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PMID: 8887627 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Basal phosphorylation of the PEST domain in the I(kappa)B(beta) regulates its functional interaction with the c-rel proto-oncogene product.

Molecular and cellular biology ·Vol. 16 ·No. 11 ·1996-11-00 ·Pages 5974-84

Chu ZL, McKinsey TA, Liu L, Qi X, Ballard DW

Abstract

The product of the c-rel proto-oncogene (c-Rel) belongs to the NF-kappaB/Rel family of polypeptides and has been implicated in the transcriptional control of cell proliferation and immune function. In human T lymphocytes, c-Rel is sequestered in the cytoplasmic compartment by constitutively phosphorylated inhibitors, including I(kappa)B(alpha) and I(kappa)B(beta). Studies with bacterially expressed forms of these inhibitory proteins revealed that unphosphorylated I(kappa)B(alpha) but not I(kappa)B(beta) assembles with c-Rel and inhibits its DNA binding activity. Furthermore, latent I(kappa)B(beta)-c-Rel complexes derived from mammalian cells were sensitive to phosphatase treatment, whereas I(kappa)B(alpha)-c-Rel complexes were resistant. We have identified a constitutive protein kinase in unstimulated T cells that associates with and phosphorylates I(kappa)B(beta) in vitro. The substrate specificity, electrophoretic mobility, and antigenic properties of this I(kappa)B(beta)-associated kinase (BAK) suggest identity with casein kinase II (CKII), an enzyme known to mediate basal phosphorylation of I(kappa)B(alpha). Phosphorylation of recombinant I(kappa)B(beta) by either BAK or CKII restored the capacity of this inhibitor to antagonize the DNA binding activity of c-Rel. Peptide mapping and mutational analyses localized the bulk of the basal phosphorylation sites in I(kappa)B(beta) to the C-terminal PEST domain, which contains two potential acceptors for CKII-mediated phosphoryl group transfer (Ser-313 and Ser-315). Point mutations introduced into the full-length inhibitor at Ser-313 and Ser-315 led to a significant reduction in the phosphorylation of I(kappa)B(beta) and severely impaired its c-Rel inhibitory function in vivo. Taken together, these findings strongly suggest that basal phosphorylation of the PEST domain of I(kappa)B(beta) at consensus CKII sites is required for the efficient formation of latent I(kappa)B(beta)-c-Rel complexes.

MeSH Terms
Amino Acid Sequence Binding Sites Calcium-Calmodulin-Dependent Protein Kinase Type 2 Calcium-Calmodulin-Dependent Protein Kinases/metabolism Cell Line DNA-Binding Proteins/chemistry,metabolism Enhancer Elements, Genetic Humans I-kappa B Proteins Kinetics Molecular Sequence Data NF-KappaB Inhibitor alpha Oligodeoxyribonucleotides Peptide Fragments/metabolism Phosphorylation Protein-Tyrosine Kinases/metabolism Proto-Oncogene Mas Proto-Oncogene Proteins/chemistry,metabolism Proto-Oncogene Proteins c-rel Proto-Oncogenes Recombinant Proteins/chemistry,metabolism Substrate Specificity T-Lymphocytes Tumor Cells, Cultured
Chemicals
DNA-Binding Proteins I kappa B beta protein I-kappa B Proteins MAS1 protein, human NFKBIA protein, human Oligodeoxyribonucleotides Peptide Fragments Proto-Oncogene Mas Proto-Oncogene Proteins Proto-Oncogene Proteins c-rel Recombinant Proteins NF-KappaB Inhibitor alpha Protein-Tyrosine Kinases Calcium-Calmodulin-Dependent Protein Kinase Type 2 Calcium-Calmodulin-Dependent Protein Kinases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Chu Z L
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
McKinsey T A
Liu L
Qi X
Ballard D W
References (75)
75 references, click to expand
  1. A calcium-dependent antibody for identification and purification of recombinant proteins.
    Biotechniques. 1989 Jun;7(6):580-9 PMID: 2698650
  2. The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the rel oncogene product.
    Cell. 1990 Sep 7;62(5):1007-18 PMID: 2203531
  3. The v-rel oncogene encodes a kappa B enhancer binding protein that inhibits NF-kappa B function.
    Cell. 1990 Nov 16;63(4):803-14 PMID: 2225078
  4. NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells.
    Proc Natl Acad Sci U S A. 1990 Dec;87(24):10028-32 PMID: 2263603
  5. Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B.
    Science. 1991 Mar 22;251(5000):1490-3 PMID: 2006423
  6. Activation of the interleukin-2 receptor alpha gene: regulatory role for DNA-protein interactions flanking the kappa B enhancer.
    New Biol. 1989 Oct;1(1):83-92 PMID: 2518691
  7. A novel NF-kappa B complex containing p65 homodimers: implications for transcriptional control at the level of subunit dimerization.
    Mol Cell Biol. 1993 Dec;13(12):7826-35 PMID: 8246997
  8. High levels of c-rel expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro.
    Cell. 1993 Dec 3;75(5):899-912 PMID: 8252626
  9. Promoter analysis of the gene encoding the I kappa B-alpha/MAD3 inhibitor of NF-kappa B: positive regulation by members of the rel/NF-kappa B family.
    EMBO J. 1993 Dec 15;12(13):5043-9 PMID: 8262046
  10. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B.
    Cell. 1994 Sep 9;78(5):773-85 PMID: 8087845
  11. Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha.
    Nucleic Acids Res. 1994 Sep 11;22(18):3787-92 PMID: 7937093
  12. Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity.
    Cell. 1991 Jun 28;65(7):1281-9 PMID: 1829648
  13. The rel-associated pp40 protein prevents DNA binding of Rel and NF-kappa B: relationship with I kappa B beta and regulation by phosphorylation.
    Genes Dev. 1991 Aug;5(8):1464-76 PMID: 1907941
  14. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.
    Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8972-6 PMID: 1924358
  15. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates.
    Methods Enzymol. 1991;201:110-49 PMID: 1943760
  16. Purified I kappa B-beta is inactivated upon dephosphorylation.
    J Biol Chem. 1992 Jan 5;267(1):239-46 PMID: 1309735
  17. The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression.
    Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1875-9 PMID: 1542686
  18. A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells.
    Mol Cell Biol. 1992 Apr;12(4):1736-46 PMID: 1372388
  19. 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.
    Proc Natl Acad Sci U S A. 1992 May 15;89(10):4333-7 PMID: 1533932
  20. Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos.
    Science. 1992 May 15;256(5059):1014-8 PMID: 1589769
  21. Kappa B site-dependent activation of the interleukin-2 receptor alpha-chain gene promoter by human c-Rel.
    Mol Cell Biol. 1992 Sep;12(9):4067-75 PMID: 1508203
  22. The c-rel protooncogene product represses NF-kappa B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus.
    Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1023-7 PMID: 8430069
  23. Effect of CD28 signal transduction on c-Rel in human peripheral blood T cells.
    Mol Cell Biol. 1994 Dec;14(12):7933-42 PMID: 7969133
  24. The interleukin 2 CD28-responsive complex contains at least three members of the NF kappa B family: c-Rel, p50, and p65.
    Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1696-700 PMID: 8383323
  25. NF-kappa B and Rel: participants in a multiform transcriptional regulatory system.
    Int Rev Cytol. 1993;143:1-62 PMID: 8449662
  26. Activation of primary human T-lymphocytes through CD2 plus CD28 adhesion molecules induces long-term nuclear expression of NF-kappa B.
    Cell Growth Differ. 1993 Apr;4(4):329-39 PMID: 8098618
  27. Characterization of protein kinase activities associated with p53-large-T immune complexes from SV40-transformed rat cells.
    Oncogene. 1993 Aug;8(8):2193-205 PMID: 8393163
  28. NF-kappa B subunit-specific regulation of the interleukin-8 promoter.
    Mol Cell Biol. 1993 Oct;13(10):6137-46 PMID: 8413215
  29. The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors.
    Genes Dev. 1993 Nov;7(11):2064-70 PMID: 8224838
  30. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain.
    Genes Dev. 1993 Nov;7(11):2135-48 PMID: 8224842
  31. CD28 signaling causes a sustained down-regulation of I kappa B alpha which can be prevented by the immunosuppressant rapamycin.
    J Biol Chem. 1994 Dec 2;269(48):30077-80 PMID: 7982907
  32. 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.
    Mol Cell Biol. 1995 Feb;15(2):872-82 PMID: 7823953
  33. Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses.
    Cell. 1995 Jan 27;80(2):321-30 PMID: 7834752
  34. Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family.
    Cell. 1995 Jan 27;80(2):331-40 PMID: 7834753
  35. Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B.
    Mol Cell Biol. 1995 Mar;15(3):1302-11 PMID: 7862124
  36. NF-kappa B: a lesson in family values.
    Cell. 1995 Feb 24;80(4):529-32 PMID: 7867060
  37. I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B.
    Cell. 1995 Feb 24;80(4):573-82 PMID: 7867065
  38. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation.
    Science. 1995 Mar 10;267(5203):1485-8 PMID: 7878466
  39. Structure, regulation and function of NF-kappa B.
    Annu Rev Cell Biol. 1994;10:405-55 PMID: 7888182
  40. Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65.
    Mol Cell Biol. 1995 Apr;15(4):2166-72 PMID: 7891711
  41. Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation.
    Mol Cell Biol. 1995 May;15(5):2809-18 PMID: 7739562
  42. Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor.
    Mol Endocrinol. 1995 Feb;9(2):243-54 PMID: 7776974
  43. 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.
    EMBO J. 1995 Jun 15;14(12):2876-83 PMID: 7796813
  44. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway.
    Genes Dev. 1995 Jul 1;9(13):1586-97 PMID: 7628694
  45. Constitutive phosphorylation of I kappa B alpha by casein kinase II.
    Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7637-41 PMID: 7644469
  46. Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression.
    Genes Dev. 1995 Aug 15;9(16):1965-77 PMID: 7649478
  47. I kappa B-alpha-mediated inhibition of nuclear transport and DNA-binding by Rel proteins are separable functions: phosphorylation of C-terminal serine residues of I kappa B-alpha is specifically required for inhibition of DNA-binding.
    Oncogene. 1995 Sep 7;11(5):811-23 PMID: 7675442
  48. N- and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NF-kappa B activity.
    Mol Cell Biol. 1995 Oct;15(10):5339-45 PMID: 7565683
  49. Mechanistic aspects of NF-kappa B regulation: the emerging role of phosphorylation and proteolysis.
    Immunity. 1995 Sep;3(3):263-72 PMID: 7552992
  50. Mechanisms of enhanced nuclear translocation of the transcription factors c-Rel and NF-kappa B by CD28 costimulation in human T lymphocytes.
    Ann N Y Acad Sci. 1995 Sep 7;766:220-3 PMID: 7486663
  51. Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway.
    Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10599-603 PMID: 7479848
  52. Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination.
    Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11259-63 PMID: 7479976
  53. Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation.
    Genes Dev. 1995 Nov 15;9(22):2723-35 PMID: 7590248
  54. Constitutive NF-kappa B activation, enhanced granulopoiesis, and neonatal lethality in I kappa B alpha-deficient mice.
    Genes Dev. 1995 Nov 15;9(22):2736-46 PMID: 7590249
  55. Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation.
    Mol Cell Biol. 1996 Mar;16(3):899-906 PMID: 8622692
  56. Both amino- and carboxyl-terminal sequences within I kappa B alpha regulate its inducible degradation.
    Mol Cell Biol. 1996 Mar;16(3):1058-65 PMID: 8622650
  57. Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB.
    Mol Cell Biol. 1996 May;16(5):2083-90 PMID: 8628274
  58. Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation.
    Mol Cell Biol. 1996 Apr;16(4):1295-304 PMID: 8657102
  59. Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.
    Mol Cell Biol. 1996 Apr;16(4):1401-9 PMID: 8657113
  60. Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha.
    Mol Cell Biol. 1996 Jul;16(7):3554-9 PMID: 8668171
  61. The NF-kappa B and I kappa B proteins: new discoveries and insights.
    Annu Rev Immunol. 1996;14:649-83 PMID: 8717528
  62. PEST sequences and regulation by proteolysis.
    Trends Biochem Sci. 1996 Jul;21(7):267-71 PMID: 8755249
  63. Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B.
    Mol Cell Biol. 1996 Oct;16(10):5444-9 PMID: 8816457
  64. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
    Anal Biochem. 1976 May 7;72:248-54 PMID: 942051
  65. Inhibition of casein kinase II by heparin.
    J Biol Chem. 1980 Sep 10;255(17):8038-41 PMID: 6447694
  66. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.
    Science. 1986 Oct 17;234(4774):364-8 PMID: 2876518
  67. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.
    Mol Cell Biol. 1987 Jan;7(1):379-87 PMID: 3031469
  68. Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides.
    J Biol Chem. 1987 Jul 5;262(19):9136-40 PMID: 3474230
  69. Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.
    J Clin Invest. 1987 Aug;80(2):357-66 PMID: 3038955
  70. Use of eukaryotic expression technology in the functional analysis of cloned genes.
    Methods Enzymol. 1987;152:684-704 PMID: 3657593
  71. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
    Anal Biochem. 1987 Nov 1;166(2):368-79 PMID: 2449095
  72. The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV.
    Cell. 1988 Jun 3;53(5):827-36 PMID: 2836068
  73. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme.
    J Biol Chem. 1989 May 15;264(14):8222-9 PMID: 2722778
  74. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells.
    Nucleic Acids Res. 1989 Aug 11;17(15):6419 PMID: 2771659
  75. UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein.
    Mol Cell Biol. 1989 Nov;9(11):5169-81 PMID: 2557547
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.16.11.5974
Published
1996-11-00
Pages
5974-84
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC231600
Subset
IM
Grants
NIAID NIH HHS · R01 AI33839 · United States
External Links
PubMed source DOI: 10.1128/MCB.16.11.5974 PMC Full Text View journal details
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