution were to the of of kinetic were performed in kinase at at The are A containing IKKβ and full-length IκBα was with a containing to and a The were at various by the of to a of were by using a were by using and by using a The of IκBα was to the at and the data was a function of The of at were to by using the A is the exponential phase is the is the exponential phase and is the linear rate mechanism of catalyzed by human PubMed Scopus Google Scholar, investigation of the and of human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. the kinetic mechanism of catalyzed by 2009; PubMed Scopus Google Scholar). The were then a function of ATP and was to the maximum observed rate (kp) of IκBα phosphorylation catalyzed by IKKβ and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) J.A. of catalyzed by a 2008; PubMed Scopus Google A containing IKKβ and either full-length IκBα or the IκBα amino acid substitution mutant was with a containing and a The were and was The of full-length IκBα phosphorylation were then to The pre–steady-state kinetic of the GST-IκBα(1–54) substrates were for the full-length IκBα substrates the IKKβ phosphorylation of the GST-IκBα(1–54) substrates a linear the of GST-IκBα(1–54) were to A is the exponential phase is the and is the observed exponential phase The contains data within the The that of interest with the of this to and for of the S. S. A. A. G. and A. A. and S. S. and A. A. S. and was supported by of and to S. 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