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T S Papas

Publications and source records attributed to T S Papas.

At least 55 records · Page 3Linked to original sources
PubMed1993

c-Ets-1 protein is hyperphosphorylated during mitosis.

The ets-1 and ets-2 proto-oncogene products can serve as transcription factors and become phosphorylated in response to Ca(2+)-mediated signals. We have examined expression of Ets proteins during the cell cycle in cells synchronized by centrifugal elutriation or nocodazole-induced mitotic block. Both methods revealed the presence of a hyperphosphorylated isoform of Ets-1 during the mitotic phase. This isoform showed a characteristic mobility shift and was observed during mitosis in each of four cell lines (three human T-cell lines and a human astrocytoma) that express ets-1. In elutriated cells, only a small portion of the Ets-1 in cells from the G2/M fractions was hyperphosphorylated, while in nocodazole-arrested cells more of the Ets-1 was shifted. When cells were released from nocodazole arrest, this isoform disappeared within 1-2 h as cells completed mitosis and entered G1. This suggests that hyperphosphorylated Ets-1 is present transiently during early mitosis, before or around the time of the metaphase-anaphase transition. Exposure of unsynchronized cells to okadaic acid resulted in a dramatic hyperphosphorylation of virtually all Ets-1, suggesting that changes in cellular phosphatase activity are important for cell cycle regulation of Ets-1. Hyperphosphorylated Ets-1 appears to arise from multiple phosphorylations on serine in the exon 7-encoded domain of the protein and did not appear to alter sequence-specific DNA-binding activity. Although enhanced phosphorylation of Ets-2 was detected in nocodazole-arrested cells, no Ets-2 hyperphosphorylation was seen.

Amino Acid Sequence↗
PubMed1993

Human ERG-2 protein is a phosphorylated DNA-binding protein--a distinct member of the ets family.

We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein. ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T. ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins. Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment. ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms. The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination. The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure. The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method. Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells. These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.

Base Sequence↗
PubMed1993

Transactivation of GATA-1 promoter with ETS1, ETS2 and ERGB/Hu-FLI-1 proteins: stabilization of the ETS1 protein binding on GATA-1 promoter sequences by monoclonal antibody.

Ets family proteins activate transcription via binding to the GGAA core sequence located in the promoter/enhancer elements of many cellular and viral genes. GATA-1 is an erythroid-specific transcription factor. The promoter of the chicken GATA-1 gene contains multiple ets binding sites (EBS), two of them are present in palindromic form. The GATA-1 promoter has been shown to be activated by the E26 virus. In this study, we have analysed whether the palidromic EBS of the chicken GATA-1 promoter is a target for binding and activation by members of the cellular ets gene family products. The results herein indicate that both EBS in the palindrome are required for DNA-binding because mutations in either site reduces the activity by at least 95%. Moreover, DNA binding of ETS1 to the EBS palindrome is dramatically stabilized in the presence of a specific monoclonal antibody whose epitope maps between amino acid positions 240-260. Although each of the single sites bind, the efficiency of binding is extremely low. Furthermore, for efficient binding the two sites must be in an inverted configuration because of the fact that the oligonucleotide containing the left and right EBS in the same orientation binds 10-fold less than the oligonucleotide containing the EBS palindrome. Additionally, we show that the transcription of a reporter gene (CAT) either linked to the GATA-1 EBS palindrome or GATA-1 promoter can be activated by cotransfection with ETS1, alternatively-spliced ETS1, ETS2 or ERGB/Hu-FLI-1 expression vectors.

Animals↗
PubMed1992

Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.

Amino Acid Sequence↗
PubMed1992

Differential expression of S19 ribosomal protein, laminin-binding protein, and human lymphocyte antigen class I messenger RNAs associated with colon carcinoma progression and differentiation.

Three complementary DNA encoding S19 ribosomal protein (S19), laminin-binding protein (LBP), and HLA class I (HLA-I) genes were isolated from a colon tumor-enriched subtraction library. To evaluate this mRNA expression, surgically removed colon tumors as well as matched normal tissue and human colon carcinoma cell lines showing various differentiation states, anchorage dependence, and proliferation states were examined by Northern blot analysis. The mRNA level of S19 mRNA (0.6 kilobase) was higher in primary colon carcinoma tissue than in matched normal colon tissue in 5 of 6 cases. In 2 of 4 cases, the expression of LBP mRNA (1.2 kilobases) was higher in carcinoma than in normal tissue. In 12 human colon cell lines, the level of LBP mRNA was higher in poorly differentiated cells. On the other hand, HLA-I mRNA (1.7 kilobases) was higher in well-differentiated cells. Although the S19 mRNA was expressed in both well- and poorly differentiated cells, a concomitant increase with tumor progression was observed in two pairs of cell lines derived from the same patients (SW480 and SW620; COLO201 and COLO205). Anchorage dependence of butyrate-treated HT29 colon carcinoma cells was correlated with lower levels of S19 and LBP mRNAs and higher levels of HLA-I mRNA expression compared with untreated cells. While the expression of S19 and LBP mRNAs was not changed due to cell growth states, HLA-I mRNA levels were found to be low in proliferating HT29 cells but highly induced in contact-inhibited cells. In summary, therefore, high expression of S19 and LBP combined with low expression of HLA-I were well correlated with colon carcinoma cells of higher malignant potential.

Amino Acid Sequence↗
PubMed1992

Characterization and uses of monoclonal antibody derived against DNA binding domain of the ets family of genes.

A monoclonal antibody recognizing ets proteins from a variety of species has been developed. This antibody recognizes ets1, ets2, erg, and other related proteins. It has a high affinity for the ets1 protein. The epitope for the pan ets mAb consists of about 13 amino acids. This antibody can be used to isolate and characterize new members of ets gene family derived from a c-DNA expression library, as well as to identify other "ets motif" binding proteins.

Amino Acid Sequence↗
PubMed1992

The sea urchin erg homolog defines a highly conserved erg-specific domain.

A genomic clone, isolated from a phage library prepared from the DNA of the sea urchin Lytechinus variegatus, was shown by sequence analysis to be a homolog of the ets family genes, ERG and Fli-1. It contains an open reading frame of which the coding region begins at a consensus 3' splice site and extends for 173 amino acid residues. The first 84 amino acids are homologous with all members of the ets gene family, while the remainder of the sequence is only homologous with the human ERG and murine Fli-1 genes. This latter region, designated R, represents a highly conserved erg-specific domain.

Amino Acid Sequence↗
PubMed1992

The ERGB/Fli-1 gene: isolation and characterization of a new member of the family of human ETS transcription factors.

All cellular ets proteins contain a region of high amino acid identity to those found in the last two exons of the ets-1 gene (C domain). We have identified and characterized a new member of the human ETS gene family, ERGB. The ERGB gene shows extensive amino acid identity to the human ERG and the mouse Fli-1 genes. The ERGB gene is found to be transcriptionally active in a variety of human cell lines and tissues, in contrast to the more restrictive expression pattern of the ERG gene. The ERGB gene encodes for a 3.2-kilobase mRNA containing an open reading frame of 451 amino acids. The ERGB gene, like human ETS1, is located on chromosome 11 and is transposed to chromosome 4 as a result of the translocation t(4;11) associated with leukemia. Pulse-field gel analysis suggests that ETS1 and ERGB are more than 200 kilobases apart. Similar to the other members of the ets family (ets 1, ets 2), this new member is also able to trans-activate transcription of a reporter gene linked to the ETS-binding sequences derived from either the GATA-1 promoter or an optimal Ets-binding site.

Amino Acid Sequence↗
PubMed1992

Molecular characterization and structural organization of D-elg, an ets proto-oncogene-related gene of Drosophila.

We have continued the molecular analysis of D-elg, a member of the Drosophila ets gene family. Based on the characterization of cDNA and genomic sequences, the D-elg gene contains five exons and four introns and produces a mRNA with an open reading frame of 464 amino acids. Consistent with this analysis, in vitro translation of a near full-length D-elg cRNA yields a protein with a molecular weight of approximately 56 kDa. D-elg shows significant homology to other ets proteins in the amino-terminal A domain and strong homology in the carboxy-terminal ETS domain. The D-elg protein is most similar to the alpha-subunit of the mouse GA-binding protein.

Amino Acid Sequence↗
PubMed1992

Genomic dispersal of the ets gene family during metazoan evolution.

Evolutionary homologs of the ets proto-oncogene have been discovered in the genomes of widely divergent eucaryote species from Drosophila to sea urchin to vertebrates. The prototype mammalian ets-1 and ets-2 genes are divided into three coding domains that differ in their rate of accumulation of sequence divergence. An analysis of sequence divergence of ets gene homologs in various species has produced a phylogenetic history of the ets gene family in the context of metazoan evolutionary radiation. A minimum of five duplication events of ets primordial genes were evident, namely (1) a duplication that separates primitive ets genes (Drosophila precursor of 74E, mouse PU.1 and human ELK1) from the ets-1, ets-2, erg ancestor; (2) and (3) two duplications that established separate ets, erg and elg/GABP-alpha lineages which occurred prior to invertebrate-vertebrate divergence; (4) divergence of ets-1 and ets-2 gene family also associated with vertebrate-invertebrate divergence; (5) duplication of ets-1 and ets-2 in Xenopus laevis to produce two ets-1 genes and two ets-2 genes during genomic tetraploidation in the recent ancestry of this species.

Amino Acid Sequence↗
PubMed1991

Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.

Amino Acid Sequence↗
PubMed1991

Nef proteins of the human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV) are structurally similar to leucine zipper transcriptional activation factors.

Analysis of the predicted amino acid sequences of the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and of the related simian immunodeficiency virus (SIV) nef gene products (Nef) reveals the presence of a conserved leucine zipper-like repeat with the characteristic 4,3 arrangement of mainly hydrophobic amino acids in the middle (core) region of the proteins, but lacking the basic (DNA binding) domain characteristic of DNA-binding leucine zipper (bZIP) proteins. Also, at the C-terminus of the Nef proteins is a highly acidic sequence (net charge of -5 to -8) stretched over about 40 amino acids, and contains two predicted alpha-helices separated by a beta-turn linker sequence with sequence homology to known activation domains of acidic transcriptional activation factors. Moreover, within this acidic region of transcriptional activators and the homologous sequence within the second Nef alpha-helix, is a potential transcriptional activation consensus sequence (TACS) bounded by a pair of acidic amino acids (aspartic or glutamic acids) at the N-terminus and a highly invariant phenylalanine (hydrophobic), often followed by an acidic (aspartic) residue, at the C-terminus of the sequence. These findings strongly implicate Nef proteins as belonging to a class of non-DNA-binding leucine zipper acidic transcription factors, and provide a structural basis for new approaches to studying Nef function.

Amino Acid Sequence↗
PubMed1991

D-elg, a member of the Drosophila ets gene family: sequence, expression and evolutionary comparison.

We have cloned a cDNA from the Drosophila elg gene, a new member of the ets family of genes. The D-elg gene is located at 97D on chromosome 3R and is expressed as a 2.0kb RNA in the embryos, pupae and adults, with no detectable expression in third instar larvae. D-elg expression is observed in all cells of early stage embryos, prior to transcriptional activation of the zygotic genome, and is maintained throughout embryogenesis with no regional localization. The cDNA encodes a predicted protein of 15.4kD that has an 86 amino acid sequence with 72% similarity to the carboxy terminal region of the Drosophila ets-2 gene. Comparison with all known ets genes allows us to define a minimal region required for assignment to the ets gene family.

Amino Acid Sequence↗
PubMed1991

High-affinity DNA-protein interactions of the cellular ETS1 protein: the determination of the ETS binding motif.

ETS1 protein purified from CEM cells was used to select its optimum DNA-binding sequence (pu) G/CCaGGA-AGTc (py). The sequence CCGGAAGT (ETS1-3) was preferred 5:1 over CAGGAAGT (PEA3). Quantitative electrophoretic mobility-shift assays (EMSA) indicated that the purified ETS1 protein binds to either ETS1-3 or PEA3 oligonucleotide probes with high affinity (Ka = 0.5-4.0 x 10(10) M-1) and that the purified ETS1 has different binding capacities for ETS1-3 and PEA3 oligonucleotide probes. The ETS1 protein binds 2-5 times more ETS1-3 than PEA3. Competitive binding experiments showed that the ETS1-3 and PEA3 probes effectively compete for the binding of ETS1-3. However, changing the core DNA-binding sequence from GGAA to AGAA eliminates competition. Since the human ETS1 protein selected the same DNA sequence from a mixture of random oligonucleotides as did the Drosophila E74A protein (one of the most divergent members of the ETS family), this strongly suggests that all proteins containing the ETS 85 amino acid domain (sequences which define the ETS family) will bind to the same sequence.

Base Sequence↗
PubMed1991

Positive and negative factors regulate the transcription of the ETS2 gene via an oncogene-responsive-like unit within the ETS2 promoter region.

The DNA-protein interactions in the ETS2 promoter have been studied. Three distinct sequence motifs have been identified, each of which interacts with at least two distinctive protein complexes. The GC motif, possessing mirror symmetry, interacts with two ubiquitously identifiable complexes (S and S2); the PEA3 motif interacts with a ubiquitous (H1) and a tissue-specific (H3) complex; the H2 (an AP1-like) motif interacts also with a ubiquitous (H2a) and a tissue-specific (H2b) complex. Mutational analysis and correlation of the presence of defined complexes with the ETS2 mRNA levels indicate that the S, S2, H1, and H2b complexes have positive effects on ETS2 transcription, whereas the H3 and H2a have negative effects. The organization of the PEA3 with the AP1-like motif in the ETS2 promoter resembles the oncogene-responsive unit previously identified in the polyoma virus enhancer region. Our data suggest that cooperation between these two motifs is vital for ETS2 promoter function.

Base Sequence↗