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N R Rice

Publications and source records attributed to N R Rice.

At least 19 recordsLinked to original sources
PubMed1995

Identification of an I kappa B alpha-associated protein kinase in a human monocytic cell line and determination of its phosphorylation sites on I kappa B alpha.

Nuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an inactive form through interaction with I kappa B. Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-kappa B in association with the induction of I kappa B alpha phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alpha. LPS stimulation transiently enhanced the I kappa B alpha-bound kinase activity in THP-1 cells. Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of I kappa B alpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS-induced NF-kappa B activation as well as inducible phosphorylation of endogenous I kappa B alpha in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of I kappa B alpha and subsequent dissociation of the NF-kappa B.I kappa B alpha complex.

Amino Acid Sequence↗
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

Distinct combinations of NF-kappa B subunits determine the specificity of transcriptional activation.

The nuclear factor that binds to the kappa light-chain enhancer of B cells (NF-kappa B) is a transcription factor that regulates the expression of a variety of cellular and viral genes. NF-kappa B is composed of distinct subunits, and at least four independent genes (p105, p100, p65, and c-rel) have been isolated that encode related proteins that bind kappa B sites. Because it is possible that specific interactions of different subunits can allow selective gene activation, we have characterized the specificity of transcriptional activation by various combinations of these subunits. When tested alone, an approximately 49-kDa form (p49) of the p100 protein bound weakly to kappa B, but p49 associated with p65 to bind efficiently to this site. Furthermore, p49 acted in combination with either p65 or a Rel/VP16 fusion protein to activate kappa B-dependent transcription in Jurkat T leukemia cells. The p49/p65 or p49/Rel combination stimulated transcription mediated by the canonical kappa B site but did not stimulate reporter genes containing interleukin 2 receptor alpha or major histocompatibility complex kappa B elements, despite its ability to bind to these sites. Transactivation mediated by the p49/p100 and p65 NF-kappa B proteins is therefore sensitive to minor changes in the sequence of the kappa B site. Specificity determined by the association of NF-kappa B subunits provides a mechanism to selectively regulate variant kappa B sites associated with different cellular and viral genes.

Base 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↗
PubMed1992

Kappa B site-dependent activation of the interleukin-2 receptor alpha-chain gene promoter by human c-Rel.

The cis-acting control elements of the interleukin-2 receptor alpha-chain (IL-2R alpha) gene contain a potent kappa B-like enhancer whose activity can be induced by various mitogenic stimuli. Recent cloning of the p50 and p65 subunits of the kappa B-binding protein NF-kappa B complex revealed a striking sequence homology of these proteins with the c-rel proto-oncogene product (c-Rel). On the basis of this homology, we examined the potential role of c-Rel in controlling IL-2R alpha transcription. We now demonstrate that the recombinant human c-Rel protein binds to the kappa B element in the IL-2R alpha promoter and results in alteration of the DNA structure in the adjacent downstream regulatory elements containing the CArG box and the GC box. We found that human c-Rel can activate transcription from the IL-2R alpha promoter, but not the kappa B-containing human immunodeficiency virus type 1 promoter, upon cotransfection into Jurkat T cells. Furthermore, truncation of the carboxyl terminus of c-Rel results in a c-Rel mutant (RelNA) that (i) localizes exclusively in the nucleus and (ii) acts in synergy with wild-type c-Rel in activating transcription from the kappa B site of the IL-2R alpha promoter. Finally, induction of surface IL-2R alpha expression coincides with the induced levels of endogenous c-Rel and induced c-Rel binding to the IL-2R alpha kappa B site. Our study identified c-Rel as one component of the Rel/NF-kappa B-family proteins involved in the kappa B-dependent activation of IL-2R alpha gene expression. Furthermore, our results suggest that a Re1NA-like cellular factor (e.g., NF-kappa B p50 or p49 subunit) acts in synergy with c-Re1 during T-cell activation.

Base Sequence↗
PubMed1992

v-rel- and c-rel-protein complexes bind to the NF-kappa B site in vitro.

Previous work by others has revealed homology between the rel oncogene and the transcription factor NF-kappa B. Further, in vitro-translated v-rel protein and c-rel protein are able to bind to an oligonucleotide containing the kappa B binding site. Unlike the in vitro-translated product, cellular Rel protein exists in high molecular weight complexes with several other proteins. In this report we show that immunopurified cellular complexes containing the v-rel protein and/or the c-rel protein are also able to bind to an oligonucleotide containing the kappa B site. These cellular complexes are heterogeneous in size, and all sizes appeared to have binding activity. UV cross-linking demonstrated that the Rel proteins themselves were bound to the DNA. Thus it is likely that the Rel proteins play a direct role in transcriptional regulation.

Animals↗
PubMed1991

Alterations at the rel locus in human lymphoma.

The rel proto-oncogene has been mapped to chromosome region 2p11.2-14, a site associated with nonrandom rearrangements in non-Hodgkin's lymphoma. We have characterized an abnormal rel mRNA from a cell line derived from a diffuse large cell lymphoma, in which the evolutionarily conserved N-terminal half of the rel coding region was fused with the C-terminal coding region of an unrelated gene. In addition, rearrangement or amplification of the rel locus was found in the lymphomatous tissue of two follicular and one diffuse large cell lymphoma. The findings suggest involvement of rel in the pathogenesis of large cell lymphoma.

Amino Acid Sequence↗
PubMed1991

The v-rel and c-rel proteins exist in high molecular weight complexes in avian and murine cells.

We reported previously that the v-rel protein (p59v-rel) exists in a high molecular weight complex with at least four other proteins in the cytoplasm of v-rel-transformed chicken pre-B lymphoid cells (Simek, S. & Rice, N.R., J. Virol., 62, 4730-4736, 1989). One of these proteins is the chicken c-rel protein, but the identities of the others (of about 36 kDa, 115 kDa, and 124 kDa) are unknown. In this report we extend that observation to additional v-rel-transformed cell lines of both pre-B and B cell phenotypes. We also introduced and expressed v-rel in several other avian cell lines (a chicken T cell line, chick embryo fibroblasts, and quail fibroblasts) and found that in these cells p59v-rel was complexed with the same proteins as observed in the v-rel-transformed cells. Thus, the associated proteins are not limited to pre-B cells, but occur and complex with p59v-rel in B cells, T cells, and fibroblasts. We next examined five uninfected avian cells and tissues and found that, with only one exception, p75c-rel was complexed with p36, p115, and p124. Thus, in most cases complex formation is not limited to or dependent on the presence of the transforming v-rel protein, but also occurs with the normal c-rel protein. To determine whether a mammalian c-rel protein is similarly associated with other proteins, we screened murine cell lines for the presence of c-rel mRNA. In agreement with our earlier findings, we found the highest expression in mature B cells, although several pre-B and myeloid cell lines were also strongly positive. Using one of the B cell lines, we detected the murine c-rel protein. We found that, like its avian counterpart, it is a protein of about 75 kDa and is associated with proteins of 36 kDa and 115 kDa. Sephacryl S-400 chromatography revealed that both the avian and murine complexes are of high molecular weight, with an average size of about 400 kDa.

Animals↗
PubMed1990

Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins.

We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells.

Amino Acid Sequence↗
PubMed1990

Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.

Amino Acid Sequence↗
PubMed1989

Viral DNA in horses infected with equine infectious anemia virus.

The amount and distribution of viral DNA were established in a horse acutely infected with the Wyoming strain of equine infectious anemia virus (EIAV). The highest concentration of viral DNA were found in the liver, lymph nodes, bone marrow, and spleen. The kidney, choroid plexus, and peripheral blood leukocytes also contained viral DNA, but at a lower level. It is estimated that at day 16 postinoculation, almost all of the viral DNA was located in the tissues, with the liver alone containing about 90 times more EIAV DNA than the peripheral blood leukocytes did. Assuming a monocyte-macrophage target, each infected cell contained multiple copies of viral DNA (between 6 and 60 copies in liver Kupffer cells). At day 16 postinoculation, most of the EIAV DNA was not integrated into host DNA, but existed in both linear and circular unintegrated forms. In contrast to acute infection, viral DNA was not detectable in tissues from asymptomatic horses with circulating antibody to EIAV.

Animals↗
PubMed1989

A human rel proto-oncogene cDNA containing an Alu fragment as a potential coding exon.

Two rel-containing cDNA clones were isolated from a library derived from the Daudi human cell line, which is known to express c-rel mRNA. Clone #1 appeared to contain the entire c-rel coding sequence, which differs from v-rel in having three additional N-terminal residues and 111 additional C-terminal residues. In addition, Clone #1 had an internal 32 amino acid exon not found in v-rel or in turkey c-rel. Clone #2 was truncated at its 5' end and did not contain this new exon. Analysis of a genomic clone of human c-rel revealed that the new exon was a portion of an inverted Alu repeat. The occurrence of potential splice sites and of open reading frames in the inverted consensus Alu sequence suggests that the incorporation of Alu fragments as potential coding exons could be a relatively common event in human mRNAs. Whether such messages can be translated is unknown: antiserum raised against a peptide at the predicted C-terminus of the c-rel protein precipitated p82hc-rel, but antiserum raised against a peptide located in the Alu exon did not.

Base Sequence↗
PubMed1988

p59v-rel, the transforming protein of reticuloendotheliosis virus, is complexed with at least four other proteins in transformed chicken lymphoid cells.

Previous studies have identified the protein product of v-rel, the oncogene carried by reticuloendotheliosis virus (REV), as a 59,000-dalton phosphoprotein located predominantly in the cytosol of transformed chicken lymphoid cells. In immune precipitates of p59v-rel, there is a closely associated protein kinase activity. In chicken lymphoid cells that do not contain REV, p68c-rel is found free in the cytosol not associated with other proteins and not detectably phosphorylated. In this study, we found that immune precipitates of 59v-rel from REV-transformed cells contain at least four other proteins, of approximate molecular weights 124, 115, 68, and 36 kilodaltons (kDa). The 124-, 115-, and 36-kDa proteins are apparently unrelated to p59v-rel in sequence, and their coprecipitation suggests that they are complexed with p59v-rel. The coprecipitating 68-kDa protein was found to be p68c-rel, which, like the other three proteins, precipitates by virtue of its association with p59v-rel. Glycerol gradient analysis suggested the presence of more than one type of complex: one containing p115, p68c-rel, p59v-rel, and p36, and another containing p124, p115, p59v-rel, and possibly p68c-rel. In vitro kinase activity was found in all size classes, coinciding with the distribution of p115 and p59v-rel. The complex(es) was stable under a variety of conditions, including a wide range of ionic strengths, chelators, and detergents, and through multiple cycles of immune precipitation and elution. This suggests a specific and functionally significant interaction among the members that may be of direct relevance to the mechanism of REV-induced transformation.

Animals↗
PubMed1988

Detection and characterization of the protein encoded by the chicken c-rel protooncogene.

We have identified the protein encoded by chicken c-rel, the cellular homolog of the v-rel oncogene carried by reticuloendotheliosis virus. The protein has been detected in two avian lymphoid cell lines and in chick embryo fibroblasts. It has a molecular weight of 68,000, suggesting that the viral gene (which encodes a protein of 59,000 molecular weight) is a truncated form of its cellular homolog. The c-rel protein is found in the soluble cytoplasmic fraction of chicken lymphoid cells and is not associated in any stable way with other cellular proteins. Unlike the viral protein, p68c-rel is not detectably phosphorylated.

Acetylglucosaminidase↗
PubMed1987

Characterization of equine infectious anemia virus long terminal repeat.

The long terminal repeats (LTRs) of equine infectious anemia virus (EIAV) were examined with respect to their ability to function as transcriptional promoters in various cellular environments. Nucleotide sequence analyses of the LTRs derived from two unique proviral clones revealed the requisite consensus transcription and processing signals. One of the proviruses possessed a duplication of a 16-base-pair sequence in the CCAAT box region of the LTR which was absent in the other provirus. To assess its functional activity, each LTR was coupled to the bacterial chloramphenicol acetyltransferase gene and transfected onto various cell lines, including matched cultures of EIAV-infected and uninfected cells. The levels of chloramphenicol acetyltransferase activity directed by the EIAV LTRs were between 250 and 900 times greater in EIAV-infected cells compared with their uninfected counterparts. Thus, EIAV expression appears to be activated by a virus-induced trans-activation phenomenon analogous to that recently shown to amplify expression of certain other lentiviruses.

Base Sequence↗
PubMed1987

Expression of the bovine leukemia virus X region in virus-infected cells.

Bovine leukemia virus, like its closest relatives the human T-cell leukemia virus types I and II, contains a 1.8-kilobase X region between the env gene and the 3' long terminal repeat. In this communication, we report the detection and characterization of a subgenomic mRNA from which this X region is presumably translated. This mRNA was produced by a complex splicing mechanism which resulted in juxtaposition of the 5' end of the env gene and the two overlapping X-region open reading frames. Translation of this mRNA could yield at least two distinct proteins depending on which initiation codon is used. Detection of the protein encoded by the BLV X-region long open reading frame has been reported (N. Sagata, J. Tsuzuku-Kawamura, M. Nagayoshi-Aida, F. Shimizu, K.-I. Imagawa, and Y. Ikawa, Proc. Natl. Acad. Sci. USA 82:7879-7883, 1985). Using synthetic peptide antisera, we detected a protein encoded by the short open reading frame in virus-infected cells. The protein migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 19,000. It is a nuclear phosphoprotein.

Base Sequence↗
PubMed1987

Detection of c-rel-related transcripts in mouse hematopoietic tissues, fractionated lymphocyte populations, and cell lines.

A portion of the human cellular homolog of v-rel, the transforming gene of the leukemogenic retrovirus reticuloendotheliosis virus, strain T, was used to survey RNAs from several mouse tissues, selected lymphocyte populations, and hematopoietic cell lines for c-rel expression. Relatively high levels of a high-molecular-weight transcript were observed in peripheral B and T cells, whereas lower levels were detectable in functionally immature thymocytes. These results suggested that, unlike c-myb and c-ets, the c-rel proto-oncogene plays a role in later stages of lymphocyte differentiation.

Animals↗