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C V Dang

Publications and source records attributed to C V Dang.

At least 19 recordsLinked to original sources
PubMed1992

Opposite orientations of DNA bending by c-Myc and Max.

The control of gene transcription requires specific protein-protein and protein-DNA interactions. c-Myc, the protein product of the c-myc protooncogene, is a member of the basic helix-loop-helix leucine-zipper class of transcription factors. Although c-Myc is able to bind to a specific core hexanucleotide DNA sequence (CACGTG), its precise function in modulating transcription remains unclear. The recent discovery of Max, a basic helix-loop-helix leucine-zipper partner protein for c-Myc, suggests that the ability of c-Myc to regulate transcription is modulated by the presence of Max. By taking advantage of the altered mobility of protein-bound DNA in the mobility-shift assay, we demonstrate the homo- and heterodimeric complexes of c-Myc and Max are able to cause increased DNA flexure as measured by the circular permutation assay. Based on phasing analysis, c-Myc and Max homodimers bend DNA in opposite orientations, whereas c-Myc-Max heterodimers cause a smaller bend, in an orientation similar to that induced by Max homodimers. To address the possibility that the apparent opposite orientation of bending was the result of DNA unwinding by one of the proteins, we measured the ability of c-Myc and Max homodimers to affect DNA unwinding; we were unable to show any specific unwinding caused by c-Myc or Max. In addition to demonstrating that members of the basic helix-loop-helix leucine-zipper class of transcription factors are able to induce DNA bending, these results suggest that different transcription factor dimers are able to bind to identical DNA sequences and yet have distinct structural effects.

Animals↗
PubMed1992

Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins.

A yeast genetic system was developed to study how the basic regions of basic-helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG. The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis. Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG. The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region. However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function. In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position. The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites.

Amino Acid Sequence↗
PubMed1992

DNA binding by the Myc oncoproteins.

The c-Myc protein is a potential activator of transcription, with the ability to bind in a heterodimer form with Max to DNA sequences containing the core hexanucleotide sequence CAC(G/A)TG. These properties are shared with L-Myc, a homologous oncoprotein expressed in small cell lung carcinoma cells; with N-Myc, expressed in neuroblastoma cells; and with avian v-Myc, the c-Myc homolog expressed by a chicken retrovirus. The c-Myc, and probably v-Myc, proteins also have nonspecific DNA binding function, which may improve the kinetics of specific DNA binding. Curiously, this domain appears not to be conserved in L-Myc or N-Myc [22]. The data that have accumulated to date are consistent with a model in which a c-Myc/Max heterodimer positively regulates the transcription of growth-related genes, with Max homodimer functioning as a negative regulator of the same genes (Fig. 4) [55]. Max is expressed constitutively at low levels, whereas c-Myc is expressed at low levels in quiescent cells, but high levels of c-Myc are induced by mitogenic stimulation [56]. Thus, in proliferating cells c-Myc/Max heterodimers might bind to the regulatory elements of growth-related genes, where the c-Myc TAD might stimulate transcription. Conversely, in quiescent cells with little c-Myc present, Max homodimers might predominate. They might bind to exactly the same regulatory elements, but due to the apparent absence of a TAD in Max [36], transcription might be repressed. Validation of this model will require the demonstration of clear regulation of a physiological promoter of a growth-related gene by c-Myc/Max. Although it is widely believed that Myc proteins function as transcriptional activators, this hypothesis has only been conclusively supported recently [57, 58]. A theory that c-Myc plays a role in DNA replication is not as well substantiated at this point. It is even possible that Myc might be involved in both transcription and replication. Although the function of these fascinating proteins has been enigmatic for a decade, the rate of progress in our understanding of Myc function is accelerating. Such progress will undoubtedly lead to a deeper appreciation of this protein, which lies at the crossroads of cellular proliferation and oncogenesis.

Amino Acid Sequence↗
PubMed1992

Function of the c-Myc oncoprotein.

The c-Myc protein, the product of the c-myc proto-oncogene, is a nuclear phosphoprotein with DNA binding properties. Deregulated c-myc expression participates in the development of experimentally induced tumors, and its expression appears to be abnormal in many naturally occurring malignancies. Although the precise molecular mechanism of c-Myc activity in oncogenesis and in normal cell proliferation is unknown, recent advances have uncovered a series of molecular and cellular properties of c-Myc. These properties include nuclear localization, transcriptional activation, oligomerization nonspecific and specific DNA binding. Recently, the c-Myc protein was found to heterodimerize with Max, a protein that cooperates with c-Myc to bind specifically to a core DNA sequence, CAC(G/A)TG. These characteristics suggest that c-Myc participates in the regulation of gene transcription in normal and neoplastic cells.

Animals↗
PubMed1992

Max: functional domains and interaction with c-Myc.

The product of the c-myc proto-oncogene is a DNA-binding protein, the deregulated expression of which is associated with a variety of malignant neoplasms. The cDNA for the max gene was recently cloned as a result of the ability of its protein product to interact with the c-Myc protein. We studied bacterially produced Max, c-Myc, and a series of truncated c-Myc proteins. Full-length c-Myc alone cannot bind DNA. However, a truncated c-Myc protein comprising the basic, helix-loop-helix, and leucine zipper regions can bind specifically to DNA bearing the sequence GGGCAC(G/A)TGCCC. Max protein, either alone or in a heteromeric complex with full-length c-Myc, binds to the same core sequence. Using a novel combination of chemical and photo-cross-linking analysis, we demonstrate that either Max or a c-Myc/Max heteromeric complex binds to DNA virtually exclusively in a dimeric structure. Using fusion proteins in cultured cells, we establish a number of functional characteristics of Max. First, we show that Max can interact with c-Myc intracellularly in a manner dependent on the integrity of the helix-loop-helix and leucine zipper motifs. Second, a nuclear localization domain that contains the sequence PQSRKKLR is mapped to the carboxy-terminal region of Max. Third, Max lacks a transcriptional activation domain that is functional in Chinese hamster ovary cells when fused to a heterologous DNA-binding domain. These data suggest that Max may serve as a cofactor for c-Myc in transcriptional activation or, by itself, as a transcriptional repressor.

Amino Acid Sequence↗
PubMed1992

Activation domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells.

Members of the Myc family of proteins share a number of protein motifs that are found in regulators of gene transcription. Conserved stretches of amino acids found in the N-terminal transcriptional activation domain of c-Myc are required for cotransforming activity. Most of the Myc proteins contain the basic helix-loop-helix zipper (bHLH-Zip) DNA-binding motif which is also required for the cotransforming activity of c-Myc. L-Myc, the product of a myc family gene that is highly amplified in many human lung carcinomas, was found to cotransform primary rat embryo cells with an activated ras gene. However, L-Myc cotransforming activity was only 1 to 10% of that of c-Myc (M. J. Birrer, S. Segal, J. S. DeGreve, F. Kaye, E. A. Sausville, and J. D. Minna, Mol. Cell. Biol. 8:2668-2673, 1988). We sought to determine whether functional differences between c-Myc and L-Myc in either the N-terminal or the C-terminal domain could account for the relatively diminished L-Myc cotransforming activity. Although the N-terminal domain of L-Myc could activate transcription when fused to the yeast GAL4 DNA-binding domain, the activity was only 5% of that of a comparable c-Myc domain. We next determined that the interaction of the C-terminal bHLH-Zip region of L-Myc or c-Myc with that of a Myc partner protein, Max, was equivalent in transfected cells. A Max expression vector was found to augment the cotransforming activity of L-Myc as well as that of c-Myc. In addition, a bacterially synthesized DNA-binding domain of L-Myc, like that o c-Myc, heterodimerizes with purified Max protein to bind the core DNA sequence CACGTG. To determine the region of L-Myc responsible for its relatively diminished cotransforming activity, we constructed chimeras containing exons 2 (constituting activation domains) and 3 (constituting DNA-binding domains) of c-Myc fused to those of L-Myc. The cotransforming potencies of these chimeras were compared with those of full-length L-Myc of c-Myc in rat embryo cells. The relative cotransforming activities suggest that the potencies of the activation domains determine the cotransforming efficiencies for c-Myc and L-Myc. This correlation supports the hypothesis that the Myc proteins function in neoplastic cotransformation as transcription factors.

Animals↗
PubMed1991

c-myc oncoprotein function.

Genetic alterations of the c-myc locus in various malignancies and the ability of c-myc to transform cultured cells and induce tumors in transgenic animals attest to its central role in many neoplasms. By dissecting the c-Myc protein, a number of critical functional domains of c-Myc have been identified and characterized; these findings suggest a model for c-Myc function and intracellular activity (Fig. 4). c-Myc is synthesized in the cytoplasm and undergoes oligomerization another protein such as Max. Its nuclear localization signal allows c-Myc to be targeted to and retained in the nucleus, where the protein seeks out and binds to specific DNA sites, perhaps facilitated by c-Myc's ability to bind non-specifically to DNA. Once bound to specific DNA sequences, c-Myc then activates or inhibits transcription of a number of target genes, with consequent alterations in cell growth and differentiation. Continued studies of c-Myc and its partner Max should further elucidate the mechanisms by which c-Myc can contribute both to the regulation of normal cell growth and the alteration in that regulation in neoplasia.

Amino Acid Sequence↗
PubMed1991

The upstream stimulatory factor binds to and activates the promoter of the rat class I alcohol dehydrogenase gene.

The gene encoding rat class I alcohol dehydrogenase (ADH) is expressed primarily in the liver. Recent studies in our laboratories indicate that multiple cellular factors present in the rat liver interact with various regions of the promoter of this gene. One of the regions contains the sequence 5'-CACATG-3' that has an "E box" homology to which a number of transcription factors containing the basic helix-loop-helix motif bind. We now demonstrate that the human transcription factor, upstream stimulatory factor (USF), a basic helix-loop-helix-containing protein, binds to and activates the promoter of the rat class I ADH gene. Electrophoretic mobility shift assays of labeled oligonucleotide containing the 5'-CACATG-3' sequence within the ADH promoter revealed the formation of multiple DNA-protein complexes when nuclear extracts obtained from adult rat liver were used. The binding of proteins to the DNA could be competed away with an oligonucleotide specifying a sequence within the adenovirus major late promoter (MLP) that had previously been shown to bind USF. Similar complexes were observed when electrophoretic mobility shift assays of labeled MLP oligonucleotide were performed with rat liver nuclear extracts. Conversely, nuclear extracts isolated from HeLa cells, cells known to have abundant USF, contain factors that interact with the sequence present in the ADH promoter. This interaction could be competed efficiently by the MLP oligonucleotide. USF synthesized in an in vitro transcription and translation system also binds to the ADH promoter as well as to the MLP. In addition, antiserum directed against USF recognizes factors present in the rat liver nuclear extracts that interact with the ADH promoter. Furthermore, transcription directed from both the ADH and the adenovirus major late promoters was inhibited by an oligonucleotide representing the USF-binding site within the ADH promoter in a cell-free in vitro transcription system. Lastly, an ADH promoter-reporter gene construct was transactivated by an eukaryotic expression vector containing USF in HepG2 cells co-transfected with the two constructs. These experiments demonstrate that USF is present in the rat liver and that it binds to and activates the promoter of the rat class I ADH gene in a sequence-specific manner.

Alcohol Dehydrogenase↗
PubMed1991

Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization.

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

Amino Acid Sequence↗
PubMed1990

Fibrinogen Baltimore I: polymerization defect associated with a gamma 292Gly----Val (GGC----GTC) mutation.

Fibrinogen Baltimore I is one of the very first congenital abnormal fibrinogens reported over several decades ago; however, the molecular defect of this dysfibrinogen has eluded identification. In fact, several reports misidentified the functional defect of Baltimore I, which has impaired fibrin monomer polymerization. Reversed-phase high-performance liquid chromatography analysis of lysyl endopeptidase digest of the purified Baltimore I gamma-chain showed an abnormal peptide not found in the co-existing normal gamma-chain of this heterozygote. Amino acid sequencing of this peptide indicated that gamma-chain Gly292 is replaced by valine. This observation was confirmed, and the genetic defect was determined by direct nucleotide sequencing of a polymerase chain reaction product containing codon gamma 292, which is mutated: GGC----GTC. The molecular defect of Fibrinogen Baltimore I lies in a region of the gamma-chain required for fibrin polymerization, suggesting that the integrity of gamma Gly292 is critical for fibrin assembly.

Adult↗
PubMed1990

Polymerization defect of fibrinogen Baltimore III due to a gamma Asn308----Ile mutation.

Fibrinogen Baltimore III, a congenital abnormal fibrinogen with impaired fibrin monomer polymerization, displays a normal gamma-chain and a gamma-variant that has an apparently lower relative molecular weight (mol wt) than normal on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Reverse phase high-performance liquid chromatography (HPLC) analysis of the lysyl endopeptidase digest of the purified gamma-chains of fibrinogen Baltimore III revealed the presence of a peptide that is not found in the digest of the normal fibrinogen gamma-chain. Amino acid sequence analysis of this peptide indicated that the gamma-chain residue 308, asparagine, is replaced by isoleucine. Concanavalin A bound both normal and variant gamma-chains of fibrinogen Baltimore III, indicating that the carbohydrate moiety is not altered and is not responsible for the increase in electrophoretic mobility of the Baltimore III gamma-chain. This study suggests that the integrity of gamma Asn308 is critical for fibrin monomer polymerization, since alteration to either a basic (fibrinogen Kyoto I, Asn----Lys) or hydrophobic (Asn----Ile) residue results in significantly delayed polymerization of fibrinogen to fibrin.

Amino Acid Sequence↗
PubMed1990

An amino-terminal c-myc domain required for neoplastic transformation activates transcription.

The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.

Amino Acid Sequence↗
PubMed1990

Definition of the activities and properties of c-myc required to inhibit cell differentiation.

Previous studies have shown that high levels of c-myc inhibit cell differentiation. The goal of this study was to define the activities and properties of c-myc that are necessary and/or sufficient for this effect. A series of mutant human c-myc genes were stably transfected into the 3T3-L1 preadipocyte cell line and assayed for their capacity to block differentiation into adipocytes. Results of the differentiation tests were then correlated with other known activities and properties of the mutants. Our studies show that the ability of c-myc to inhibit 3T3-L1 cell differentiation requires its transforming activity, and the ability of c-myc to bind sequence-nonspecific DNA and to form oligomers is not sufficient for this effect. Thus, the ability of c-myc to inhibit cell differentiation may be central to its role as a transforming oncogene.

Adipose Tissue↗
PubMed1990

A potential transcriptional activation element in the p53 protein.

The human p53 gene codes for a 393 amino acid nuclear phosphoprotein. p53 is most commonly described as a tumor suppressor, or anti-oncogene, although its role in vivo remains unclear. We report that GAL4-p53 fusion protein can activate transcription of a CAT reporter gene downstream of a GAL4-DNA binding site. We tested both the amino terminal 160 amino acids and the carboxyl terminal 233 amino acids of the p53 protein and found that the transcriptional activating (TA) region was restricted to the amino terminal fragment. These results imply that p53 may be a transcriptional activating factor (TAF); furthermore, these data lend support to the hypothesis of p53 as a positive regulator of transcription which might mediate its tumor suppressor role by inducing expression of a set of genes with a negative effect on cellular growth.

Animals↗
PubMed1989

Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins.

Protein import into the cell nucleus requires specific binding of nuclear proteins to the nuclear pore complex. Based on amino acid sequence "motifs" of known nuclear targeting signals, we identified peptides within a number of nuclear proteins with likely nuclear targeting potential and tested their function by transfecting into cells fusion genes that produce the cytoplasmic "reporter" protein, pyruvate kinase (PK), joined to the test sequence. Sequences within c-myb (PLLKKIKQ), N-myc (PPQKKIKS), p53 (PQPKKKP), and c-erb-A (SKRVAKRKL) oncoproteins that direct PK hybrids into the nucleus were identified. A peptide (GRKKRRQRRRAP) of the human immunodeficiency virus (HIV) tat protein (Tat), which contains two short basic regions, targets fusion proteins to the nucleolus. The COOH-terminal basic Tat region (QRRRAP) does not target PK hybrid proteins into the nucleus, but mutation of two basic amino acids in this region decreases but does not abolish nucleolar accumulation mediated by the entire Tat nucleolar targeting sequence. Moreover, the c-Myc nuclear targeting sequence fused to the COOH-terminal basic Tat region (PAAKRVKLDQRRRAP) effectively localizes PK hybrids to the nucleus and nucleolus. A similar sequence (FKRKHKKDISQNKRAVRR) in the human heat-shock protein HSP70 also localizes PK to the nucleus and nucleolus.

Amino Acid Sequence↗
PubMed1989

Fibrinogen sialic acid residues are low affinity calcium-binding sites that influence fibrin assembly.

Calcium ions occupy low (n congruent to 10; Kd congruent to 1 mM) and high (n = 3; Kd congruent to 1 microM) affinity sites on fibrinogen and facilitate fibrin monomer polymerization. We have previously localized two of the three high affinity Ca2+ sites to gamma 311-gamma 336. However, optimal enhancement of fibrin monomer polymerization occurs only at physiological millimolar Ca2+ concentrations which are two orders of magnitude higher than the concentration required for occupancy of the high affinity Ca2+-binding sites. In this study, we show that removal of fibrinogen sialic acid residues results in loss of low affinity Ca2+-binding sites. Clotting of asialofibrinogen appears to be Ca2+-independent and results in fiber bundles thicker in diameter than normal fibrin bundles as determined by turbidometry and scanning and transmission electron microscopy. By using a Ca2+-sensitive electrode, free sialic acid is shown to bind Ca2+ (Kd congruent to 1 mM). These observations suggest that the high affinity fibrinogen D-domain Ca2+-binding sites may play a role in the tertiary structure of the D-domain, whereas, sialic acid residues are low affinity sites whose occupancy by Ca2+ at physiological calcium concentration facilitates fibrin polymerization.

Asialoglycoproteins↗
PubMed1989

Phosphorylation of threonyl- and seryl-tRNA synthetase by cAMP-dependent protein kinase. A possible role in the regulation of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) synthesis.

Threonyl-tRNA synthetase has been shown to be phosphorylated in reticulocytes (Dang, C. V., Tan, E. M., and Traugh, J. A., (1988) FASEB J. 2, 2376-2379). Upon incubation of reticulocytes with 8-bromo-cAMP, phosphorylation of threonyl-tRNA synthetase is stimulated approximately 2-fold, an increase similar to that observed with ribosomal protein S6. To analyze the effects of phosphorylation on activity, threonyl-tRNA synthetase has been purified to apparent homogeneity from rabbit reticulocytes utilizing a four-step purification procedure with the simultaneous purification of seryl-tRNA synthetase. Both synthetases are phosphorylated in vitro by the cAMP-dependent protein kinase. Prior to phosphorylation, the two synthetases produce significant amounts of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) in the presence of the cognate amino acid and ATP, with activities comparable to that of lysyl-tRNA synthetase. Phosphorylation has no effect on aminoacylation, but an increase in Ap4A synthesis of up to 6-fold is observed with threonyl-tRNA synthetase and 2-fold with seryl-tRNA synthetase. Thus, cAMP-mediated phosphorylation of specific aminoacyl-tRNA synthetases appears to be a potential mode of regulation of Ap4A synthesis in mammals.

8-Bromo Cyclic Adenosine Monophosphate↗