Ontogeny of the v-erbA oncoprotein from the thyroid hormone receptor: an alteration in the DNA binding domain plays a role crucial for v-erbA function.

The avian erythroblastosis virus v-erbA oncogene is imprecisely derived from a cellular gene (c-erbA) encoding a thyroid hormone receptor: the v-erbA protein has sustained both small terminal deletions and internal amino acid sequence changes relative to c-erbA. We report here that one of these missense differences between v- and c-erbA proteins, located in a zinc finger DNA binding domain, has dramatic effects on the biological activities of the encoded protein. Back mutation of the viral coding sequence to resemble c-erbA at this site severely impairs erythroid transformation and produces subtle changes in DNA binding by the encoded protein, suggesting that differences in DNA binding by the viral and cellular proteins may be involved in the activation of v-erbA as an oncogene.     

Sequence-specific DNA binding by the v-erbA oncogene protein of avian erythroblastosis virus.

The v-erbA oncogene, a transduced copy of a thyroid hormone receptor, plays an important role in establishment of the transformed cell phenotype induced by avian erythroblastosis virus. The ability of thyroid hormone receptors to bind to specific sites on chromatin and to thereby modify the expression of adjacent target genes is a crucial element in their mechanism of action in the normal cell. The v-erbA protein also bound at high affinity to a set of DNA fragments recognized by the rat thyroid hormone receptor, but the relative affinity of the v-erbA protein for the different binding sites was distinct from that previously reported for the thyroid hormone receptors.     

Purification of a novel UV-damaged-DNA binding protein highly specific for (6-4) photoproduct.

UV damage-specific binding proteins are considered to play important roles in early responses of cells irradiated with UV, including damage recognition in the DNA repair process. We have surveyed nuclear and cytoplasmic proteins which bind selectively to UV-irradiated DNA using an electrophoretic mobility shift assay. We detected four distinct binding activities with different mobilities in fractions separated from HeLa cells by heparin chromatography. Three of them were found in nuclear extracts and one in cytoplasmic extracts. We purified one of the binding factors from nuclear extracts to homogeneity, which was designated NF-10 (the 10th fraction of nuclear extract on heparin chromatography). It migrated as a 40 kDa polypeptide in SDS-PAGE, and bound to UV-irradiated double- stranded DNA but not to unirradiated DNA. The binding pattern of the NF-10 protein to DNA irradiated with UV corresponded to the induction kinetics of (6-4) photoproduct. Removal of (6-4) photoproducts from UV- irradiated DNA by (6-4) photoproduct-specific photolyase diminished the binding of NF-10 protein. These results suggest that the NF-10 protein binds to UV-damaged DNA through (6-4) photoproduct. Immunoblot analysis using a monoclonal antibody revealed that the NF-10 protein was expressed in cell lines from all complementation groups of xeroderma pigmentosum, indicating that the NF-10 protein is a novel UV-damaged-DNA binding protein.     

Cyclic AMP-binding protein and estrogen receptor: antagonism during nuclear translocation in a hormone-dependent mammary tumor

Incubation of nuclei from hormone-dependent rat mammary tumors with its cytosol activated with 5 nM 17β-estradiol resulted in a 4-fold increase of nuclear estrogen binding activity over the control nuclei. The presence of 100 nM cAMP in the activated cytosol inhibited this nuclear uptake of estrogen receptor by 50%. Conversely, incubation of the nuclei with cytosol activated with 100 nM cAMP increased nuclear cAMP binding and cAMP-dependent protein kinase activity 4-fold, while the presence of 5 nM 17β-estradiol in the activated cytosol inhibited the nuclear cAMP binding and the protein kinase activity by 50%. No competition was found between estrogen and cAMP for each other's cytoplasmic binding proteins or the nuclear acceptor sites. These data suggest that a mutual antagonism exists between the cAMP-binding protein and estrogen receptor during their nuclear translocation.     

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.     

Nuclear localization of herpesvirus proteins: potential role for the cellular framework.

Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : IV. HeLa Tumor Strain Cells

Interferometric and photometric measurements have been made on HeLa cells, a strain of cells originally derived from a human carcinoma. From a study of the relations between successive physical measurements on individual cells, it was confirmed that, whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results on nuclear metabolism agree with others previously reported in cell strains derived from tumors; they contrast with results from freshly prepared normal cells, where the net syntheses of DNA, nuclear RNA, and protein are closely associated during interphase. Cytoplasmic measurements on HeLa cells showed that much of the net synthesis of cytoplasmic RNA is associated with DNA replication as in normal cells, and they failed to detect transfer from the nucleus of a stable RNA component synthesized independently from DNA replication. In auxiliary experiments, an inhibition of the onset of DNA synthesis was produced by a dose of X-rays; under these conditions it was shown that the major part of the accumulation of nuclear protein was independent of DNA replication and that the accumulation of nuclear RNA was equivalent to or slightly less than that of nuclear protein. About half the accumulation of cytoplasmic RNA was inhibited when DNA synthesis was blocked.     

Molybdate and the 1,25-dihydroxyvitamin D3 receptor from chick intestine

The nature of the 1,25-dihydroxyvitamin D3 receptor from chick intestine was examined in regard to its response to sodium molybdate. Sodium molybdate (10 mM) stabilized the receptor from crude nuclear extract but not that from the supernatant or cytoplasmic fraction, suggesting the molybdate may act by binding to the DNA binding region of the receptor. At a concentration of 50 mM, sodium molybdate prevented aggregation of the nuclear receptor. This concentration of sodium molybdate also inhibited the receptor from binding to DNA cellulose while the same ionic strength KCl (90 mM) did not. These properties also suggest that molybdate interacts with the DNA binding region. Purification of the receptor using DNA cellulose chromatography has also been improved by using a sodium molybdate gradient (0-0.2 M) instead of the KCl gradient used previously.     

Genetic dissection of functional domains within the avian erythroblastosis virus v-erbA oncogene.

The avian erythroblastosis virus v-erbA locus potentiates the oncogenic transformation of erythroid and fibroblast cells and is derived from a host cell gene encoding a thyroid hormone receptor. We report here the use of site-directed mutagenesis to identify and characterize functional domains within the v-erbA protein. Genetic lesions introduced into a putative hinge region or at the extreme C-terminus of the v-erbA coding domain had no significant effect on the biological activity of this polypeptide. In contrast, mutations introduced within the cysteine-lysine-arginine-rich center of the v-erbA coding region, a DNA-binding domain in the thyroid and steroid hormone receptors, abolished or severely compromised the ability of the viral protein to function. Our results suggest that the mechanism of action of the v-erbA protein in establishing the neoplastic phenotype is closely related to its ability to interact with DNA, presumably thereby altering expression of host target genes by either mimicking or interfering with the action of the normal c-erbA gene product.     

The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1

Rabies virus P protein inhibits alpha interferon (IFN-alpha)- and IFN-gamma-stimulated Jak-STAT signaling by retaining phosphorylated STAT1 in the cytoplasm. Here, we show that P also blocks an intranuclear step that is the STAT1 binding to the DNA promoter of IFN-responsive genes. As P is a nucleocytoplasmic shuttling protein, we first investigated the effect of the cellular distribution of P on the localization of STAT1 and consequently on IFN signaling. We show that the localization of STAT1 is correlated with the localization of P: in cells expressing a nuclear form of P (the short P3 isoform or the complete P in the presence of the export inhibitor leptomycin B), STAT1 is nuclear, whereas in cells expressing a cytoplasmic form of P, STAT1 is cytoplasmic. However, the expression of nuclear forms of P inhibits the signaling of both IFN-gamma and IFN-alpha, demonstrating that the retention of STAT1 in the cytoplasm is not the only mechanism involved in the inhibition of IFN signaling. Electrophoretic mobility shift analysis indicates that P expression in the cell extracts of infected cells or in stable cell lines prevents IFN-induced DNA binding of STAT1. The loss of the DNA binding of STAT1 and ISGF3 was also observed when purified recombinant P or P3 was added to the extracts of IFN-gamma- or IFN-alpha-treated cells, indicating that P directly affects the DNA binding activity of STAT1. Then products of the rabies virus P gene are able to counteract IFN signaling by creating both cytoplasmic and nuclear blocks for STAT1.     

In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function.

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.     

c-abl has a sequence-specific enhancer binding activity.

The enhancers of several distinct viruses contain a common functional element, termed EP. This element binds ubiquitous cellular proteins and generates specific complexes in gel retardation analysis. Ultraviolet cross-linking and Southwestern analysis showed that a 140 kd polypeptide is the major EP DNA-binding protein. Using a combination of DNA binding and immunological techniques, we have identified the c-abl protein in a nuclear complex that binds to the EP element. abl was found to have both a specific and high affinity DNA binding activity. The ability to bind DNA is abolished in the mutant abl protein, p210bcr-abl, consistent with its cytoplasmic localization in chronic myelogenous leukemia.