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A Israël

Publications and source records attributed to A Israël.

At least 55 records · Page 3Linked to original sources
PubMed1995

Ubiquitin-mediated processing of NF-kappa B transcriptional activator precursor p105. Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation.

In most cases, the transcriptional factor NF-kappa B is a heterodimer consisting of two subunits, p50 and p65, which are encoded by two distinct genes of the Rel family. p50 is translated as a precursor of 105 kDa. The C-terminal domain of the precursor is rapidly degraded, forming the mature p50 subunit consisted of the N-terminal region of the molecule. The mechanism of generation of p50 is not known. It has been suggested that the ubiquitin-proteasome system is involved in the process; however, the specific enzymes involved and the mechanism of limited proteolysis, in which half of the molecule is spared, have been obscure. Palombella and colleagues (Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785) have shown that ubiquitin is required for the processing in a cell-free system of a truncated, artificially constructed, 60-kDa precursor. They have also shown that proteasome inhibitors block the processing both in vitro and in vivo. In this study, we demonstrate reconstitution of a cell-free processing system and demonstrate directly that: (a) the ubiquitin-proteasome system is involved in processing of the intact p105 precursor, (b) conjugation of ubiquitin to the precursor is an essential intermediate step in the processing, (c) the recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of p53, is also required for the limited processing of the p105 precursor, and (d) a novel, approximately 320-kDa species of ubiquitin-protein ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3 alpha, the "N-end rule" ligase.

Animals↗
PubMed1995

Cooperatively between an upstream TATA-like sequence and a CAA repeated element mediates E1A-dependent negative repression of the H-2Kb class I gene.

In primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12, repression of transcription mediated by the far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presence of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs. A gel shift competition study demonstrated that the TATA-binding protein (TBP) or a TBP-like protein can bind to both the upstream TATA-like sequence and the regular TATA box on the H-2Kb basal promoter. Moreover, employing immunoselection and cyclic amplification and selection of targets (CASTing) methods with nuclear extracts derived from Ad12-E1A transformants, we have identified a high affinity binding site in the H-2Kb class I promoter for E1A-associated DNA-binding proteins. The sequences of the binding sites were identified and were found to contain both the upstream TATA-like motif and the CAA repeated motifs. Our results suggest that the TATA-like sequence in the far upstream region of the H-2Kb gene is one of the elements that is required for Ad12-E1A-mediated negative repression.

Adenovirus E1A Proteins↗
PubMed1995

Activation of NF-kappa B by the Tax protein of HTLV-1.

The Tax protein, encoded by the human T cell leukemia virus HTLV-1, is responsible for transcriptional activation of the viral genome through conserved 21bp repeats located in its promoter. Tax also activates the transcription of cellular genes such as interleukin 2, interleukin 2 receptor (IL2R), GM-CSF, vimentin, c-fos, c-jun as well as the major histocompatibility complex class I genes. Tax does not bind DNA directly, but seems to activate transcription indirectly by enhancing the activity of the transcription factors that recognize responsive elements located in the promoters of the Tax-responsive genes, or by forming ternary complexes with these factors and DNA. One class of target sites for Tax are the kappa B sequences which are bound by members of the rel/NF-kappa B family. It has been previously shown that Tax is able to induce nuclear translocation of NF-kappa B. The activity of the NF-kappa B transcription factor is normally controlled through cytoplasmic retention by either of two types of molecules: the inhibitor I kappa B alpha/MAD3 or the p105 and p100 precursors of the p50 and p52 DNA-binding subunits. Treatment of cells with classical NF-kappa B inducers like TNF, IL-1, PMA or LPS results in MAD-3 degradation followed by nuclear translocation of NF-kappa B. On the other hand, the mechanisms involved in the dissociation of the cytoplasmic p105/p100-containing complexes are largely unknown. We demonstrate here that Tax can induce translocation of members of the NF-kappa B family retained in the cytoplasm through interaction with either p105 or p100. On the other hand Tax induces no apparent degradation of MAD-3. These results suggest that Tax activates NF-kappa B essentially through the p105/p100-retention pathway.

Animals↗
PubMed1995

N- and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NF-kappa B activity.

The proteolytic degradation of the inhibitory protein MAD3/I kappa B alpha in response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-kappa B. Analysis of the expression of human I kappa B alpha protein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous I kappa B alpha is degraded in response to inducers of NF-kappa B activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-norleucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyperphosphorylated form of the human I kappa B alpha protein. By expressing mutant forms of the human protein in this cell line, we have been able to delineate the sequences responsible for both the ligand-induced phosphorylation and the degradation of I kappa B alpha. Our results show that deletion of the C terminus of the I kappa B alpha molecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of I kappa B alpha maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. We propose that treatment of 70Z/3 cells with either phorbol myristate acetate or lipopolysaccharide induces a kinase activity which phosphorylates serines 32 and that these phosphorylations target the protein for rapid proteolytic degradation, possibly by the ubiquitin-26S proteasome pathway, thus allowing NF-kappa B to translocate to the nucleus and to activate gene expression.

Amino Acid Sequence↗
PubMed1994

Inhibition of the DNA-binding activity of Drosophila suppressor of hairless and of its human homolog, KBF2/RBP-J kappa, by direct protein-protein interaction with Drosophila hairless.

We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless [Su(H)] gene. Deletion studies of the RBP-J kappa and Su(H) proteins allowed us to define a DNA-binding domain conserved during evolution. Because Su(H) mutant alleles exhibit dose-sensitive interactions with Hairless (H) loss-of-function mutations, we have investigated whether the RBP-J kappa or Su(H) proteins directly interact with the H protein in vitro. We show here that H can inhibit the DNA binding of both Su(H) and RBP-J kappa through direct protein-protein interactions. Consistent with this in vitro inhibitory effect, transcriptional activation driven by Su(H) in transfected Drosophila S2 cells is inhibited by H. These results support a model in which H acts, at least in part, as a negative regulator of Su(H) activity. This model offers a molecular view to the antagonistic activities encoded by the H and Su(H) genes for the control of sensory organ cell fates in Drosophila. We further propose that a similar mechanism might occur in mammals.

Amino Acid Sequence↗
PubMed1994

Tax induces nuclear translocation of NF-kappa B through dissociation of cytoplasmic complexes containing p105 or p100 but does not induce degradation of I kappa B alpha/MAD3.

The activity of the NF-kappa B transcription factor is controlled through cytoplasmic retention by either of two types of molecules: the inhibitor I kappa B alpha/MAD3 or the p105 and p100 precursors of the p50 and p52 DNA-binding subunits. Treatment of cells with classical NF-kappa B inducers such as tumor necrosis factor, interleukin-1, phorbol myristate acetate, and lipopolysaccharide results in MAD3 degradation followed by nuclear translocation of NF-kappa B. On the other hand, the mechanisms involved in the dissociation of the cytoplasmic p105/p100-containing complexes are largely unknown. The Tax protein encoded by human T-cell leukemia virus type 1 is a potent activator of viral and cellular gene transcription. It does not bind DNA directly but seems to activate transcription indirectly either by enhancing the activities of the transcription factors that recognize responsive elements located in the promoters of the Tax-responsive genes or by forming ternary complexes with these factors and DNA. It has been previously shown that Tax is able to induce nuclear translocation of NF-kappa B. We demonstrate here that Tax can induce translocation of members of the NF-kappa B family retained in the cytoplasm through their interaction with either p105 or p100. On the other hand, Tax induces no apparent degradation of MAD3, although experiments using cycloheximide indicate that it decreases the half-life of MAD3. However, this activity is shared by a mutant of Tax which is unable to activate NF-kappa B. These results suggest that Tax activates NF-kappa B essentially through the p105/p100 retention pathway.

Animals↗
PubMed1993

Negative regulation of the gene for H-2Kb class I antigen by adenovirus 12-E1A is mediated by a CAA repeated element.

Both positive and negative regulatory elements responsive to the product of the adenovirus type 12 E1A gene are located in the promoter region of the gene for mouse H-2Kbm1 major histocompatibility complex (MHC) class I antigen (1). We have analyzed the negative regulatory element that is affected by E1A and identified a target CAA repeated motif, CAA(A)CAAA, within -1725 to -1705 and -1591 to -1568 in a 316-bp sequence located in the far upstream region of H-2Kb promoter (-1837 to -1522; +1 refers to the cap site). The extent of cell surface expression of the MHC class 1 antigen was significantly decreased in the case of transfectants obtained by introducing an expression plasmid that included MHC class 1 cDNA with the CAA repeated element, as compared with that of a plasmid with mutated CAA repeats. We have also characterized the nuclear proteins that bind to this motif. The analysis of the effects of mutations during competition assays of in vivo and gel retardation competition assays demonstrated that the CAA repeated element is essential not only for E1A-dependent repression of transcription but also for the cell surface expression of the product of the mouse H-2Kb class I gene, presumably through nuclear proteins that specifically bind to it.

Adenovirus E1A Proteins↗
PubMed1993

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.

In order to characterize the regulation of the gene encoding the I kappa B-alpha/MAD3 inhibitor of the transcription factor NF-kappa B, we have isolated a human genomic clone and sequenced the promoter of this gene. The MAD3 promoter exhibits a potential TATA element upstream of one of the two major transcription sites, and contains several potential NF-kappa B binding sequences, suggesting that the gene is positively regulated by members of this family. Transfection experiments demonstrate that the MAD3 promoter can be activated by various combinations of members of the rel/NF-kappa B family, as well as by phorbol esters and tumor necrosis factor. Specific deletion of one of the kappa B motifs, located 37 bp upstream of the TATA box, abolishes responses to PMA and TNF. This kappa B motif binds NF-kappa B (p50/relA), p50/c-rel and relA/c-rel heterodimers as well as KBF1 (p50 homodimer). These results help to explain the previously observed transient nature of the NF-kappa B response: following NF-kappa B activation, the expression of the inhibitor is increased, therefore the extent of nuclear translocation of the active complex is reduced, resulting in a decreased activation of its target genes.

Base Sequence↗
PubMed1993

Two factors, IRF1 and KBF1/NF-kappa B, cooperate during induction of MHC class I gene expression by interferon alpha beta or Newcastle disease virus.

The expression of class I genes of the Major Histocompatibility Complex is stimulated by IFN. The promoter of these genes contains an interferon response sequence (IRS) which overlaps the major enhancer. These elements are recognized by several protein factors, including IRF-1, which binds the IRS, and KBF1/NF-kappa B, which binds the enhancer. We demonstrate here that infection by Newcastle Disease Virus (NDV) results in an increased expression of class I genes, by a mechanism partially different from that of IFN, but that in both cases the cooperative action of IRF1 and KBF1/NF-kappa B is required. In F9 embryonal carcinoma cells, where KBF1/NF-kappa B activity cannot be detected, both types of stimuli are ineffective.

Animals↗
PubMed1992

The precursor of NF-kappa B p50 has I kappa B-like functions.

The C-terminal half of the p105 precursor of the NF-kappa B p50 subunit contains ankyrin-like repeats similar to those in I kappa B molecules, which are known to retain NF-kappa B complexes in the cytoplasm. We demonstrate that in various cell lines p105 is found associated with either c-rel or p65 in the cytoplasm and serves I kappa B-like functions. p105 retains c-rel or p65 in the cytoplasm in cotransfection experiments in COS cells. It also inhibits DNA binding by c-rel in gel retardation assays. Stable interaction of p105 with c-rel or p65 requires the putative dimerization domain in the conserved rel homology region of p105, as well as a second contact with the I kappa B-related C-terminal part of p105. Pulse-chase experiments indicate that cytoplasmic complexes of p105 with c-rel or p65 give rise to cytoplasmic as well as nuclear p50-c-rel and p50-p65, respectively, probably through processing of p105. Thus, p105, like the I kappa Bs, controls the subcellular localization and hence the transcriptional activity of at least two other members of the rel/NF-kappa B family.

Biological Transport↗
PubMed1992

The characterization of the promoter of the gene encoding the p50 subunit of NF-kappa B indicates that it participates in its own regulation.

In order to characterize the regulation of the gene encoding the p50 subunit of the transcription factor NF-kappa B, we have isolated a human genomic clone, and sequenced the promoter of this gene. By in situ hybridization we have mapped the gene encoding the p50 subunit of NF-kappa B to the 4q23-4q25 region of the human genome and the H1-H3 region of the murine chromosome 3. The p50 promoter lacks TATA and CAAT elements, but contains NF-kappa B, AP-1 and HIP-1 binding sequence. The kappa B motif binds NF-kappa B, KBF1, and heterodimers of p50 and c-rel, suggesting that the gene is regulated by members of this family. Co-transfection experiments demonstrate that the p50 promoter can be activated by either of the two subunits of NF-kappa B (p50 and p65), and more strongly by the combination of both. The promoter of p50 can be activated by phorbol esters and tumor necrosis factor alpha but not by forskolin and these responses are mediated through the NF-kappa B binding sequence. The involvement of NF-kappa B in the regulation of the p50 gene is also supported by the inhibition of the PMA activation of the promoter by an NF-kappa B transdominant negative mutant, as well as the product of the v-rel oncogene.

Animals↗
PubMed1992

NF-kappa B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats.

Molecular cloning of the subunits of the transcription factor NF-kappa B and its inhibitor l kappa B revealed regions of sequence homology with two different classes of proteins: the Rel/dorsal family and a heterogeneous group of proteins containing ankyrin-like repeats. Both the Rel/dorsal homology domain and the ankyrin-like repeats appear to play important roles in protein-protein interactions that regulate localization and activity of the NF-kappa B subunits.

Amino Acid Sequence↗
PubMed1992

Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II (NF-kappa B) element.

We have biochemically and functionally characterized a new transcription factor, NP-TCII, which is present in nuclei from unstimulated T and B lymphocytes but is not found in nonhematopoietic cells. This factor has a DNA-binding specificity similar to that of NF-kappa B but is unrelated to this or other Rel proteins by functional and biochemical criteria. It can also be distinguished from other previously described lymphocyte-specific DNA-binding proteins.

Animals↗
PubMed1991

Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection.

Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is regulated in part by cellular factors and is stimulated by activation of latently infected T cells. T-cell activation also correlates with the induction of the factor NF-kappa B which binds to two adjacent sites in the HIV-1 long terminal repeat. This factor consists of two DNA-binding subunits of relative molecular mass 50,000 (50K) associated with two 65K subunits. It is located in the nucleus in mature B cells, but is present in other cell types as an inactive cytoplasmic complex. External stimuli, including those that activate T cells, result in nuclear translocation of active NF-kappa B. The cloning of the complementary DNA for the 50K subunit helped to identify an exclusively cytoplasmic 105K precursor (p105) (V.B., P.K. and A.I., manuscript submitted). The expression of active NF-kappa B might therefore also be regulated by the extent of processing of p105. Because HIV-1 requires active NF-kappa B for efficient transcription, we tested the effect of HIV-1 infection on the processing of the human 105K precursor. We show here that the HIV-1 protease can process p105 and increases levels of active nuclear NF-kappa B complex.

Cell Line↗
PubMed1991

Kappa B binding proteins are constitutively expressed in an IL-2 autocrine human T cell line.

The IL-2 and the IL-2-R alpha genes are both expressed transiently in normal T lymphocytes after Ag or mitogen activation. In contrast, the human T cell line, IARC 301, expresses these two genes constitutively and we have previously demonstrated that its growth depends on the autocrine production of this T cell growth factor and high affinity IL-2R. To dissect the molecular basis for the unusual persistent expression of the IL-2 and IL-2-R alpha genes in these IARC 301 T cells, we have analyzed the interactions of constitutively expressed nuclear proteins with the 5' flanking regions of the IL-2 and IL-2-R alpha genes using both DNase I footprinting and gel retardation techniques. We have found that a region in both genes (-276 to -250 for IL-2-R alpha and -203 to -183 for IL-2), which corresponds to a kappa B enhancer element, is specifically protected by nuclear proteins from IARC 301. In agreement with this finding, both the IL-2 and IL-2-R alpha promoters are active in transient transfection assays in IARC 301 cells. In contrast, mutation of the kappa B enhancer results in markedly attenuated activities of both promoters. Two proteins binding the kappa B sequence, NF-kappa B and KBF1, are constitutively expressed in IARC 301 nuclei and induced by PMA and PHA in Jurkat. They bind to the kappa B motifs with different relative affinities that may reflect their different contribution in the expression of various promoters.

Base Sequence↗
PubMed1991

Cytoplasmic retention, DNA binding and processing of the NF-kappa B p50 precursor are controlled by a small region in its C-terminus.

The transcription factor NF-kappa B binds to DNA as a heterodimer composed of two subunits of 50 kDa (p50) and 65 kDa (p65). p50 contains a DNA binding and dimerization domain and represents a truncated form of a 105 kDa (p105) precursor molecule. We show here that in different cell types the p105 precursor as well as the processed p50 coexist in the cytoplasm, but that only the latter enters the nucleus. The cytoplasmic retention of the precursor molecule is controlled by a small region in its C-terminal part. We show that this region is responsible for the observed lack of DNA binding of the p50 precursor and controls the extent of processing of the precursor to the mature form. We also present evidence that a stretch of four basic amino acids, similar to a sequence found in the other proteins belonging to the rel/NF-kappa B family, is required for translocation of the processed p50 protein into the nucleus and thus could be the target for the retention mechanism.

Amino Acid Sequence↗