IκB Kinases Phosphorylate NF-κB p65 Subunit on Serine 536 in the Transactivation Domain*

Recent investigations have elucidated the cytokine-induced NF-κB activation pathway. IκB kinase (IKK) phosphorylates inhibitors of NF-κB (IκBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-κB. We have examined the possibility that IKK can phosphorylate the p65 NF-κB subunit as well as IκB in the cytokine-induced NF-κB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-α in a time dependent manner similar to IκB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKβ efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-κB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-κB·IκB complex.

[1]  T. Deerinck,et al.  The IKKβ Subunit of IκB Kinase (IKK) is Essential for Nuclear Factor κB Activation and Prevention of Apoptosis , 1999, The Journal of experimental medicine.

[2]  I. Verma,et al.  IKK1-deficient mice exhibit abnormal development of skin and skeleton. , 1999, Genes & development.

[3]  Inder M. Verma,et al.  Severe Liver Degeneration in Mice Lacking the IκB Kinase 2 Gene , 1999 .

[4]  S. Akira,et al.  Limb and skin abnormalities in mice lacking IKKalpha. , 1999, Science.

[5]  T. Deerinck,et al.  Abnormal Morphogenesis But Intact IKK Activation in Mice Lacking the IKKα Subunit of IκB Kinase , 1999 .

[6]  W. Toriumi,et al.  Functional Interactions of Transforming Growth Factor β-activated Kinase 1 with IκB Kinases to Stimulate NF-κB Activation* , 1999, The Journal of Biological Chemistry.

[7]  D. Goeddel,et al.  Embryonic Lethality, Liver Degeneration, and Impaired NF-κB Activation in IKK-β-Deficient Mice , 1999 .

[8]  Quan Zhao,et al.  Mitogen-activated Protein Kinase/ERK Kinase Kinases 2 and 3 Activate Nuclear Factor-κB through IκB Kinase-α and IκB Kinase-β* , 1999, The Journal of Biological Chemistry.

[9]  J. Ninomiya-Tsuji,et al.  The kinase TAK1 can activate the NIK-IκB as well as the MAP kinase cascade in the IL-1 signalling pathway , 1999, Nature.

[10]  M. Mann,et al.  IκB Kinase (IKK)-Associated Protein 1, a Common Component of the Heterogeneous IKK Complex , 1999, Molecular and Cellular Biology.

[11]  W. Greene,et al.  The Proto-Oncogene Cot Kinase Participates in CD3/CD28 Induction of NF-κB Acting through the NF-κB-Inducing Kinase and IκB Kinases , 1999 .

[12]  A. Baldwin,et al.  Activation of Nuclear Factor-κB-dependent Transcription by Tumor Necrosis Factor-α Is Mediated through Phosphorylation of RelA/p65 on Serine 529* , 1998, The Journal of Biological Chemistry.

[13]  R. Gaynor,et al.  HTLV-I Tax Protein Binds to MEKK1 to Stimulate IκB Kinase Activity and NF-κB Activation , 1998, Cell.

[14]  S. Ghosh,et al.  Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. , 1998, Molecular cell.

[15]  H. Nakano,et al.  Differential regulation of IκB kinase α and β by two upstream kinases, NF-κB-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1 , 1998 .

[16]  H. Sakurai,et al.  TGF-beta-activated kinase 1 stimulates NF-kappa B activation by an NF-kappa B-inducing kinase-independent mechanism. , 1998, Biochemical and biophysical research communications.

[17]  G. Haegeman,et al.  p38 and Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase Pathways Are Required for Nuclear Factor-κB p65 Transactivation Mediated by Tumor Necrosis Factor* , 1998, The Journal of Biological Chemistry.

[18]  Mike Rothe,et al.  IκB Kinase-β: NF-κB Activation and Complex Formation with IκB Kinase-α and NIK , 1997 .

[19]  Matthias Mann,et al.  IKK-1 and IKK-2: Cytokine-Activated IκB Kinases Essential for NF-κB Activation , 1997 .

[20]  E. Zandi,et al.  The IκB Kinase Complex (IKK) Contains Two Kinase Subunits, IKKα and IKKβ, Necessary for IκB Phosphorylation and NF-κB Activation , 1997, Cell.

[21]  G. Nabel,et al.  A new member of the I kappaB protein family, I kappaB epsilon, inhibits RelA (p65)-mediated NF-kappaB transcription , 1997, Molecular and cellular biology.

[22]  David M. Rothwarf,et al.  A cytokine-responsive IκB kinase that activates the transcription factor NF-κB , 1997, Nature.

[23]  D. Goeddel,et al.  Identification and Characterization of an IκB Kinase , 1997, Cell.

[24]  A. Israël,et al.  Regulation of IkappaBbeta degradation. Similarities to and differences from IkappaBalpha. , 1997, The Journal of biological chemistry.

[25]  A. Israël,et al.  I kappa B epsilon, a novel member of the IκB family, controls RelA and cRel NF‐κB activity , 1997 .

[26]  D. Wallach,et al.  MAP3K-related kinase involved in NF-KB induction by TNF, CD95 and IL-1 , 1997, Nature.

[27]  T. Maniatis,et al.  Activation of the IκBα Kinase Complex by MEKK1, a Kinase of the JNK Pathway , 1997, Cell.

[28]  David Baltimore,et al.  NF-κB: Ten Years After , 1996, Cell.

[29]  D. Thomas,et al.  Identification of lysine residues required for signal-induced ubiquitination and degradation of I kappa B-alpha in vivo. , 1996, Oncogene.

[30]  M. Karin,et al.  Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation , 1996, Molecular and cellular biology.

[31]  Jill K Thompson,et al.  Role of IB Ubiquitination in Signal-induced Activation of NF-B in Vivo(*) , 1996, The Journal of Biological Chemistry.

[32]  T. Maniatis,et al.  Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[33]  E M Schwarz,et al.  Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. , 1995, Genes & development.

[34]  T. Maniatis,et al.  Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. , 1995, Genes & development.

[35]  M. Schmitz,et al.  Transactivation domain 2 (TA2) of p65 NF-kappa B. Similarity to TA1 and phorbol ester-stimulated activity and phosphorylation in intact cells. , 1995, The Journal of biological chemistry.

[36]  H. Pahl,et al.  Phosphorylation of human I kappa B‐alpha on serines 32 and 36 controls I kappa B‐alpha proteolysis and NF‐kappa B activation in response to diverse stimuli. , 1995, The EMBO journal.

[37]  H. Erdjument-Bromage,et al.  IκB-β regulates the persistent response in a biphasic activation of NF-κB , 1995, Cell.

[38]  M. Czisch,et al.  Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation. , 1994, The Journal of biological chemistry.

[39]  M. Nichols,et al.  Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription. , 1992, The EMBO journal.

[40]  M. Karin,et al.  Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73 , 1991, Nature.

[41]  P. Baeuerle,et al.  The p65 subunit is responsible for the strong transcription activating potential of NF‐kappa B. , 1991, The EMBO journal.

[42]  S. Haskill,et al.  Characterization of an immediate-early gene induced in adherent monocytes that encodes IκB-like activity , 1991, Cell.

[43]  P. Baeuerle,et al.  Function and activation of NF-kappa B in the immune system. , 1994, Annual review of immunology.