NF-<i>κ</i>B/Rel family members are physically associated phosphoproteins

Li, C.-C. H.; Korner, Mira; Ferris, Douglas K.; Chen, Eying; Dai, Ruitong; Longo, Dan L.

doi:10.1042/bj3030499

Cited by 49 publications

(40 citation statements)

References 35 publications

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“…NFkB complexes are normally sequestered in the cytoplasm as precursors or by binding IkB (Beg et al, 1992;Finco and Baldwin, 1995), an association that is known to be disrupted by PKC/Ca 2+ mediated phosphorylation and proteolysis of IkB and p105 (the p50 precursor) (Israel, 1995;Li et al, 1995;Verma et al, 1995). Phosphorylation of p50 also results in a higher level of DNA binding stability (Li et al, 1994), and increased p105 transcription is mediated by PMA (Meyer et al, 1991;Cogswell et al, 1993). All of these mechanisms are potentially involved in the induction of NFkB binding observed.…”

Section: Discussionmentioning

confidence: 99%

“…The induction of AP1 binding by PMA and ionomycin was probably mediated by PKC/Ca 2+ dependent phosphorylation/ dephosphorylation and consequent increased binding a nity (Boyle et al, 1991;Karin, 1995), and by increased transcription of Fos/Jun family members (Makover et al, 1991;Rincon and Flavell, 1994;Janknecht, 1995). Interestingly, both NFkB and AP1 binding would be expected to increase shortly after stimulation of the cells by PMA/ionomycin (Boyle et al, 1991;Li et al, 1994), and, in the case of NFkB be inhibited thereafter by newly synthesized IkB (Finco and Baldwin, 1995). The data presented here clearly show that both NFkB and AP1 binding activities remain high in cells treated with PMA/ionomycin for 4 h, a time point at which we estimated the GM ± CSF promoter was most active (data not shown).…”

Section: Discussionmentioning

confidence: 99%

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ETS1, NFκB and AP1 synergistically transactivate the human GM – CSF promoter

et al. 1997

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Activation of helper T cells results in coordinate expression of a number of cytokines involved in di erentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GM ± CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFkB, AP1 and ETS-like binding motifs have been identi®ed in the promoter region of the GM ± CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GM ± CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GM ± CSF reporter construct in unstimulated Jurkat cells, providing that either NFkB or AP1 transcription factors are supplied by co-transfection. We con®rm that binding of endogenous NFkB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFkB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are interdependent. Our results suggest that constitutive ETS1, and inducible NFkB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

ETS1, NFκB and AP1 synergistically transactivate the human GM – CSF promoter

et al. 1997

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“…Rel/NF-kB proteins have been observed to be positively regulated by phosphorylation events both constitutively and inducibly in vivo (Li et al, 1994b). Hyperphosphorylation of p50 is required for phorbol ester and hemagglutinin induced nuclear translocation in Jurkat cells, and phosphorylated p50 accounts for most of the observed nuclear p50 population (Li et al, 1994a).…”

Section: Phosphorylationmentioning

confidence: 99%

“…The adenovirus Ad12 exploits hypophosphorylation of p50 to downregulate the transcription of the major histocompatability complex I gene allowing the cell to evade cytotoxic T cellmediated lysis. While preliminary phosphoamino analysis indicates that p50 is phosphorylated on serine residues (Hayashi et al, 1993;Li et al, 1994b), it still remains to be determined which phosphorylated residues of p50 are responsible for the observed e ects on DNA binding. Nevertheless, it is apparent that phosphorylation cannot be enhancing DNA binding by a direct mechanism since the additional negative charge resulting from a phosphorylation can only provide repulsive forces when binding to a negatively-charged DNA molecule.…”

Section: Phosphorylationmentioning

confidence: 99%

Regulation of DNA binding by Rel/NF-κB transcription factors: structural views

1999

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Rel/NF-kB transcription factors form homo-and heterodimers with di erent DNA binding site speci®cities and DNA binding a nities. Several intracellular pathways evoked by a wide range of biological factors and environmental conditions can lead to the activation of Rel/NF-kB dimers by signaling degradation of the inhibitory IkB protein. In the nucleus Rel/NF-kB dimers modulate the expression of a variety of genes including those encoding cytokines, growth factors, acute phase response proteins, immunoreceptors, other transcription factors, cell adhesion molecules, viral proteins and regulators of apoptosis. The primary focus of this review is on structural and functional aspects of Rel/NFkB:DNA complexes and their formation. The salient features of the Rel/NF-kB dimer:DNA structure are described, as are modes of transcriptional regulation by phosphorylation, altered DNA binding properties, varying protein conformations, and interactions with IkB proteins.

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“…The NF-B p50 subunit is known to be phosphorylated on serine residues, and much less significantly on threonine residues, but not on tyrosine residues (35,36). Previous studies have demonstrated the biological significance of p50 phosphorylation on NF-B DNA binding.…”

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confidence: 99%

Phosphorylation of Serine 337 of NF-κB p50 Is Critical for DNA Binding

Hou

Guan

Ricciardi

2003

Journal of Biological Chemistry

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It has been demonstrated that phosphorylation of the p50 subunit of NF-B is required for efficient DNA binding, yet the specific phospho-residues of p50 have not been determined. In this study, we substituted all of the serine and conserved threonine residues in the p50 Rel homology domain and identified three serine residues, Ser 65 , Ser 337 , and Ser 342 , as critical for DNA binding without affecting dimerization. Although substitution with negatively charged aspartic acid at each of these positions failed to restore DNA binding, substitution with threonine, a potential phospho-acceptor, retained DNA binding for residues 65 and 337. In particular, Ser 337 , in a consensus site for protein kinase A (PKA) and other kinases, was shown to be phosphorylated both in vitro and in vivo. Importantly, phosphorylation of Ser 337 by PKA in vitro dramatically increased DNA binding of p50. This study shows for the first time that the DNA binding ability of NF-B p50 subunit is regulated through phosphorylation of residue Ser 337 , which has implications for both positive and negative control of NF-B transcription.The transcription factor NF-B acts as a central regulator of inflammatory, immune, and stress responses by controlling gene expression of cytokines, chemokines, immunoreceptors, antigen-presenting proteins, growth factors, transcription factors, cell adhesion molecules, stress response proteins, and apoptotic regulators (1, 2). Members of Rel/NF-B transcription factor family include Dorsal, Relish, and Dif in Drosophila and p65 (RelA), RelB, c-Rel, p50/p105, and p52/p100 in vertebrates. All of these proteins have a highly conserved DNAbinding and dimerization domain called the Rel homology domain. The vertebrate Rel family proteins can form homodimers or heterodimers that bind to 10-basepair B sites in the promoters of NF-B target genes (1, 3, 4). The most common and important NF-B transactivating species is the p50/p65 heterodimer. The p65 subunit, which contains a transactivation domain in the C terminus of the Rel homology domain, is responsible for the ability of NF-B to stimulate transcription. By contrast, p50 subunit, which lacks a transactivation domain, functions mainly in NF-B DNA binding (5-9). Another important dimeric species, the p50/p50 homodimer, serves mainly as a negative regulator of NF-B activity through competing with p50/p65 for NF-B response elements on DNA and through its association with co-repressor histone deacetylase (10, 11). The x-ray crystal structures of p50/p65 heterodimer and p50/p50 and p65/p65 homodimer binding to DNA have revealed a conformation often referred to as a "butterfly" (12-16). The DNA recognition loop (L1) in the N-terminal half of NF-B Rel homology domain mediates base-specific DNA contacts, whereas the C-terminal half is responsible for dimerization and nonspecific DNA contacts (17).NF-B activity is regulated by nuclear translocation. In most cell types, p50/p65 heterodimers exist in the cytoplasm as an inactive form associated with the inhibitor protein, I B. A w...

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NF-κB/Rel family members are physically associated phosphoproteins

Cited by 49 publications

References 35 publications

ETS1, NFκB and AP1 synergistically transactivate the human GM – CSF promoter

ETS1, NFκB and AP1 synergistically transactivate the human GM – CSF promoter

Regulation of DNA binding by Rel/NF-κB transcription factors: structural views

Phosphorylation of Serine 337 of NF-κB p50 Is Critical for DNA Binding

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