Biomedical subjects

V N Rao

Publications and source records attributed to V N Rao.

At least 19 recordsLinked to original sources

PubMed2001

EWS-ATF-1 chimeric protein in soft tissue clear cell sarcoma associates with CREB-binding protein and interferes with p53-mediated trans-activation function.

The recurrent t(12;22) (q13;q12) chromosomal translocation associated with soft tissue clear cell sarcoma results in a chimeric protein EWS-ATF-1 that acts as a constitutive transcriptional activator. The CBP/p300 transcriptional coactivator, which links various transcriptional factors to basal transcription apparatus, participates in transcriptional activation, growth and cell cycle control and differentiation. In this study, we show that EWS-ATF-1 associates constitutively with CBP both in vitro and in vivo. Both EWS and ATF-1 fusion domains are needed for this interaction. Here, we demonstrate that EWS-ATF-1 represses p53/CBP-mediated trans-activation function. Overexpression of CBP can counteract this repressive effect of EWS-ATF-1. Taken together, these findings suggest that one of the mechanisms by which EWS-ATF-1 may cause tumors is through targeting CBP/p300 resulting in the loss of function of p53. This novel mechanism may be responsible for the development of these and other related solid tumors.

Blotting, Western↗

PubMed2001

c-Fos oncogene regulator Elk-1 interacts with BRCA1 splice variants BRCA1a/1b and enhances BRCA1a/1b-mediated growth suppression in breast cancer cells.

Elk-1, a c-Fos protooncogene regulator, which belongs to the ETS-domain family of transcriptional factors, plays an important role in the induction of immediate early gene expression in response to a variety of extracellular signals. In this study, we demonstrate for the first time the in vitro and in vivo interaction of Elk-1 with BRCA1 splice variants BRCA1a and BRCA1b using GST-pull down assays, co-imunoprecipitations/Western blot analysis of cell extracts from breast cancer cells and mammalian two-hybrid assays. We have localized the BRCA1 interaction domain of Elk-1 protein to the conserved ETS domain, a motif involved in DNA binding and protein-protein interactions. We also observed binding of BRCA1 proteins to other ETS-domain transcription factors SAP1, ETS-1, ERG-2 and Fli-1 but not to Elk-1 splice variant DeltaElk-1 and c-Fos protooncogene. Both BRCA1a and BRCA1b splice variants function as growth suppressors of human breast cancer cells. Interestingly, our studies reveal that although both Elk-1 and SAP-1 are highly homologous members of a subfamily of ETS domain proteins called ternary complex factors, it is only Elk-1 but not SAP-1 that can augment the growth suppressive function of BRCA1a/1b proteins in breast cancer cells. Thus Elk-1 could be a potential downstream target of BRCA1 in its growth control pathway. Furthermore, we have observed inhibition of c-Fos promoter activity in BRCA1a transfected stable breast cancer cells and over expression of BRCA1a/1b attenuates MEK-induced SRE activation in vivo. These results demonstrate for the first time a link between the growth suppressive function of BRCA1a/1b proteins and signal transduction pathway involving Elk-1 protein. All these results taken together suggest that one of the mechanisms by which BRCA1a/1b proteins function as growth/tumor suppressors is through inhibition of the expression of Elk-1 target genes like c-Fos.

Alternative Splicing↗

PubMed1999

The second BRCT domain of BRCA1 proteins interacts with p53 and stimulates transcription from the p21WAF1/CIP1 promoter.

Inherited mutations in the breast and ovarian cancer susceptibility gene BRCA1 are associated with high risk for developing breast and ovarian cancers. Several studies link BRCA1 to transcriptional regulation, DNA repair, apoptosis and growth/tumor suppression. BRCA1 associates with p53 and stimulates transcription in both p53 dependent and p53-independent manners. BRCA1 splice variants BRCA1a (p110) and BRCA1b (p100) associates with CBP/p300 co-activators. Here we show that BRCA1a and BRCA1b proteins stimulate p53-dependent transcription from the p21WAF1/CIP1 promoter. In addition, the C-terminal second BRCA1 (BRCT) domain is sufficient for p53 mediated transactivation of the p21 promoter. Previous studies emphasized the importance of the BRCT domain, which shows homology with p53 binding protein (53BP1), in transcriptional activation, growth inhibition and tumor suppression. Our findings demonstrate an additional function for this domain in protein-protein interaction and co-activation of p53. We also found that BRCA1a and BRCA1b proteins interact with p53 in vitro and in vivo. The p53 interaction domain of BRCA1a/1b maps, in vitro, to the second BRCT domain (aa 1760-1863). The BRCT domain binds to the central domain of p53 which is required for sequence specific DNA binding. These results demonstrate for the first time the presence of a second p53 interaction domain in BRCA1 proteins and suggests that BRCA1a and BRCA1b proteins, like BRCA1, function as p53 co-activators. This BRCT domain also binds in vitro to CBP. These results suggest that one of the mechanisms by which BRCA1 proteins function is through recruitment of CBP/p300 associated HAT/FAT activity for acetylation of p53 to specific promoters resulting in transcriptional activation.

Animals↗

PubMed1998

Fli-1b is generated by usage of differential splicing and alternative promoter.

The proto-oncogene Fli-1, a member of Ets family is rearranged or activated through proviral integration in erythroleukemias, induced by Friends' Murine Leukemia Virus. The DNA binding domain (ETS domain) of Fli-1 is fused to the RNA binding domain of EWS by t(11q24:22q12) chromosomal translocation in Ewing's sarcoma and primitive neuroectodermal tumors. Screening of human cDNA libraries has identified two different 5'-termini and alternatively spliced forms of the human Fli-1 gene (Fli-1b), suggesting the possible existence of two independent promoters. The genomic sequence adjacent to the alternate exon of human Fli-1b gene shows functional promoter activity when cloned in promoter-less CAT expression vector and transfected into QT-6 cells. The transcription initiation (CAP) site and minimum promoter region necessary for function were localized. The 5'-flanking regions of human Fli-1b and mouse Fli-1 show 80% homology suggesting conserved promoter regulatory elements. The Fli-1b 5'-flanking sequence lacks canonical TATA or CCAAT boxes but contains a partially conserved TATA-like sequence at position 242. Several transcription factor binding sequences like ATF/CREB, E2A-PBX1, EBP, PEA-3, ETS-2, Sp-1, c-Myc, TBP, GATA-1 and Oct-3 were conserved in the promoter sequence. Functional promoter assays revealed that Fli-1b promoter shows very strong transcriptional activation compared to Fli-1 promoter. We also showed that variant Fli-1b has transcriptional activation properties similar to those of Fli-1. Fli-1b and Fli-1 show differential expression in various hematopoietic cell lines. This differential expression and promoter activities of Fli-1 and Fli-1b suggests that several mechanisms are involved in Fli-1 gene regulation which are mediated by many transcription factors.

Alternative Splicing↗

PubMed1998

Induction of apoptosis by Elk-1 and deltaElk-1 proteins.

Elk-1, an ets related gene codes for at least two splice variants Elk-1, which regulates c-fos transcription and deltaElk-1, both of which function as transcriptional activators. To investigate the role of Elk-1 and deltaElk-1 proteins in apoptosis; we have developed rat fibroblast cell lines and human breast cancer cell lines expressing Elk-1 and deltaElk-1. The expression of Elk-1 and deltaElk-1 proteins in the Elk-1/deltaElk-1 transfectants were analysed by immunofluorescence, immunohistochemistry, and Western blot analysis. The Elk-1 unlike deltaElk-1 transfectants showed a shortened and flattened morphology compared to the parental cells. We have found that calcium ionophore treatment of Rat-1 Elk-1, MCF-7 Elk-1, Rat-1 deltaElk-1 and MCF-7 deltaElk-1 transfectants resulted in programmed cell death. These results indicate that constitutive expression of Elk-1 and deltaElk-1 proteins triggers apoptosis in Rat-1 fibroblasts and breast cancer cells when treated with calcium ionophore.

Animals↗

PubMed1998

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells.

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.

Cell Lineage↗

PubMed1998

The BRCA2 is a histone acetyltransferase.

Patients carrying mutations in BRCA1 or BRCA2 tumor suppressor genes have shown to have high risk in developing breast and ovarian cancers. Two potential functions of BRCA2 were proposed which includes role in the regulation of transcription and also in DNA repair. Forty-five-amino acid region encoded by exon 3 of BRCA2 was shown to have transcriptional activation function. Recent studies of the several enzymes involved in acetylation and deacetylation of histone residues have revealed a possible relationship between gene transcriptional activation and histone acetylation. Since BRCA2 appear to function as a transcriptional factor, we have tested for Histone acetyl transferase (HAT) activity of BRCA2. Here, we present evidence that BRCA2 has intrinsic HAT activity, which maps to the amino-terminal region of BRCA2. Our results demonstrate that BRCA2 proteins acetylate primarily H3 and H4 of free histones. These observations suggest that HAT activity of BRCA2 may play an important role in the regulation of transcription and tumor suppressor function.

Acetylation↗

PubMed1998

Too far, too little, too late: a community-based case-control study of maternal mortality in rural west Maharashtra, India.

A total of 121 maternal deaths, identified through multiple-source surveillance in 400 villages in Maharashtra, were prospectively enrolled during 1993-95 in a population-based case-control study, which compared deaths with the survivors of similar pregnancy complications. The cases took significantly longer to seek care and to make the first health contact after the decision to seek care was taken. They also travelled significantly greater distances through a greater number of health facilities before appropriate treatment was started. Multivariate analysis showed the negative effect of excessive referrals and the protective effect of the following: residing in and not away from the village; presence of a resident nurse in the village; having an educated husband and a trained attendant at delivery; and being at the woman's parents' home at the time of illness. Other significant findings showed that deaths due to domestic violence were the second-largest cause of deaths in pregnancy, that more than two-thirds of maternal deaths were underreported in official records, and that liveborn infants of maternal deaths had a markedly higher risk of dying in the first year of life. This study points to the need for information-education-communication (IEC) efforts to increase family (especially male) preparedness for emergencies, decentralized obstetric management with effective triage, and a restructuring of the referral system.

Adult↗

PubMed1997

BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases.

BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using different BRCA1 antibodies by immunofluorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyrosine. Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc finger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that specifically interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring finger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc finger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers.

3T3 Cells↗

PubMed1997

Inhibition of apoptosis by normal and aberrant Fli-1 and erg proteins involved in human solid tumors and leukemias.

Two ets family members, namely erg and Fli-1 are fused with two EWS family members namely EWS and TLS/FUS as a result of chromosome translocation in human solid tumors and leukemias. EWS-erg and EWS-Fli-1, which are involved in greater than 95% of Ewing family of tumors, were shown to function as transcriptional activators. TLS/FUS-erg, which is involved in human myeloid leukemias also functions as a transcriptional activator. Expression of these fusion proteins (EWS-erg and EWS-Fli-1) are shown to be essential for maintaining the oncogenic and tumorigenic properties of tumor cells. Cancer is thought to be caused not only by uncontrolled cell proliferation but also by deregulation of programmed cell death. Therefore, we have studied the role of normal (Fli-1 and erg) and aberrant fusion proteins (EWS-erg, EWS-Fli-1 and TLS/FUS-erg) in apoptosis. We have found that expression of normal (Fli-1 and erg) and aberrant fusion proteins inhibit the apoptosis of NIH3T3 cells induced by either serum deprivation or by treatment with calcium ionophore. We have also observed similar suppression of apoptosis in Ewing's sarcoma cells expressing EWS-Fli-1 and EWS-erg proteins suggesting that these fusion proteins may be responsible for the decreased ability of these tumor cells to undergo apoptosis. Inhibition of the expression of these aberrant fusion proteins by antisense RNA technique resulted in increased susceptibility to apoptosis leading to the death of tumor cells. Therefore, our results suggest that one can use therapeutic agents which can down regulate the expression of fusion proteins in combination with chemotherapeutic agents as an effective treatment for these human solid tumors and leukemias.

3T3 Cells↗

PubMed1996

Induction of apoptosis by the tumor suppressor protein BRCA1.

The breast and ovarian cancer susceptibility gene BRCA1, is a nuclear phosphoprotein which functions as a tumor suppressor. To investigate the role of BRCA1 in apoptosis, we have developed mouse fibroblast cell lines and human breast cancer cell lines expressing BRCA1. The expression of BRCA1 protein in the BRCA1 transfectants were analysed by immunofluorescence and immunohistochemistry. The BRCA1 transfectants showed a flattened morphology compared to the parental cells. We show that serum deprivation or calcium ionophore treatment of BRCA1 transfectants resulted in programmed cell death. These results indicate that BRCA1 genes may play a critical role in the regulation of apoptosis. Thus, since a wide variety of human malignancies like breast and ovarian cancers have a decreased ability to undergo apoptosis, this could be due to lack/decreased levels of functional BRCA1 proteins. Treatments that are aimed at increasing the apoptotic threshold by BRCA1 gene therapy may have the potential to prevent the progression of these malignancies.

3T3 Cells↗

PubMed1996

Antisense RNA to the putative tumor suppressor gene BRCA1 transforms mouse fibroblasts.

Recently, BRCA1, a familial breast and ovarian cancer susceptible gene has been cloned and shown to be either lost or mutated in families with breast and ovarian cancers. BRCA1 has been postulated to encode a tumor suppressor, a protein that acts as a negative regulator of tumor growth. We have characterized the BRCA1 gene products by Western blot and immunoprecipitation analysis in mouse and tumor cells. Multiple BRCA1 polypeptides of approximately 225, 185, 160, 145, 100, 52 and 38 kD were identified in these cells. BRCA1 proteins were found to be localized mainly in the nucleus of normal Rat1 cells and human breast cancer cells. In order to understand the role of BRCA1 in cell transformation, we have established a stable NIH3T3 cell line expressing BRCA1 antisense RNA. The inhibition of expression of endogenous BRCA1 protein was detected in NIH3T3 transfectants by Western blot analysis. The antisense BRCA1 expressing NIH3T3 cells showed accelerated growth rate, anchorage independent growth and tumorigenicity in nude mice unlike the parental and sense transfectants. These results provide the first direct biological evidence for the possible function of BRCA1 as a tumor suppressor gene.

3T3 Cells↗

PubMed1996

The EWS-ATF-1 gene involved in malignant melanoma of soft parts with t(12;22) chromosome translocation, encodes a constitutive transcriptional activator.

Molecular characterization of malignant melanoma of soft parts or soft tissue clear cell sarcoma which shares t(12;22) chromosome translocation revealed fusion of EWS with a transcriptional factor gene ATF-1. The EWS gene, which encodes an RNA binding protein, was also shown to be involved in Ewing sarcoma, related primitive neuroectodermal tumors and desmoplastic small round cell tumors. In order to understand the functional role of EWS-ATF-1 chimeric protein in human solid tumors, we have cloned the aberrant human ATF-1 (EWS-ATF-1) cDNA and studied its DNA binding, transcriptional activation properties and compared with normal ATF-1 protein. Our results demonstrate that EWS-ATF-1 binds weakly to DNA in vitro but functions as an efficient constitutive transcriptional activator unlike the normal ATF-1 which needs to be induced with cAMP. Deletion analysis revealed that EWS-fusion domain functions as a regulatory domain for the transcriptional activation properties of EWS-ATF-1 chimeric protein. Deletion of leucine zipper domain results in a loss of transcriptional activation of EWS-ATF-1 chimeric protein suggesting that protein-protein interaction play a role in the transcriptional activation properties of EWS-ATF-1. We demonstrate that EWS-fusion domain negatively regulates the DNA binding activity of EWS-ATF-1 chimeric protein. Therefore replacement of part of the amino-terminal kinase regulatory domain of ATF-1 protein with EWS regulatory domain results in an altered DNA binding, protein-protein interactions and transcriptional activation properties of EWS-ATF-1 causing deregulated gene expression which may be responsible for the genesis of t(12;22) chromosome translocation-bearing human solid tumors. Targeting the transcriptional cofactors (CBP, etc) by EWS-fusion proteins could be one of the mechanisms of activation of EWS-fusion proteins in human neoplasia.

Activating Transcription Factor 1↗

PubMed1995

Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts.

Cytogenetic analysis of Ewing's sarcoma, primitive neuroectodermal tumors and Askin tumors revealed characteristic translocations t(11;22) or t(21;22). Molecular analysis of these translocations revealed 5'-region of EWS gene (from band 22q12) is fused to the 3'-region of either Fli-1 gene (from band 11q24) or erg gene (from band 21q22). Functional characterization of the EWS-Fli-1 and EWS-erg chimeric proteins suggested that they function as transcriptional activators. In order to develop therapeutic agents, it is essential to know whether expression of the EWS-fusion gene products is coupled to tumorigenicity of Ewing's sarcoma cells and if targeting the EWS-fusion products results in loss of tumorigenicity of Ewing's sarcoma cells. For this reason, we have made stable Ewing's sarcomas expressing antisense EWS-Fli-1 or EWS-erg expression plasmids. Expression of antisense EWS fusion transcripts resulted in a significant loss of endogenous EWS-Fli-1 and EWS-erg proteins in Ewing's sarcoma cells. These cells expressing antisense EWS fusion transcripts showed loss of anchorage independent growth and tumorigenicity in nude mice unlike the parental Ewing's sarcoma cells. These results demonstrate the necessity of a certain threshold level of expression of EWS-fusion products in the clonogenicity and tumorigenicity of Ewing's sarcoma cells and therefore emphasizes the importance of targeting the EWS-fusion products as a therapy for Ewing family of tumors.

Animals↗

PubMed1994

TLS/FUS fusion domain of TLS/FUS-erg chimeric protein resulting from the t(16;21) chromosomal translocation in human myeloid leukemia functions as a transcriptional activation domain.

EWS and TLS/FUS genes, which code for RNA binding proteins are involved in a wide variety of human solid tumors. The TLS/FUS gene is involved both in human myxoid liposarcomas which carry a characteristic chromosomal translocation, t(12;16)(q13;p11) and in human myeloid leukemias with recurrent chromosomal translocation, t(16;21)(p11:q22). The TLS/FUS gene is fused to a transcriptional repressor, CHOP (in human myxoid liposarcomas) or transcriptional activator, erg (in human myeloid leukemias). To understand better the functional role of TLS/FUS-erg in human myeloid leukemias, we have cloned the TLS/FUS and TLS/FUS-erg cDNAs and studied the functional properties of their gene products. TLS/FUS protein binds to RNA in vitro and shows preferential binding to poly G. Both the amino- and the carboxy- terminal regions of TLS/FUS containing the conserved RNA binding motifs are needed for poly G specific RNA binding activity. The TLS/FUS fusion domain (TFD) appears to regulate the DNA binding activity of TLS/FUS-erg chimeric protein which shows weaker transcriptional activation properties compared to normal erg proteins. Mutational analysis of the TLS/FUS-erg chimeric protein reveals TFD to function as a transcriptional activation domain thus replacing the amino terminal transcriptional activation domain of the erg protein. Therefore alterations in both DNA binding and transcriptional activation properties of aberrant erg proteins may be responsible for the genesis of t(16;21) chromosomal translocation-bearing human myeloid leukemias.

Chromosomes, Human, Pair 16↗

PubMed1994

The EWS gene, involved in Ewing family of tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors, codes for an RNA binding protein with novel regulatory domains.

The EWS gene, which maps to band q12 of human chromosome 22, is involved in a wide variety of human solid tumors including Ewing sarcoma, related primitive neuroectodermal tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors. In these tumors, the EWS is fused to genes encoding transcriptional activators/repressors, like Fli-1 or erg or ATF 1 or wt1. To better understand the function of the EWS protein, we cloned the EWS cDNA. Sequence analysis of this cDNA revealed differential splicing involving two exons encoding 72 amino acids. Both alternatively spliced transcripts, EWS and EWS-b, are expressed in a variety of cells. Because EWS proteins contain putative conserved RNA binding motifs, we studied the RNA binding properties of the EWS protein. The EWS-b protein binds to RNA in vitro and, specifically, to poly G and poly U. The RNA binding activity was localized to the carboxy terminal 86 amino acids, which constitute RGG box. Thus the amino terminal domain of EWS (NTD-EWS), which is involved in chromosome translocation may regulate the specificity of RNA binding activity of EWS. An EWS-erg chimeric protein, which is found in Ewing's sarcoma cells, functions as a transcriptional activator. Mutational analysis of EWS-erg chimeric protein revealed that NTD-EWS functions as a regulatory domain for the transcriptional activation properties of EWS-erg chimeric protein.

Amino Acid Sequence↗

PubMed1994

elk-1 proteins interact with MAP kinases.

Mitogen activated protein kinases (MAP) or extracellular signal regulated protein kinases (ERK) are a family of protein serine/threonine kinases that are activated very rapidly in response to many extracellular stimuli. elk-1, an ets related gene codes for two transcriptional factors elk-1, which regulates c-fos transcription and delta elk-1, both of which are substrates for MAP kinases. A part of the C-terminal transcriptional activation domain (ETA-2) which is common to both the proteins was previously shown to function as an activator of MAP kinases. In this report, in an attempt to investigate the mechanism of activation of MAP kinases, purified preparations of recombinant elk-1 and P44mpk/ERK-1/ERK-2 proteins were used to show the association of elk-1 proteins with MAP kinases. The specific interactions of elk-1 proteins with MAP kinases were confirmed by co-immunoprecipitation studies. Thus elk-1 proteins appear to regulate the activity of MAP kinases by interacting with them ensuring a conformational change and stimulating their autophosphorylation and activation property. The activation was dependent on the presence of ATP and Mg2+. In vitro phosphorylation of elk-1 protein was not regulatory for autonomous DNA binding activity of elk-1 protein. Cells which were exposed to EGF showed a rapid stimulation of an elk-1 specific kinase activity, probably MAP kinase which phosphorylated MBP and was found to be associated with immobilized GST-elk-1. Furthermore, dephosphorylation studies indicate that elk-1 proteins can activate only tyrosine phosphorylated MAP kinase. These results demonstrate the presence of an alternative pathway/mechanism (other than MAP kinase kinase, MAPKK/Mek) for the activation of MAP kinases with tyrosine phosphorylation occurring before serine/threonine autophosphorylation and activation by elk-1 proteins.

3T3 Cells↗

PubMed1994

Differentially spliced erg-3 product functions as a transcriptional activator.

ets oncogene superfamily consists of a family of transcriptional factors that functions as activators and/or repressors. Previously, we have identified a member of this ets superfamily namely erg, ets related gene. erg gene was shown to code for at least two proteins erg-1 and erg-2 because of alternative splicing and alternative usage of initiation codon. In this report we show that erg gene codes for an additional erg variant protein, erg-3 as a result of differential splicing which results in the insertion of 24 amino acids in the coding region of erg-2 protein. RNAase protection analysis revealed that erg-3 transcripts are expressed in a variety of cells. Erg-3 was also found to activate the transcription of the reporter TK-CAT gene linked to erg target sequences suggesting that erg-3 codes for a sequence specific transcriptional activator.

Amino Acid Sequence↗