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Biomedical subjects

I M Verma

Publications and source records attributed to I M Verma.

At least 145 records · Page 8Linked to original sources
PubMed1990

Cross-talk in signal transduction: TPA-inducible factor jun/AP-1 activates cAMP-responsive enhancer elements.

The product of the jun proto-oncogene has been identified as one form of the transcription factor AP-1. The p55fos protein associates with jun/AP-1 by means of a heterodimer which requires intact 'leucine zipper' domains of both proteins. The fos/jun heterodimer binds to and activates transcription from TPA-responsive promoter elements (TGACTCA), which represent one final target of the protein kinase C pathway. The other main signal transduction pathway, initiated by the activation of the adenylate cyclase, involves the transcription factor CREB. The promoter element recognized by CREB, a cyclic AMP responsive element (CRE), consist of a palyndromic sequence similar to a TRE (TGACGTCA). We show that jun efficiently trans-activates CRE sequences and that fos and jun efficiently bind and cooperate in activating CRE promoter elements. The similarity between TRE and CRE sequences may involve an interplay in transcriptional regulation and 'cross-talk' between components of the two major signal transduction pathways.

Base Sequence↗
PubMed1990

Developmental expression of the Xenopus laevis fos protooncogene.

We have isolated the Xenopus laevis homologue of the protooncogene fos. Nucleotide sequence analysis of the complementary DNA revealed a coding domain for a 370-amino acid protein which shares extensive sequence homology with the avian and mammalian fos protein. During Xenopus development, the fos mRNA is present in the oocyte and egg stages and declines to relatively low levels by the mid-blastula transition. Transient expression of fos can be seen during the late neurula and again at the tadpole stage. The fos mRNA is uniformly distributed in the vegetal and animal poles of the oocytes. In a Xenopus kidney cell line, the fos gene can be transcriptionally activated by serum growth factors and 12-O-tetradecanoylphorbol-13-acetate, and the fos mRNA can be superinduced by cycloheximide. The X. laevis fos protein migrates as a series of bands around Mr 55,000 and is localized in the nucleus. Both the Xenopus fos RNA and fos proteins exhibit a short half-life analogous to that observed with their mammalian counterpart. The Xenopus fos protein readily associates with murine jun (AP-1) oncoprotein, and the complex binds efficiently to AP-1 recognition elements. Furthermore, the Xenopus fos protein collaborates with mammalian jun protein to activate transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive element. The differential expression of fos during Xenopus development and its ability to regulate transcription in association with jun suggest a role during embryogenesis.

Amino Acid Sequence↗
PubMed1989

Retrovirus-mediated gene transfer into embryonal carcinoma and hemopoietic stem cells: expression from a hybrid long terminal repeat.

Retroviral vectors can be used as an efficient gene delivery system in a wide variety of cell types. However, in some cell types, such as embryonal carcinoma (EC) cells or normal bone-marrow cells the expression of genes introduced by retroviral vectors has been very inefficient. This expression block has severely hampered the application of retroviral vector systems in those cell types. The enhancer sequences present in the long terminal repeat (LTR) of retroviruses are known to be responsible for the tissue specificity of viral expression. Therefore, we set out to construct a vector in which this enhancer element has been replaced. A recombinant retrovirus was constructed in which the enhancer from the Moloney murine leukemia virus LTR was replaced by the enhancer of a mutant polyoma virus (PyF101) that was selected to grow on EC cells. A neomycin-resistance marker (neoR) was placed under the transcriptional control of the hybrid LTR. Following infection with this virus, neoR was expressed in EC cells, as well as in the hemopoietic progenitor cells present in normal murine bone marrow. Moreover, upon transplantation of infected bone marrow cells into lethally irradiated mice, neoR expression was sustained in hemopoietic cells of the engrafted recipients.

Animals↗
PubMed1989

Cationic liposome-mediated RNA transfection.

We have developed an efficient and reproducible method for RNA transfection, using a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome (lipofectin). Transfection of 10 ng to 5 micrograms of Photinus pyralis luciferase mRNA synthesized in vitro into NIH 3T3 mouse cells yields a linear response of luciferase activity. The procedure can be used to efficiently transfect RNA into human, rat, mouse, Xenopus, and Drosophila cells. Using the RNA/lipofectin transfection procedure, we have analyzed the role of capping and beta-globin 5' and 3' untranslated sequences on the translation efficiency of luciferase RNA synthesized in vitro. Following transfection of NIH 3T3 cells, capped mRNAs with beta-globin untranslated sequences produced at least 1000-fold more luciferase protein than mRNAs lacking these elements.

Animals↗
PubMed1989

Fos-Jun interaction: mutational analysis of the leucine zipper domain of both proteins.

Jun and Fos oncoproteins form a complex that regulates transcription from promoters containing AP-1 binding sites. The 'leucine zipper' domain of both Fos and Jun is necessary for the formation of the heterodimer, but the role of specific leucine residues is unclear. We have used site-specific mutagenesis to examine the contribution of individual leucine residues to the formation of a stable Fos-Jun protein complex and the binding of this complex to the AP-1 site. Mutation of a single leucine in either Fos or Jun had no effect on protein complex formation. Furthermore, mutations of two consecutive leucines in Jun did not interfere with heterodimer formation; however, in the case of Fos, two consecutive mutations resulted in an inability to form a heterodimer. Although mutagenesis of the first leucine of the heptad repeat had no effect on protein complex formation, this mutation in either Fos or Jun drastically reduced the affinity of the complex for DNA. Thus, both Fos and Jun contribute directly to the DNA-binding potential of the heterodimer.

Amino Acid Sequence↗
PubMed1989

Differential transcription of exon 1 of the human c-fms gene in placental trophoblasts and monocytes.

Structural analysis of the 5' end of the human c-fms gene revealed that a large intron of about 25 kilobases separates an upstream noncoding exon (exon 1) from the signal peptide-containing exon (exon 2). Northern (RNA) blot analysis, S1 nuclease mapping, and primer extensions showed that exon 1 is transcribed in placenta but not in cells of the monocytic lineage. This is due to the differential usage of promoters, separated by approximately 25 kilobases, in a cell-specific manner. One major c-fms transcript was observed in U-937 cells, whereas multiple initiation sites for transcription appeared to be utilized in placental cells. Nucleotide sequence comparisons showed that the 3' end of the human platelet-derived growth factor receptor gene lies approximately 350 base pairs upstream of the major initiation sites for c-fms transcription in placental trophoblasts.

Amino Acid Sequence↗
PubMed1989

Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos.

A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.

Cell Line↗
PubMed1989

ras-induced neuronal differentiation of PC12 cells: possible involvement of fos and jun.

Rat pheochromocytoma PC12 cells differentiate to sympathetic neuron-like cells upon treatment with nerve growth factor (NGF). The ras and src transforming proteins also induce PC12 neuronal differentiation and are likely to involve the protein kinase C signal transduction pathway. Using a number of ras mutants, we have established that the domains of oncogenic ras protein responsible for PC12 differentiation overlap those required for cellular transformation. All of the ras mutants that induced neuronal differentiation also activated c-fos transcription through the dyad symmetry element (DSE). Transforming ras protein activated an intracellular signal pathway, which led to the induction of 12-O-tetradecanoyl phorbol-13-acetate-responsive elements; activation was enhanced by coexpression of the proto-oncogene jun (encoding AP-1) and was further augmented by fos. Nuclear extracts from ras-infected PC12 cells showed an increased AP-1 DNA-binding activity. Transcriptional activation by ras was independent of the cyclic AMP-dependent pathway of signal transduction. We propose a possible involvement of fos and jun in ras-induced differentiation.

Adrenal Gland Neoplasms↗
PubMed1989

Removal of an mRNA destabilizing element correlates with the increased oncogenicity of proto-oncogene fos.

AU-sequence motifs present in the 3' untranslated region (UTR) of many rapidly inducible messenger RNAs have been proposed to mediate their selective degradation. We have analyzed by quantitative nuclease S1 analysis the mRNA decay-rates of viral (v)/c-fos deletion mutants following transfection in a transient assay system. A 67 nucleotide mRNA destabilizing element, encompassing three conserved AUUUA motifs, was identified in the c-fos 3' UTR. The transforming ability of several v/c-fos recombinants correlates with the removal of the AU-rich sequence. Insertion of a c-fos DNA fragment containing the destabilizing element into the alpha-globin 3' UTR confers high instability to the otherwise stable alpha-globin mRNA. We conclude that a major parameter of oncogenicity by the c-fos gene is predicated by the presence or absence of AT-rich sequences located in the 3' non-coding region.

Animals↗
PubMed1989

fos and jun interaction: the role of the leucine zipper.

Jun and fos oncoproteins form a complex which regulates transcription from promoters containing AP-I binding sites. The "leucine zipper" domain of both fos and jun is necessary for the formation of the heterodimer, but the role of specific leucine residues is unclear. We have used site-specific mutagenesis to examine the contribution of individual leucine residues to the formation of a stable fos/jun protein complex and the binding of this complex to the AP-I site. Mutation of a single leucine in either fos or jun had no effect on protein complex formation. Furthermore, mutations of two consecutive leucines in jun did not interfere with heterodimer formation; however, in the case of fos, two consecutive mutations resulted in an inability to form a heterodimer. Although mutagenesis of the first leucine of the heptad repeat had no effect on protein complex formation, this mutation in either fos or jun drastically reduced the affinity of the complex for DNA. Thus, both fos and jun contribute directly to the DNA binding potential of the heterodimer.

Amino Acid Sequence↗
PubMed1989

fos-jun Conspiracy: implications for the cell.

Two nuclear oncoproteins, fos and jun (AP-1), cooperate in forming a very stable heterodimeric complex that binds to the AP-1 site with increased affinity. The 'leucine zipper' domain of both fos and jun is necessary for the formation of this heterodimer. Mutations of single residues within the leucine zipper domain had no effect on protein complex formation. However, mutagenesis of the first leucine of the heptad repeat in either fos or jun basic regions and alteration of the spacing between the basic and leucine zipper domains indicate that the basic region of fos has a crucial role in determining the DNA binding affinity of the transcriptional complex. Mutations of the basic amino acids in fos protein prevent binding to TPA (phorbol ester)-responsive element (TRE) in the presence of wild-type jun protein. Thus fos protein appears to be dominant in jun-fos binding to DNA, even though fos alone cannot bind to TRE. Mutants in the basic regions of fos and jun can be exploited as dominant-negative mutants to ablate their normal cellular function.

Amino Acid Sequence↗
PubMed1988

Direct interaction between fos and jun nuclear oncoproteins: role of the 'leucine zipper' domain.

Gene expression is modulated by the specific interactions of nuclear proteins with unique regulatory sequences in the genome. Proteins involved in transcriptional regulation seem to be either transcription factors or transcription modulators and their interactions are crucial in determining whether the expression of a specific gene is activated or repressed. Recently, the product of the proto-oncogene jun has been identified as the transcription factor AP-1, whereas nuclear oncoproteins fos and myc have been implicated in transcriptional transregulation of several promoters. Furthermore, the products of the fos and jun proto-oncogenes are associated in some transcription complexes. Although the nature of the association is unclear, the two proteins co-immunoprecipitate with fos antibodies in nuclear extracts. Here, we report studies that demonstrate that the fos protein directly modulates jun function by means of a heterodimer of fos and jun proteins. The fos 'leucine zipper' domain is necessary for the DNA binding of the heterodimer; a distinct domain, localized in the C-terminal region of the fos protein, is responsible for transcriptional regulation.

DNA↗
PubMed1988

Induction of proto-oncogene JUN/AP-1 by serum and TPA.

The response of a cell to mitogens and differentiation agents involves the transcriptional induction of several cellular genes. Prominent among these so-called 'immediate early' or 'competence' genes are the nuclear oncogenes fos and myc. Although the precise function of these early response genes in growth control is not understood, it is likely that many of them are involved in the transition from G0 to G1 in the cell cycle. The findings that the products of nuclear proto-oncogenes jun and erbA are transcriptional factors supports the notion of the role of the nuclear oncoproteins in the regulation of gene expression. Recently, it has been reported that the FOS protein is associated in transcriptional complexes with the product of the jun oncogene, the transcription factor AP-1. As the fos gene is induced in response to mitogens during initiation of cell growth, we investigated whether expression of the nuclear transcription factor AP-1 is also inducible. We report that mouse c-jun gene transcription is rapidly induced by serum and phorbol-ester 12-o-tetradecanoyl phorbol 13-acetate (TPA). Furthermore, induction is transient and the mRNA is superinduced by inhibitors of protein synthesis.

Amino Acid Sequence↗
PubMed1988

fos-associated cellular p39 is related to nuclear transcription factor AP-1.

Proto-oncogene fos encodes a nuclear protein that appears to be involved in the transcriptional regulation of some genes. fos protein (p55fos) is associated with other nuclear proteins in complexes that bind to regulatory regions containing TPA-responsive promoter elements (TREs). The enhancer-binding activator protein 1 (AP-1), which is the specific binding factor of a TRE, is the product of the nuclear proto-oncogene jun. We show that one of the fos-associated proteins, p39, is immunologically and structurally related to nuclear factor AP-1. The sequence TGACTCA, which is the consensus minimal binding site of AP-1, is also recognized by gel-purified p39. In HeLa cell extracts p55fos and p39jun are associated in a nucleoprotein complex that interacts with a TRE. Cooperation between nuclear oncoproteins fos and jun is required for full activation of transcription from a TPA-responsive promoter element in transfected mammalian cells.

Enhancer Elements, Genetic↗
PubMed1988

Transcriptional autoregulation of the proto-oncogene fos.

Serum-induced transcription of the proto-oncogene fos is under negative feedback regulation mediated by the fos protein. The fos promoter region responsive to repression is also required for serum inducibility and binds a nucleoprotein complex in which the nuclear factor AP-1 is associated with fos protein.

Animals↗
PubMed1988

c-fos promoter trans-activation by the tax1 protein of human T-cell leukemia virus type I.

To understand the mechanisms of oncogenesis by human T-cell leukemia virus type I, we have investigated the ability of the tax1, protein to modulate transcription of protooncogenes. By using a transient cotransfection assay, we report that the protooncogene fos promoter is transactivated by tax1 in a variety of cell types. Two regions containing upstream sequences between positions -362/-324 and -323/-276 of the c-fos promoter responded to this activation and also conferred tax1 responsiveness to the heterologous herpesvirus thymidine kinase promoter. These two sequences include elements mediating the induction by v-sis-conditioned medium and serum, phorbol ester, or epidermal growth factor, respectively. Furthermore, expression of the endogenous c-fos gene was activated by tax1 in human T-cell leukemia virus type I-infected cell lines. In contrast, no trans-activation of the c-myc or c-Ha-ras promoter was observed.

Carcinoma, Hepatocellular↗