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.

Post‐translational activation of the higher eukaryotic transcription factor NF‐kappa B requires both phosphorylation and proteolytic degradation of the inhibitory subunit I kappa B‐alpha. Inhibition of proteasome activity can stabilize an inducibly phosphorylated form of I kappa B‐alpha in intact cells, suggesting that phosphorylation targets the protein for degradation. In this study, we have identified serines 32 and 36 in human I kappa B‐alpha as essential for the control of I kappa B‐alpha stability and the activation of NF‐kappa B in HeLa cells. A point mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. This and various other Ser32 and Ser36 mutants behaved as potent dominant negative I kappa B proteins attenuating kappa B‐dependent transactivation in response to OA, phorbol 12‐myristate 13‐acetate (PMA) and tumor necrosis factor‐alpha (TNF). While both endogenous and transiently expressed wild‐type I kappa B‐alpha were proteolytically degraded in response to PMA and TNF stimulation of cells, the S32/36A mutant of I kappa B‐alpha remained largely intact under these conditions. Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kinase system to phosphorylate and thereby destabilize I kappa B‐alpha, leading to NF‐kappa B activation.

[1]  W. Greene,et al.  Human T-cell leukemia virus type I Tax activation of NF-kappa B/Rel involves phosphorylation and degradation of I kappa B alpha and RelA (p65)-mediated induction of the c-rel gene , 1994, Molecular and cellular biology.

[2]  C. Scheidereit,et al.  Activation of NF‐kappa B in vivo is regulated by multiple phosphorylations. , 1994, The EMBO journal.

[3]  P. Dobrzanski,et al.  Rapid induction in regenerating liver of RL/IF-1 (an I kappa B that inhibits NF-kappa B, RelB-p50, and c-Rel-p50) and PHF, a novel kappa B site-binding complex , 1992, Molecular and cellular biology.

[4]  I. Verma,et al.  Tumor necrosis factor alpha-induced phosphorylation of I kappa B alpha is a signal for its degradation but not dissociation from NF-kappa B. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Karin,et al.  Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B , 1995, Molecular and cellular biology.

[6]  C. Scheidereit,et al.  Common structural constituents confer I kappa B activity to NF‐kappa B p105 and I kappa B/MAD‐3. , 1993, The EMBO journal.

[7]  R. Pearson,et al.  Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. , 1991, Methods in enzymology.

[8]  B. Kistler,et al.  Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells. , 1994, The EMBO journal.

[9]  S. Mizel,et al.  In vitro activation and nuclear translocation of NF-kappa B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C , 1989, Molecular and cellular biology.

[10]  V. Bours,et al.  The oncoprotein Bcl-3 directly transactivates through κB motifs via association with DNA-binding p50B homodimers , 1993, Cell.

[11]  N. Rice,et al.  In vivo control of NF‐kappa B activation by I kappa B alpha. , 1993, The EMBO journal.

[12]  G. Franzoso,et al.  Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Schmitz,et al.  Proteins controlling the nuclear uptake of NF-κB, Rel and dorsal , 1991 .

[14]  W. Greene,et al.  Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Tom Maniatis,et al.  The ubiquitinproteasome pathway is required for processing the NF-κB1 precursor protein and the activation of NF-κB , 1994, Cell.

[16]  M. Grilli,et al.  NF-kappa B and Rel: participants in a multiform transcriptional regulatory system. , 1993, International review of cytology.

[17]  R. Steward,et al.  Dissociation of the dorsal-cactus complex and phosphorylation of the dorsal protein correlate with the nuclear localization of dorsal , 1993, The Journal of cell biology.

[18]  B. Franza,et al.  Identification of complex formation between two intracellular tyrosine kinase substrates: human c-Rel and the p105 precursor of p50 NF-kappa B. , 1992, Oncogene.

[19]  N. Rice,et al.  The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers , 1995, Molecular and cellular biology.

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

[21]  W. Greene,et al.  I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding. , 1992, Molecular biology of the cell.

[22]  Dorsal developments , 1994, Nature.

[23]  G. Franzoso,et al.  Kinetic analysis of human T-cell leukemia virus type I Tax-mediated activation of NF-kappa B , 1994, Molecular and cellular biology.

[24]  A. Brasier,et al.  Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. , 1989, BioTechniques.

[25]  F. Bach,et al.  Cytokine‐inducible expression in endothelial cells of an I kappa B alpha‐like gene is regulated by NF kappa B. , 1993, The EMBO journal.

[26]  M. Figueiredo-Pereira,et al.  A New Inhibitor of the Chymotrypsin‐Like Activity of the Multicatalytic Proteinase Complex (20S Proteasome) Induces Accumulation of Ubiquitin‐Protein Conjugates in a Neuronal Cell , 1994, Journal of neurochemistry.

[27]  A. Israël,et al.  NF-κB and related proteins: Rel/dorsal homologies meet ankyrin-like repeats , 1992 .

[28]  M. Read,et al.  Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-kappa B proteins in human monocytic THP-1 cells. , 1993, The Journal of biological chemistry.

[29]  Y. Ben-Neriah,et al.  Rapid proteolysis of IκB-α is necessary for activation of transcription factor NF-κB , 1993, Nature.

[30]  D. Baltimore,et al.  Activation in vitro of NF-κB" by phosphorylation of its inhibitor IκB" , 1990, Nature.

[31]  S. Goodbourn,et al.  Proteolytic degradation of MAD3 (IϰBα) and enhanced processing of the NF-ϰB precursor p105 are obligatory steps in the activation of NF-ϰB , 1993 .

[32]  Tony Hunter,et al.  The regulation of transcription by phosphorylation , 1992, Cell.

[33]  M. Wigler,et al.  Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor , 1978, Cell.

[34]  A. Baldwin,et al.  Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[35]  A. Baldwin,et al.  Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation , 1993, Molecular and cellular biology.

[36]  David Baltimore,et al.  Regulation of the NF-?B/rel transcription factor and I?B inhibitor system , 1993 .

[37]  I. Verma,et al.  Direct association of pp40/I kappa B beta with rel/NF-kappa B transcription factors: role of ankyrin repeats in the inhibition of DNA binding activity. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[38]  D. Longo,et al.  NF-kappa B/Rel family members are physically associated phosphoproteins. , 1994, The Biochemical journal.

[39]  P. Morin,et al.  The IκB proteins: members of a multifunctional family , 1993 .

[40]  O. ole-MoiYoi,et al.  Evidence for the induction of casein kinase II in bovine lymphocytes transformed by the intracellular protozoan parasite Theileria parva. , 1993, The EMBO journal.

[41]  J. Ostrowski,et al.  A serine/threonine kinase activity is closely associated with a 65-kDa phosphoprotein specifically recognized by the kappa B enhancer element. , 1991, The Journal of biological chemistry.

[42]  D. Baltimore,et al.  Rel-associated pp40: an inhibitor of the rel family of transcription factors. , 1991, Science.

[43]  P. Herrlich,et al.  Infection with the intracellular protozoan parasite Theileria parva induces constitutively high levels of NF-kappa B in bovine T lymphocytes , 1989, Molecular and cellular biology.

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

[45]  P. Leder,et al.  Casein kinase II alpha transgene-induced murine lymphoma: relation to theileriosis in cattle , 1995, Science.

[46]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[47]  D. Ballard,et al.  Proteolytic Processing of NF-B/IB in Human Monocytes , 1995, The Journal of Biological Chemistry.

[48]  D. Baltimore,et al.  I kappa B: a specific inhibitor of the NF-kappa B transcription factor. , 1988, Science.

[49]  Y. Tan,et al.  Differential induction of nuclear NF-kappa B by protein phosphatase inhibitors in primary and transformed human cells. Requirement for both oxidation and phosphorylation in nuclear translocation. , 1993, The Journal of biological chemistry.

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

[51]  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.

[52]  P. Baeuerle,et al.  A proteasome inhibitor prevents activation of NF‐kappa B and stabilizes a newly phosphorylated form of I kappa B‐alpha that is still bound to NF‐kappa B. , 1994, The EMBO journal.

[53]  Steffen Jung,et al.  In Vivo Stimulation of IkB Phosphorylation Is Not Sufficient To Activate NF-kB , 1995 .

[54]  N. Rice,et al.  In vivo control of NF-xB activation by IxBa , 2022 .