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The NF-kappa B precursor p105 and the proto-oncogene product Bcl-3 are I kappa B molecules and control nuclear translocation of NF-kappa B. 总被引:23,自引:7,他引:16
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We have examined the interaction of the NF-kappa B precursor p105 with NF-kappa B subunits. Similar to an I kappa B molecule, p105 associates in the cytoplasm with p50 or p65. Through this assembly, p105 efficiently blocks nuclear transfer of either subunit. Moreover, the p105 protein inhibits DNA binding of dimeric NF-kappa B subunits in a similar, but not identical, manner to its isolated C-terminal domain, which contains an ankyrin-like repeat domain (ARD). The proto-oncogene product Bcl-3 also controls nuclear translocation of p50, but not of p65. Hence, p50 can be retained in the cytoplasm via at least three distinct interactions: through direct interactions either with its own precursor, with Bcl-3 or indirectly through I kappa B alpha or -beta when attached to p65. We discuss a function of p105 as a cytoplasmic assembly unit for homo- and heteromeric NF-kappa B complexes and of Bcl-3 as an I kappa B with novel subunit specificity. 相似文献
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The precursor of NF-kappa B p50 has I kappa B-like functions. 总被引:60,自引:0,他引:60
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The bcl-3 proto-oncogene encodes a nuclear I kappa B-like molecule that preferentially interacts with NF-kappa B p50 and p52 in a phosphorylation-dependent manner. 总被引:13,自引:7,他引:6
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G P Nolan T Fujita K Bhatia C Huppi H C Liou M L Scott D Baltimore 《Molecular and cellular biology》1993,13(6):3557-3566
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A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene. 总被引:21,自引:3,他引:18
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S K Hansen C Nerlov U Zabel P Verde M Johnsen P A Baeuerle F Blasi 《The EMBO journal》1992,11(1):205-213
The NF-kappa B subunits, p50 and p65, have extensive sequence homology with the c-rel proto-oncogene and the Drosophila morphogen dorsal. It has recently been shown that in vitro translated c-Rel can bind to DNA and form a complex with p50. However, the conditions for DNA binding of c-Rel in vivo and its DNA sequence specificity have not been established. Here we report the identification a novel heterodimeric complex that binds to a kappa B-like, phorbol ester (TPA) responsive DNA sequence, 5'-GGGAAAGTAC-3', in the 5' flanking region of the human urokinase (uPA) gene. This sequence was shown to bind two protein complexes, LC and UC. LC was indistinguishable from NF-kappa B as it reacted with antibodies recognizing the p50 subunit of NF-kappa B, and was shown by UV crosslinking to contain the p50 and p65 subunits of NF-kappa B. UC, on the other hand, strongly reacted with anti-v-Rel, but not with the anti-p50 antibodies, and was shown by crosslinking to contain 75 kDa and 85 kDa protein-DNA adducts. The 75 kDa and the 85 kDa adducts could be immunoprecipitated only by anti-p65 and anti-c-Rel antibodies, respectively, showing that c-Rel formed a heterodimer with p65. Both protein complexes were present in inactive forms in HeLa cell cytosol, and their nuclear translocation was induced by TPA. DNA binding of UC and LC could, furthermore, be inhibited by I kappa B-alpha.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65. 总被引:10,自引:4,他引:6
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We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed. 相似文献
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Two distinct NF-kappa B complexes differing in their larger subunit bind the E-selectin promoter kappa B element. 总被引:2,自引:1,他引:1
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We have investigated the proteins binding the E-selectin promoter NF-kappa B element in its natural DNA context, using probes extending beyond the NF-kappa B recognition decamer. In band shift assays, we detected two distinct NF-kappa B complexes using nuclear extracts from several cytokine-induced cells. Subunit-specific antisera as blockers of complex formation plus DNA-protein cross-linking experiments revealed the faster migrating form to contain the NF-kappa B p50 plus p65 subunits. In contrast, the slower migrating form is composed of p50 plus the p65-related p75 protein. We show as the crucial determinant in generation of the larger complex the presence of more than five basepairs extra DNA sequence downstream of the NF-kappa B-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappa B binding site is. This may be explained by a requirement for activated p50 as part of this complex. The potential for a regulatory role for the p75 containing complex on the E-selectin promoter is discussed. 相似文献
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Activation of NF-kappa B in vivo is regulated by multiple phosphorylations. 总被引:10,自引:3,他引:7
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The activation of nuclear factor kappa B (NF-kappa B) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-kappa B/I kappa B components following stimulation and show that the final step of NF-kappa B induction in vivo involves phosphorylation of several members of the NF-kappa B/I kappa B protein families. In HeLa cells as well as in B cells, TNF-alpha rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-kappa B precursors and I kappa B alpha became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-kappa B revealed constitutively phosphorylated p65 and I kappa B alpha. Phosphorylation was accompanied by induced processing of the precursors p100 and p105 and by degradation of I kappa B alpha. As an in vitro model we show that phosphorylation of p105 impedes its ability to interact with NF-kappa B, as has been shown before for I kappa B alpha. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-alpha selectively activates only specific NF-kappa B heteromers and that modifications regulate not only I kappa B molecules but also NF-kappa B molecules. In fact, cellular NF-kappa B activity was phosphorylation-dependent and the DNA binding activity of p65-containing NF-kappa B was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-kappa B translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-alpha. Thus, phosphorylation appears to be a general mechanism for activation of NF-kappa B in vivo. 相似文献
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Common structural constituents confer I kappa B activity to NF-kappa B p105 and I kappa B/MAD-3. 总被引:18,自引:4,他引:14
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The vertebrate NF-kappa B/c-rel inhibitors MAD-3/I kappa B alpha, I kappa B gamma/pdI and bcl-3 all share a conserved ankyrin repeat domain (ARD) consisting of six complete repeats, a short acidic motif and/or an incomplete seventh repeat. We present here a detailed analysis of the domain in p105/pdI and MAD-3/I kappa B involved in inhibition of DNA binding and in protein interaction with rel factors. We demonstrate that in both cases an acidic region and six ankyrin-like repeats are sufficient and required for protein interaction with the rel factors. However, for p105/pdI to achieve the high affinity needed to suppress DNA binding, an incomplete seventh repeat is required in addition. Both pdI and MAD-3 associate with rel proteins by forming heterotrimeric complexes, as shown by native gel analysis and by cross-linking. Furthermore, we demonstrate that deletion of only three amino acids in the first repeat converts the subunit specificity of the p105 ARD into that of MAD-3/I kappa B. We conclude that functionally the ARD in these molecules has a modular structure, with different subregions determining the specificity for the NF-kappa B subunits p50 and p65. 相似文献
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Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal 总被引:189,自引:0,他引:189
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Machado J Fernandez PC Baumann I Dobbelaere DA 《Microbes and infection / Institut Pasteur》2000,2(11):1311-1320
Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells. 相似文献
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DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide 总被引:140,自引:0,他引:140
The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor kappa B (NF-kappa B) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-kappa B p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c-rel and lesser similarity to NF-kappa B p50. In vitro translated p65 forms a DNA-binding complex with NF-kappa B p50, and the binding of this complex can be specifically inhibited by purified I kappa B. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by I kappa B, thus mapping the I kappa B interaction domain to the rel-homologous region and suggesting that I kappa B exerts its inhibitory effect upon NF-kappa B primarily through interaction with p65. 相似文献
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