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Query: UNIPROT:P06889 (Mol)
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Previously we have shown that nuclear extracts from mouse cells contain a heterogeneous group of polypeptides (p65, p80, p90, p100) which form distinct DNA-protein complexes on the 18 base-pair sequence element (termed Sal-box), which constitutes the murine rDNA transcription termination signal. These distinct proteins mediate cessation of RNA polymerase I (pol I) transcription elongation and release of the nascent RNA chains, indicating that they function as termination factor(s). Here, we report the biochemical analysis of the pol I-specific transcription termination factor TTFI. We show that the heterogeneity of TTFI is due to limited proteolysis of a larger, 130 kDa precursor protein (p130). The DNA-binding activity of p130 is strongly reduced as compared to the proteolytic derivatives, indicating that the DNA-binding domain is repressed within the full-length molecule. We have used limited proteolysis to purify and functionally characterize a TTFI core polypeptide (p50) which still specifically binds to the Sal-box target sequence and directs rDNA transcription termination. The equilibrium constant of purified p50 to bind specifically to DNA is 9 x 10(9) M-1. Additionally, we demonstrate that TTFI binds to DNA as a monomer and that binding induces DNA bending. This observation suggests that not only specific DNA-protein and protein-protein interactions but also conformational alterations of DNA may play a role in the termination process.
J Mol Biol 1992 Oct 05
PMID:Limited proteolysis unmasks specific DNA-binding of the murine RNA polymerase I-specific transcription termination factor TTFI. 140 80

The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.
Mol Cell Biol 1989 Jul
PMID:Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. 255 Jul 92

By raising monoclonal antibodies to the apical surface of Caco-2 cells we have identified a membrane protein (p100) that internalizes and recycles constitutively between the apical plasma membrane and endosomes in the apical cytoplasm. By applying tracers bound to the transferrin receptor, which internalizes and recycles back to the basolateral border, we demonstrate that the apical endosomes containing p100 include a subset of multivesticular bodies (MVB), which are also accessible to proteins arriving from the basolateral endosome. Tracers bound to EGF receptors and alpha-2-macroglobulin, which internalize from the basolateral border and are degraded, probably in lysosomes, also pass through the p100-containing MVB. These studies therefore suggest that the apical cytoplasm of Caco-2 cells contains a population of MVB capable of receiving membrane proteins trafficking in from both apical and basolateral borders and then routing them to a variety of cell surface and intracellular destinations. The differential distribution of apical and basolateral tracers within the 50-nm-diameter tubules connected to these p100-positive apical MVB suggests that the destination of proteins trafficking from the MVB back to apical and basolateral surfaces is determined by the tubules to which they gain access.
Mol Biol Cell 1995 May
PMID:Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells. 754 32

Epstein-Barr virus nuclear antigen 2 (EBNA 2) activates transcription of specific genes and is essential for B-lymphocyte transformation. EBNA 2 has an acidic activation domain which interacts with general transcription factors TFIIB, TFIIH, and TAF40. We now show that EBNA 2 is specifically bound to a novel nuclear protein, p100, and that p100 can coactivate gene expression mediated by the EBNA 2 acidic domain. The EBNA 2 acidic domain was used to affinity purify p100. cDNA clones encoding the p100 open reading frame were identified on the basis of peptide sequences of the purified protein. Antibody against p100 coimmunoprecipitated p100 and EBNA 2 from Epstein-Barr virus-transformed lymphocyte extracts, indicating that EBNA 2 and p100 are complexed in vivo. p100 overexpression in cells specifically augmented EBNA 2 acidic domain-mediated activation. The coactivating effect is probably mediated by p100 interaction with TFIIE. Bacterially expressed p100 specifically adsorbs TFIIE from nuclear extracts, and in vitro-translated p56 or p34 TFIIE subunit can independently bind to p100. p100 also appears to be essential for normal cell growth, since cell viability was reduced by antisense p100 RNA and restored by sense p100 RNA expression.
Mol Cell Biol 1995 Sep
PMID:The Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE. 765 91

The enhancer of Moloney murine leukemia virus (Mo-MuLV) contains an array of transcriptional control elements that direct viral gene expression in diverse cell types. The murine transcription factor Ets-1 was shown to bind to the LVb and LVc elements of the enhancer by DNase I protection and methylation interference assays. Enhancers containing disrupted Ets-1 binding sites were tested in transient expression assays in the murine T-cell line EL4.E1; alterations in the LVb element affected constitutive enhancer activity, while mutation of either the LVb or LVc element disrupted phorbol ester-induced enhancer activity. Members of the ets gene family of proteins display similar DNA-binding properties; therefore, we speculated that ets proteins other than Ets-1 also might bind these elements. Crude nuclear extracts of EL4.E1 cells were assayed to identify the protein(s) that potentially functions at the LVb element. The predominant binding activity was not Ets-1 but rather two independent DNA-protein complexes that comigrated in mobility shift assays. UV cross-linking and denaturing gel electrophoresis sized the two DNA-binding species, which we denoted p55 and p100. Immunoprecipitation combined with UV cross-linking identified p55 as the alpha subunit of GA-binding protein. The DNA-binding properties of p100 and several ets proteins were compared. Similarities suggested that p100 is also an ETS domain protein, possibly Elf-1. This strategy could be used to identify other ETS domain proteins in crude nuclear extracts. These findings suggest multiple ETS domain proteins could regulate gene expression of Mo-MuLV.
Mol Cell Biol 1994 Nov
PMID:Identification of ETS domain proteins in murine T lymphocytes that interact with the Moloney murine leukemia virus enhancer. 793 72

The prothoracic glands of fifth instar Manduca sexta larvae respond to stimulation by the brain neuropeptide, prothoracicotropic hormone (PTTH), with a several-fold increase in the rate of ecdysteroid synthesis. Previous studies have shown that this response requires protein synthesis and that the action of PTTH can be mimicked by dibutyryl cAMP (dbcAMP) and the Ca2+ ionophore, A23187. To further understand the role of protein synthesis in the response of prothoracic glands to PTTH, patterns of protein synthesis in stimulated glands were examined using glands incubated in vitro with [35S]methionine. All three agents caused an increase in the rate of ecdysteroid synthesis as well as an increase of up to 300% in the synthesis and/or accumulation of three proteins (p100, p70, and p"50") within 2 h of stimulation. Changes in these three proteins were specific to the prothoracic gland, were not elicited by non-brain peptides and were not simply a result of increased general protein synthesis in the gland. Exposure of the glands to A23187 alone, or concurrently with dbcAMP, resulted in increased synthesis of p100, p70, p"50" and ecdysteroids but decreased general protein synthesis. Increased synthesis of these proteins could be detected within 15 min after initiating PTTH stimulation. The behavior of these three proteins makes them candidates for modulators of ecdysteroid synthesis in the prothoracic gland. The results suggest also that PTTH may activate two biochemical pathways in the gland: one path leading to increased synthesis of the p100, p70, and p"50" proteins and increased ecdysteroid synthesis, and the second leading to increased general protein synthesis. This second trophic effect is vulnerable to intracellular Ca2+ changes that do not inhibit the first pathway.
Insect Biochem Mol Biol 1994 Feb
PMID:Changes in general and specific protein synthesis that accompany ecdysteroid synthesis in stimulated prothoracic glands of Manduca sexta. 811 26

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of the adult T-cell leukemia, an aggressive and often fatal malignancy of activated human CD4 T cells. HTLV-I encodes an essential 40-kDa protein termed Tax that not only transactivates the long terminal repeat of this retrovirus but also induces an array of cellular genes. Tax-mediated transformation of T cells likely involves the deregulated expression of various cellular genes that normally regulate lymphocyte growth produced by altered activity of various endogenous host transcription factors. In particular, Tax is capable of modulating the expression or activity of various host transcription factors, including members of the NF-kappa B/Rel and CREB/ATF families, as well as the cellular factors HEB-1 and p67SRF. An additional distinguishing characteristic of HTLV-I infection is the profound state of viral latency that is present in circulating primary leukemic T cells. In this study, we demonstrate that HTLV-I Tax can physically associate with p100, the product of the Rel-related NF-kappa B2 gene, both in transfected cells and in HTLV-I-infected leukemic T-cell lines. Furthermore, the physical interaction of Tax with p100 leads to the inhibition of Tax-induced activation of the HTLV-I and human immunodeficiency virus type 1 long terminal repeats, reflecting p100-mediated cytoplasmic sequestration of the normally nuclearly expressed Tax protein. In contrast, a mutant of Tax that selectively fails to activate nuclear NF-kappa B expression does not associate with p100. Together, these results suggest that the cytoplasmic interplay of Tax and p100 may play an important role in the initiation and maintenance of HTLV-1 latency observed in adult T-cell leukemia.
Mol Cell Biol 1994 Feb
PMID:Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF-kappa B2 p100 gene product: implications for viral latency. 828 13

NF-kappa B is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappa B activity is induced by numerous stimuli, such as phorbol esters, B- and T-cell mitogens, the cytokines tumor necrosis factor and interleukin-1, and serum growth factors. The standard model for the induction of NF-kappa B activity involves the release of the transcription factor from a cytoplasmic inhibitor termed I kappa B, allowing translocation of NF-kappa B to the nucleus. I kappa B contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappa B and related binding activities are members of the Rel multigene family. Two such subunits, p50 and p52 (also called p50B), are proteolytically processed from precursors of 105 kDa (also called p105 and NFKB1) and 100 kDa (also called p100, NFKB2, and Lyt-10), respectively. Both contain N-terminal Rel-homologous domains as well as multiple copies of C-terminal ankyrin repeats. We show here that NF-kappa B p100 is a component of the previously identified DNA-binding activity H2TF1. In addition, we show that p100 is localized in the cytoplasm in HeLa cells, where it is associated with c-Rel, p50, or p65 (RelA). In transient-transfection assays, p100 represses the ability of NF-kappa B p65 to activate a kappa B-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappa B probe, as measured by an electrophoretic mobility shift assay, and a loss of nuclear p65 immunoreactivity, as measured by immunoblotting. This loss of nuclear p65 is paralleled by a gain of p65 DNA-binding activity and immunoreactivity in the cytoplasm. We interpret these data as demonstrating that p100 functions as an I kappa B-like molecule to sequester Rel family members in the cytoplasm. Proteolytic processing of p100 to the activator p52 is predicted to generate several new forms of Rel family heterodimers and therefore represents a form of regulation of NF-kappa B activity distinct from the classic I kappa B pathway.
Mol Cell Biol 1993 Oct
PMID:NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule. 841 11

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.
Mol Cell Biol 1993 Mar
PMID:Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3). 844 77

Proteolytic degradation of the C-terminal region of NF-(kappa)B precursors to their active DNA binding forms represents an important regulatory step in the activation of NF-(kappa)B. NF-(kappa)B2(p100) is found ubiquitously in the cytoplasm; however, the site and mechanism of processing to p52 have not previously been defined. We show by deletion mapping that processing of NF-(kappa)B2(p100) terminates at alanine 405 to generate p52 and is prevented by specific inhibitors of the multicatalytic proteinase complex. Although the C-terminal I(kappa)B-like domain of NF-(kappa)B2(p100) was constitutively phosphorylated, disruption of this phosphorylation by mutagenesis demonstrated that it was not required as a signal to mediate processing. Mutational analysis further showed that cleavage of NF-(kappa)B2 is not dependent on a specific sequence motif adjacent to alanine 405, the ankyrin repeats, or other C-terminal sequences but is directed by structural determinants amino terminal to the cleavage site, within the Rel homology domain and/or the glycine hinge region. The level of processing of NF-(kappa)B2(p100) was much lower than that of NF-(kappa)B1(p105) and differed from that of I(kappa)B-alpha, suggesting differential control of processing of NF-(kappa)B/I(kappa)B family members.
Mol Cell Biol 1996 Nov
PMID:Differential regulation of NF-kappaB2(p100) processing and control by amino-terminal sequences. 888 65


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