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V N Rao

Publications and source records attributed to V N Rao.

At least 37 records · Page 2Linked to original sources
PubMed1993

EWS/Fli-1 chimeric protein is a transcriptional activator.

Fli-1, an ets related gene, was found to be rearranged in 75% of erythroleukemias induced by Friend murine leukemia virus. We have shown previously that the Fli-1 gene codes for a sequence specific transcriptional activator which contains two autonomous transcriptional activation domains, one at the amino terminal region and the other at the carboxy terminal region. Recently human Fli-1 gene was shown to be involved in Ewing's sarcoma and related subtypes of primitive neuroectodermal tumors which share t(11;22) (q24;q12) chromosome translocation. In these tumors the carboxyl terminal region of Fli-1 was found to be fused with the amino terminal region of a putative RNA binding protein, EWS. Because part of the amino terminal transcriptional activation domain of Fli-1 was replaced with the amino terminal domain of the EWS (NTD-EWS) which shares homology with RNA polymerase II, it was speculated that NTD-EWS may interfere with RNA pol II function. Alternatively, NTD-EWS could also contribute to the transcriptional activation function of EWS/Fli-1 chimeric protein by providing either a modulatory/regulatory domain or a novel transcriptional activation domain. Here we show that EWS/Fli-1 chimeric protein functions as a transcriptional activator. Deletion analysis reveals that the EWS domain functions as a modulatory/regulatory domain for the transcriptional activation properties of the carboxy terminal transcriptional activation domain of EWS/Fli-1. We therefore propose that replacement of the amino terminal transcriptional activation domain of the Fli-1 protein with the regulatory domain of NTD-EWS results in the activation of the carboxy terminal transcriptional activation domain of Fli-1 which may be the molecular mechanism involved in these human tumors.

3T3 Cells↗
PubMed1993

Elk-1 proteins are phosphoproteins and activators of mitogen-activated protein kinase.

Mitogen-activated protein kinases (MAP kinases) or meiosis-activated myelin basic protein kinase (p44mpk) are known to be activated by a mechanism involving dual phosphorylation at both tyrosine and serine/threonine in response to many extracellular stimuli. There has been considerable speculation as to whether MAP kinases are autophosphorylated and activated by an upstream protein kinase (MAP kinase kinase) or an activator of autophosphorylation or both. Here we report that the ets-related proteins elk-1 and delta elk-1 to be potential physiological substrates and activators of MAP kinases. Our results demonstrate for the first time that MAP kinase activators can also be non-kinase proteins that enhance the autophosphorylation and activation of MAP kinase. These findings could establish a general mechanism wherein specific MAP kinase activator protein(s) may function by interacting with MAP kinases ensuring a conformational change and stimulating their autophosphorylation and activation property. Our results also suggest that the amino-terminal truncated elk-1 proteins are better activators of MAP kinase than full length proteins indicating the presence of a potential negative regulatory region which may control the kinase activator function of elk-1 proteins. Our results suggest differential regulation of elk-1 and delta elk-1 proteins in fibroblasts stimulated by epidermal growth factor implicating a key role for these proteins in the signal transduction pathway. These results establish the presence of an alternative pathway for activation of MAP kinases. Thus we propose that elk-1 proteins may represent key intermediates which would transmit signals arriving at the surface of the cell from activated receptors to downstream MAP kinases in the cytoplasm to reach the transcriptional factors in the nucleus.

3T3 Cells↗
PubMed1993

Delta elk-1, a variant of elk-1, fails to interact with the serum response factor and binds to DNA with modulated specificity.

The ets oncogene superfamily codes for a family of transcriptional factors that are involved in gene regulation not only by autonomous DNA binding but also by indirect DNA binding through interaction with cellular factors. We have previously shown that a member of this superfamily, elk-1, is a sequence specific transcriptional activator, which forms a serum response factor (SRF) dependent ternary complex with serum response element (SRE) similar to p62TCF. We describe here an alternatively spliced variant of elk-1 named delta elk-1, which has lost the SRF interaction domain, negative regulatory DNA binding domain, and part of the elk-1 DNA binding domain. This variant elk-1 protein has lost the capacity to form a SRF dependent ternary complex with SRE and to activate fos transcription. Since this splice variant lacks part of the ets DNA binding domain, it binds to DNA with a specificity that is different from that of the full length elk-1 protein. Therefore differential splicing within the DNA binding and protein-protein interaction domains of transcriptional factors can generate proteins with modulated DNA binding specificities and transcriptional regulation. Thus it is conceivable that variant elk-1 might function by competing for some of the elk-1 target sequences (like SRE) and thereby block the transcriptional activation of fos by SRF and elk-1. Alternately, variant elk-1 protein may be the repressor, recruited by the SRE bound SRF for c-fos repression, or it may have an altogether different function. Therefore, elk-1 appears to fall in the category of genes that encode activators and repressors through the mechanism of differential splicing.

3T3 Cells↗
PubMed1993

Transcriptional activation domains of elk-1, delta elk-1 and SAP-1 proteins.

elk-1, an ets related gene codes for a sequence specific DNA binding transcriptional activator which in association with serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE). Recently the C-terminal region of both elk-1 and delta elk-1 proteins was shown to undergo phosphorylation by MAP kinases and function as an activator of MAP kinases. Here we show that delta elk-1 and two other elk-1 related proteins SAP-1a and SAP-1b, like elk-1, can function as transcriptional activators. In this report we have localized the transcriptional activation domain of the SAP-1 proteins (STA) to a large portion of the carboxy terminal region and have identified two autonomous transcriptional activation domains in the elk-1 protein, one at the amino (ETA-1) and the other at the carboxy terminal region (ETA-2). delta elk-1 protein contains only the ETA-2 domain indicating differential usage of activation domains as a result of alternative splicing. We can speculate that the ETA-1 domain can function in vivo independent of ETA-2, but the ETA-2 domain can function either in the absence of ETA-1 (as seen in delta elk-1) or in the presence of accessory proteins like SRF. The role of SRF in the activation of the ternary complex might be to bind to the ETA-1 domain, somehow conceal it's activation domain and in the process unmask the ETA-2 domain (for phosphorylation by MAP kinases) and activate transcription. The ETA-1 domain may be functioning as a negative regulatory transcriptional activation domain for ETA-2. These observations suggest that the elk-1 family of proteins may not only regulate fos and MAP kinases but also other elk-1 target genes that are essential for cellular growth control.

Animals↗
PubMed1993

Analysis of the DNA-binding and transcriptional activation functions of human Fli-1 protein.

Three of the ets oncogene superfamily members v-ets, Spi-1/PU.1 and Fli-1, have been shown to be directly involved in retroviral-mediated acute erythroleukemias. The Fli-1 gene was found to be rearranged in 75% of the erythroleukemias induced by Friend murine leukemia virus (F-MuLV), suggesting that it could play a key role in cellular transformation. We have previously isolated and characterized the human Fli-1 gene and have found it to be highly homologous (80%) to the human erg-2 gene. Human Fli-1 was also shown to be rearranged in Ewing's sarcoma cases, in which the amino-terminal region of the Fli-1 gene was replaced with a novel coding region of a putative RNA-binding protein, EWS. In this report, we show that the recombinant Fli-1 protein expressed in bacteria binds to DNA in a sequence-specific manner. It appears that Fli-1 and erg proteins fall into the category of ets proteins that recognize limited ets target sequences, unlike c-ets-1, ets-2 and Elk-1. The Fli-1 gene was found to activate the transcription of the reporter gene that was linked to Fli-1 target sequences, suggesting that Fli-1 is a sequence-specific transcriptional activator. Deletion analysis revealed the presence of two autonomous transcriptional activation domains, one at the amino-terminal region (amino-terminal transcriptional activation domain, ATA) and the other at the carboxy-terminal region (carboxy-terminal transcriptional activation domain, CTA). Secondary structural analysis of ATA and CTA domains revealed the presence of helix-loop-helix (H-L-H) and/or turn-loop-turn (T-L-T) regions. From these results it appears that a portion of the Fli-1 ATA domain (H-L-H region) was replaced by the amino-terminal domain of EWS gene in Ewing's sarcoma cases. Therefore alteration in the transcriptional activation function of Fli-1 may be responsible for human malignancies such as sarcomas, leukemias and lymphomas in which this gene is rearranged.

Base Sequence↗
PubMed1993

Characterization of the DNA binding and transcriptional activation domains of the erg protein.

erg, an ets related gene encodes a sequence specific DNA binding transcriptional activator protein. We have identified four functional domains of erg protein that are responsible for DNA binding, transcriptional activation and negative regulation of transcriptional activation. Deletion analysis revealed that the 3'-ets domain of the erg protein is sufficient for DNA binding activity. Analysis of these deletion mutants also revealed the presence of two autonomous transcriptional activation domains, one at the amino and the other at the carboxyterminal region. This aminoterminal transcriptional activator domain (5'-ets domain) is conserved in six of the ets genes suggesting that it (ETA, ets Transcriptional Activation domain) may contribute to a common function among these genes. The transcriptional activation function of the carboxy terminal transcriptional activation domain (CTA) was inhibited by the presence of a Negative Regulatory Transcriptional activation domain (NRT), which is located at the amino terminal region of erg DNA binding domain. These results may help in understanding the structure/function relationship of other erg/ets related proteins.

3T3 Cells↗
PubMed1992

Structure and expression of human Fli-1 gene.

Three ets family members, v-ets, spleen focus forming virus proviral integration 1/Pu.1, and Friend leukemia integration 1 (Fli-1), were shown to be involved in retroviral mediated acute leukemias suggesting that ets family members play a crucial role in transformation. Mouse Fli-1 was shown to be involved in 75% erythroleukemias induced by Friend murine leukemia virus suggesting the possibility that Fli-1 may play a critical role in cellular transformation. Since Fli-1 maps to the mouse chromosome region syntenic with human chromosome 11q23-24, it is tempting to speculate that human Fli-1 may be involved in human sarcomas, leukemias, and lymphomas involving human chromosome 11q23-24. We have isolated complementary DNA clones representing the human homologue of Fli-1 gene. Nucleotide sequence analysis revealed that the human Fli-1 gene codes for a 452-residue protein the predicted amino sequence of which shows 80% homology to the human erg-2 protein previously described. A 3.5-kilobase transcript of the human Fli-1 gene was observed in different cells. Sequence analysis revealed two domains of ets homology, one at the 5' and the other at the 3' end of the Fli-1 gene. This 3'-ets homology domain, which is mainly responsible for DNA binding activity, is seen in all the ets family members; however, the 5'-ets homology region is conserved in only five genes, Fli-1, c-ets-1, ets-2, GABP-alpha, and erg, suggesting a common biological function which is shared among these genes. Interestingly, mouse and human Fli-1 transcripts contain highly homologous 5'-untranslated region suggesting that this conserved region may play an important role in the posttranscriptional regulation of the Fli-1 transcript.

Amino Acid Sequence↗
PubMed1992

Myb and Ets proteins cooperate in transcriptional activation of the mim-1 promoter.

In the generation of the acutely transforming avian retrovirus E26, both myb and ets genes have been transduced, leading to the production of a Gag-Myb-Ets fusion protein. This co-occurrence of v-myb and v-ets oncogenes suggests that the two might have a functional relationship. To look for such a relationship, we tested the transcriptional activation activity of Myb alone or with coexpressed Ets-1 or Ets-2. Using the promoter of the v-Myb-inducible mim-1 gene as a target, we found that full-length c-Myb gene products were poor activators of transcription, while an oncogenic (truncated) form of this protein was a strong trans-activator. However, coexpression of Ets-2 with full-length or truncated forms of Myb greatly increased trans-activation. Coexpression of Ets-1, Fos, Jun, or Myc with Myb did not increase trans-activation of the mim-1 promoter. The ability of Myb and Ets-2 to transactivate was cooperative, since Ets-2 alone gave little or no activation. Bacterially synthesized Ets-2 protein was found to bind specifically to the mim-1 promoter, suggesting that it may be a target for both Myb and Ets proteins. Thus, Myb and Ets proteins can cooperate in transcriptional activation, and their co-occurrence in the E26 virus may reflect a functional relationship between these two oncoproteins. Truncated forms of Myb may have a reduced need for cooperating factors such as Ets-2, and this might constitute an important mechanism associated with oncogenic activation.

Amino Acid Sequence↗
PubMed1992

elk-1 domains responsible for autonomous DNA binding, SRE:SRF interaction and negative regulation of DNA binding.

The ets oncogene superfamily consists of a family of sequence-specific DNA-binding transcriptional activator proteins. We have previously identified, cloned and characterized one of the divergent ets-related members elk-1 and shown that it codes for a sequence-specific DNA-binding transcriptional activator. We have also shown that elk-1 forms SRF (Serum Response Factor) dependent ternary complex with SRE (Serum Response Element), similar to p62TCF. In this report, we have mapped the DNA-binding domain of the elk-1 protein (EDB, elk-1 DNA Binding domain) to the 76 amino acid ets homology region. We have also mapped the SRF interaction domain of the elk-1 protein (ESI, elk-1 SRF Interaction domain) to the carboxy-terminal region of the EDB domain. Ternary complex formation by elk-1 requires both EDB and ESI domains of the elk-1 protein. Our results also show that the EDB domain of the elk-1 protein (residues 1-89) binds SRE autonomously, unlike full-length elk-1 protein, suggesting the presence of a potential Negative Regulatory DNA binding domain (NRD) which prevents the binding of elk-1 protein to SRE. Interaction of SRF with the ESI domain allows the elk-1 protein to bind to SRE. Thus elk-1 belongs to a class of transcriptional factors that are involved in gene regulation not only by autonomous DNA binding but also by indirect DNA binding through recruitment by cellular factors.

DNA↗
PubMed1992

A divergent ets-related protein, elk-1, recognizes similar c-ets-1 proto-oncogene target sequences and acts as a transcriptional activator.

The ets oncogene superfamily consists of a family of sequence-specific DNA-binding proteins that activate transcription. We have previously identified two new members of the ets oncogene superfamily, namely elk-1 and elk-2. In this report we show that the recombinant elk-1 protein expressed in bacteria, like the c-ets-1 proto-oncogene, binds in a sequence-specific manner to Moloney murine sarcoma virus long terminal repeat, E74 target sequences and the PEA3 motif (polyoma enhancer), but does not bind to PU box sequences. Thus analysis of the DNA-binding specificity of ets-related proteins supports the view that different members show similar DNA-binding specificity, which is a general feature of the homeobox proteins. Our data using the chloramphenicol acetyltransferase gene linked to a thymidine kinase promoter containing multimers of the elk-1 target sequence indicates that elk-1 functions as a transcriptional activator. Interestingly, although elk-1 is the most divergent of all the members of the ets gene family, it shows very close similarities with c-ets-1 in some of its sequence-specific DNA-binding specificities. Here, we propose a new function for the elk-1 gene to act as a transcriptional activator of retroviruses and DNA tumor viruses.

Base Sequence↗
PubMed1991

erg, an ets-related gene, codes for sequence-specific transcriptional activators.

E26 is a replication-defective avian acute leukemia virus which causes erythroblastosis and myeloblastosis in chickens. It carries two distinct oncogenes, v-myb and v-ets, both of which contribute to its transforming properties. Several genes related to the ets oncogene (c-ets-1, ets-2, erg, elk-1, elk-2, PU.1/Spi-1, E74 and Fli-1) have been described. Previously we have shown that the erg gene (ets-related gene) codes for at least two proteins (erg-1 and erg-2) because of alternative splicing and alternative usage of the initiation codon. We have expressed erg-1 and erg-2 proteins in Escherichia coli and have used these recombinant proteins to show that they bind to DNA in a sequence-specific manner. erg proteins exhibited different sequence specificity and affinity for the oligonucleotides recognized by c-ets-1, ets-2, some of PU.1/Spi-1 and elk-1, suggesting that the DNA-binding specificities of erg and other members may overlap but are not necessarily identical. The erg gene was found to transactivate a reporter gene that was linked to erg target sequences. These results suggest that erg-1 and erg-2 are sequence-specific transcriptional activators like the other members of the ets oncogene superfamily which represent a distinct class of transcriptional activators.

Animals↗
PubMed1990

Localization and modulation of the DNA-binding activity of the human c-ets-1 protooncogene.

The avian acute leukemia virus (E26) induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. The viral protein responsible for transformation is a gag-myb-ets fusion protein that is located in the nucleus of the transformed cells. The cellular homologue of v-ets (c-ets-1) is highly expressed in lymphoid cells and differs from the v-ets gene at its carboxy terminal region. Here, we show that both the c-ets-1 and v-ets gene products are DNA-binding proteins and their DNA-binding activity is located in the carboxy terminal (46 amino acid residues) region. It appears that this DNA-binding activity is modulated by the extreme carboxy terminal region. The amino acid sequences of the putative ets DNA-binding domain at its carboxy terminal region showed a helix-turn-helix secondary structure. Exchanging the nonhomologous extreme carboxy terminal regions of c-ets-1 with v-ets gene sequences showed differences in DNA-binding affinity, indicating that these differences may be partly responsible for the activation of the ets oncogene.

Amino Acid Sequence↗
PubMed1989

elk, tissue-specific ets-related genes on chromosomes X and 14 near translocation breakpoints.

The myb-ets-containing acute leukemia virus, E26, transforms myeloblasts and erythroblasts in culture and causes a mixed erythroid and myeloid leukemia in chicks. Genes (ets-1, ets-2, and erg) with variable relatedness to the v-ets oncogene of the E26 virus have been identified, cloned, and characterized in several species. Two new members (elk-1 and elk-2) of the ets oncogene superfamily have now been identified. Nucleotide sequence analysis of the elk-1 cDNA clone revealed that this gene encodes a 428-residue protein whose predicted amino acid sequence showed 82% similarity to the 3' region of v-ets. The elk or related sequences appear to be transcriptionally active in testis and lung. The elk cDNA probe detects two loci in the human genome, elk-1 and elk-2, which map to chromosome regions Xp11.2 and 14q32.3, respectively. These loci are near the translocation breakpoint seen in the t(X;18) (p11.2;q11.2), which is characteristic of synovial sarcoma, and the chromosome 14q32 breakpoints seen in ataxia telangiectasia and other T cell malignancies. This suggests the possibility that rearrangements of elk loci may be involved in pathogenesis of certain tumors.

Amino Acid Sequence↗
PubMed1988

Structure, expression and alternative splicing of the human c-ets-1 proto-oncogene.

The avian acute leukemia virus E26 induces a mixed erythroid-myeloid leukemia in chicken and carries two distinct oncogenes, v-myb and v-ets. It was shown that there exist two ets loci in humans, c-ets-1 and c-ets-2, which were mapped to human chromosome 11 and 21, respectively. The human c-ets-1 proto-oncogene locus was shown to translocate from chromosome 11 to 9 or 4 in certain patients with acute monocytic leukemia. In order to understand the functional role of c-ets-1 proto-oncogene, we have made a cDNA library from MOLT-4 cells and isolated seven c-ets-1 positive cDNA clones. Analysis of two cDNA clones revealed a complete open reading frame of the c-ets-1 proto-oncogene. Except for 81 nucleotides at the 5'-end (downstream of initiation codon) and 39 nucleotides at the 3'-end (upstream of termination codon), the c-ets-1 cDNA is approximately 82% homologous to v-ets oncogene sequences and encodes a 441-residue protein (c-ets-1a) which contains contiguous 5' and 3' regions of the v-ets oncogene as in the case of chicken. Sequence analysis also revealed alternative splicing, giving rise to another related c-ets-1 polypeptide (c-ets-1b) lacking 87 amino acids. Our results suggest that differential RNA splicing allows the synthesis of at least two related c-ets-1 polypeptides from a single c-ets-1 proto-oncogene.

Amino Acid Sequence↗
PubMed1988

The human erg gene maps to chromosome 21, band q22: relationship to the 8; 21 translocation of acute myelogenous leukemia.

There is accumulating evidence to support that genes on chromosome 21 play an important role in the development of pathologies associated with leukemia, Down's syndrome, and Alzheimer's disease. We have previously described erg, a human gene related to the ets oncogene. In this study, we have regionally assigned the erg gene to chromosome 21q22.3 by using somatic cell hybrids and in situ hybridization analysis. In light of this chromosome assignment, the relationship of erg to the 21q translocation breakpoint characteristic of acute myelogenous leukemia (AML) was considered. By using a DNA probe that is specific for the erg gene, a panel of rodent-human cell hybrids was analyzed by the Southern technique to study specific chromosome translocations occurring in acute myeloblastic leukemia. The erg gene was found to translocate from chromosome 21 to 8 in the t(8; 21) (q22; q22), a non-random translocation found in patients with acute myelogenous leukemia of the subgroup M2 (AML-M2). The localization of the erg gene to chromosome 21q22 raises the possibility that this gene may be involved in the pathogenesis of AML-M2.

Animals↗
PubMed1987

erg, a human ets-related gene on chromosome 21: alternative splicing, polyadenylation, and translation.

The avian acute leukemia virus E26 induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. Recently, a novel gene named erg, closely related to the v-ets oncogene, was identified in human COLO 320 cells and the nucleotide sequence of its approximately 5.0-kilobase transcript, erg 1 was determined. In the present study, the nucleotide sequence of the alternatively spliced transcript, erg 2, was found to differ from erg 1 by a splicing event that causes a coding frameshift near the amino terminus, resulting in an additional 99-amino acid insertion at the amino-terminus. Expression of complementary DNAs for the two transcripts in vitro resulted in synthesis of polypeptides of approximately 41 and 52 kilodaltons, suggesting the use of alternative translation initiation codons in the case of erg proteins. The erg gene was localized by somatic cell genetic analysis to human chromosome 21. It is proposed that alternative sites of splicing and polyadenylation, together with alternative sites of translation initiation, allow the synthesis of two related polypeptides from a single erg gene transcriptional unit.

Amino Acid Sequence↗