Human BCR Gene Research Articles

P160 Bcr mediates platelet-derived growth factor activation of extracellular signal-regulated kinase in vascular smooth muscle cells.

The human Bcr gene was originally identified by its presence in the chimeric Bcr/Abl oncogene, which is causative for chronic myeloblastic leukemia. Because Bcr encodes a serine/threonine protein kinase, we studied its kinase activity and determined the role of Bcr in the PDGF signaling pathway to ERK1/2 activation and DNA synthesis in rat aortic smooth muscle cells (RASMCs). In RASMCs, platelet-derived growth factor-BB (PDGF) stimulated Bcr kinase activity, with a maximum at 1 minute. Because phosphatidylinositol 3'-kinase (PI3-K) is essential for Bcr/Abl leukemogenesis, we evaluated the role of mouse PDGF-beta-receptor binding sites for PI3-K (Y708, Y719) and for phospholipase C-gamma (Y977, Y989) in PDGF-mediated Bcr kinase activation. The mutant PDGF receptor Y708F/Y719F but not Y977F/Y989F showed significantly reduced Bcr kinase activity. To determine the role of Bcr in PDGF-mediated signal transduction events leading to ERK1/2 and its downstream Elk1 transcription activation, wild-type (WT) and kinase-negative (KN) Bcr were transiently expressed in RASMCs. Bcr WT enhanced, whereas Bcr KN inhibited, PDGF-stimulated ERK1/2 and Elk1 transcriptional activity. Overexpression of Bcr also enhanced PDGF-induced Ras/Raf-1 activity and DNA synthesis, but this regulation is independent of the kinase activity of Bcr. Finally, we found that Bcr expression was increased in the neointimal layer after balloon injury of rat carotid artery. These results demonstrated the importance of Bcr in PDGF-mediated events, such as activation of Ras, Raf-1, ERK1/2, and Elk1, and stimulation of DNA synthesis.

Targeting neighbouring poly(purine.pyrimidine) sequences located in the human bcr promoter by triplex-forming oligonucleotides.

Most poly(purine.pyrimidine) [poly(R.Y)] sequences in eukaryotic genomes are interrupted by one or more base pair inversions. When the inversions are centrally located, the poly(R.Y) sequences can be regarded as the sum of two abutting sites, each potentially capable of forming a triple helix. Employing band-shift, footprinting and modeling methods we examined the formation of triple helices at a critical 27 bp poly(R.Y) sequence interrupted by two adjacent CG inversions, and located in the promoter of the human bcr gene at transcription initiation. We designed several 13-mer and 14-mer triplex-forming oligonucleotides (TFOs) capable of binding the bcr abutting sites, thereby generating different base juxtapositions at the triple helical junction, to examine whether triplex formation occurs in a cooperative manner. It is found that in 50 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 2 mM spermine, 37 degrees C, the 13-and the 14-mer TFOs bind to one half of the bcr site with Delta G between -30 and -35 kJ x mol-1. However, when different 13-mer/14-mer combinations of TFOs were directed against the abutting poly(R x Y) sites, triplex formation has been found to be enhanced only for the triple helical junction formed by the 5'-A-T-3' base juxtaposition, in keeping with a partial stacking suggested from modeling analysis. On the other hand, a longer 24-mer TFO, binding noncooperatively to the same abutting sites, forms a much more stable triplex (Delta G = -51 kJ x mol-1), notwithstanding the two T x CG triads in the middle. Modeling investigations reveal that there is no continuity or propagation of base stacking involving adjacent bases of the third strand at the site of base inversion as well as on the 5' side. The data indicate that the entropy penalty of forming a triplex with two oligonucleotides is much higher than the energy gained from base stacking interactions at the triplex junction formed between the two TFOs.

Formation of Triple Helices at Irregular Poly (R·Y) Sites Located in Critical Positions in the Human BCR Promoter

Two polypurine sequences interrupted respectively by one and two adjacent pyrimidines have been identified in the promoter of the human bcr gene. Although these targets are irregular they are recognised and tightly bound by AG and GT motif triplex-forming oligonucleotides. Thermodynamic and kinetic data are presented.

Formation of stable DNA triple helices within the human bcr promoter at a critical oligopurine target interrupted in the middle by two adjacent pyrimidines.

Antigene strategies based on the use of triplex-forming oligonucleotides (TFO) as artificial repressors are constrained by the need for genomic targets with a polypurine-polypyrimidine [poly (R.Y)] DNA motif. In this study, we demonstrate that both A/G and G/T motif oligonucleotides recognize and bind strongly to a critical polypurine sequence interrupted in the middle by two adjacent cytosines and located in the promoter of the human bcr gene at the transcription initiation. The interaction between the designed TFO and this irregular poly (R.Y) target has been studied using a number of techniques, including electrophoretic mobility shift assay (EMSA), circular dichroism (CD), DNase I, and dimethyl sulfate (DMS) footprinting. Although CD shows that the 24-mer TFO self-aggregate in solution, they bind to the bcr target at 37 degrees C, forming stable triplexes that do not dissociate during electrophoretic runs performed up to 50 degrees C in 50 mM Tris-acetate, pH 7.4, 10 mM MgCl2, 50 mM NaCl (buffer A). We used EMSA to determine the equilibrium dissociation constants (Kd) for the reaction T <==> D + TFO at 37 degrees C, either in buffer A or in 50 mM Tris-acetate, pH 7.4, 10 mM MgCl2, 5 mM NaCl (buffer B). The triplexes were found to be more stable in buffer B, a behavior that can be rationalized in terms of monovalent and divalent cation competition for binding to DNA. Footprinting experiments showed that the TFO interact with the irregular poly (R.Y) target in a highly sequence-specific way and that the A/G motif oligonucleotide, juxtaposing T to the double CG inversions of the target, formed the most stable triplex (e.g., 1 microM TFO promoted strong footprints at 37 degrees C). These triplexes, except the one containing two A.C.G mismatched triads, are not destabilized under near physiologic conditions, that is, in 50 mM Tris-acetate, pH 7.4, 80 mM KCl, 20 mM NaCl, 2 mM spermidine. Moreover, we found that guanine N7 in T.C.G and guanine N7 in A.C.G are both accessible to DMS and that the first is less reactive than the second. In conclusion, the results of this study indicate that a critical sequence in the human ber promoter may be used as a potential binding site for TFO designed to repress artificially the transcription of the fused bcr/abl gene expressed in leukemia cells.

Co-expression with BCR Induces Activation of the FES Tyrosine Kinase and Phosphorylation of Specific N-terminal BCR Tyrosine Residues

The human BCR gene encodes a protein with serine/threonine kinase activity and regulatory domains for the small G-proteins RAC and CDC42. Previous work in our laboratory has established that BCR is a substrate for c-FES, a non-receptor tyrosine kinase linked to myeloid growth and differentiation. Tyrosine phosphorylation led to the association of BCR with the RAS guanine nucleotide exchange complex GRB2-SOS in vivo via the GRB2 SH2 domain, linking BCR to RAS signaling (Maru, Y., Peters, K. L., Afar, D. E. H., Shibuya, M., Witte, O. N., and Smithgall, T. E. (1995) Mol. Cell. Biol. 15, 835-842). In the present study, we demonstrate that BCR Tyr-246 and at least one of the closely spaced tyrosine residues, Tyr-279, Tyr-283, and Tyr-289 (3Y cluster), are phosphorylated by FES both in vitro and in 32Pi-labeled cells. Mutagenesis of BCR Tyr-177 to Phe completely abolished FES-induced BCR binding to the GRB2 SH2 domain, identifying Tyr-177 as an additional phosphorylation site for FES. Co-expression of BCR and FES in human 293T cells stimulated the tyrosine autophosphorylation of FES. By contrast, tyrosine phosphorylation of BCR by FES suppressed BCR serine/threonine kinase activity toward the 14-3-3 protein and BCR substrate, BAP-1. These data show that tyrosine phosphorylation by FES affects the interaction of BCR with multiple signaling partners and suggest a general role for BCR in non-receptor protein-tyrosine kinase regulation and signal transduction.

Sequence and Analysis of the Human ABL Gene, the BCR Gene, and Regions Involved in the Philadelphia Chromosomal Translocation

The complete human BCR gene (152-141 nt) on chromosome 22 and greater than 80% of the human ABL gene (179-512 nt) on chromosome 9 have been sequenced from mapped cosmid and plasmid clones via a shotgun strategy. Because these two chromosomes are translocated with breakpoints within the BCR and ABL genes in Philadelphia chromosome-positive leukemias, knowledge of these sequences also might provide insight into the validity of various theories of chromosomal rearrangements. Comparison of these genes with their cDNA sequences reveal the positions of 23 BCR exons and putative alternative BCR first and second exons, as well as the common ABL exons 2-11, respectively. Additionally, these regions include the alternative ABL first exons 1b and 1a, a new gene 5' to the first ABL exon, and an open reading frame with homology to an EST within the BCR fourth intron. Further analysis reveals an Alu homology of 38.83 and 39.35% for the BCR and ABL genes, respectively, with other repeat elements present to a lesser extent. Four new Philadelphia chromosome translocation breakpoints from chronic myelogenous leukemia patients also were sequenced, and the positions of these and several other previously sequenced breakpoints now have been mapped precisely, although no consistent breakpoint features immediately were apparent. Comparative analysis of genomic sequences encompassing the murine homologues to the human ABL exons 1b and 1a, as well as regions encompassing the ABL exons 2 and 3, reveals that although there is a high degree of homology in their corresponding exons and promoter regions, these two vertebrate species show a striking lack of homology outside these regions.

Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.

No evidence for genomic imprinting of the human BCR gene

Chronic myeloid leukemias and 5% to 20% of acute lymphoid leukemias are characterized by the Philadelphia chromosome, a reciprocal chromosomal translocation, t(9;22)(q34;q11), generating BCR-ABL and ABL-BCR fusion genes. Cytogenetic studies have recently shown a preferential involvement of the paternally derived chromosome 9 and the maternally derived chromosome 22 in this translocation, indicating that imprinting might be involved in the formation or selection of the translocation. In this study, we have identified a BamHI polymorphism in the coding region of BCR exon 1, allowing us to investigate whether both BCR alleles are transcribed. By using a reverse transcriptase-polymerase chain reaction assay, we show that both BCR alleles are expressed in the peripheral blood cells of normal individuals.

The hematopoietically expressed vav proto-oncogene shares homology with the dbl GDP-GTP exchange factor, the bcr gene and a yeast gene ( CDC24) involved in cytoskeletal organization : J.M. ADAMS ET AL. Oncogene 7, 611–618

The vav proto-oncogene encodes a protein of unknown function that is rendered oncogenic by loss of a short N-terminal domain. A correction reported here to the vav sequence reveals that a central domain of some 230 amino acids is similar to the products of three genes: the human dbl oncogene, now known to encode a GDP-GTP exchange factor for the Ras-like polypeptide CDC42Hs; the CDC24 gene of Saccharomyces cerevisiae, which participates with CDC42Sc in organization of the cytoskeleton for budding; and the human bcr gene, which recombines with the abl oncogene in certain forms of leukemia. Furthermore, the N-terminal portion of Vav (and of CDC24) is similar to that of certain proteins that associate with filamentous structures. These similarities suggest that Vav, and perhaps also Bcr, may function as a GDP-GTP exchange factor for a Ras-like molecule such as CDC42Hs, and that its action may coordinate cytoplasmic architecture with the cell cycle. Reported evidence that the vav proto-oncogene is widely expressed in hematopoietic cells but not other cell types is extended here by detection of vav mRNA in 49 of 50 murine hematopoietic cell lines representing diverse hematopoietic lineages, and by in situ hybridization in embryos showing expression confined to the only hematopoietic tissue, fetal liver. Thus, like Dbl in other cell types, Vav may function throughout the hematopoietic compartment to govern a Ras-like signal transduction pathway.

Unique organization of the human BCR gene promoter

The promoter of the human BCR gene, regulating the transcription of the chimeric BCR/ABL mRNA in leukemia, has been isolated and characterized. A region of 1.1 kb immediately 5' to the transcription start site was analyzed in detail by sequencing, DNase 1 footprinting, gel retardation and functional studies. These experiments localized a minimal promoter to a 650 bp sequence, composed of 270 bp of 5' flanking sequences and 380 bp of exon 1 transcribed sequences. The promoter region includes a TTTAA box, one Sp1 site and a novel protein-binding sequence absolutely necessary for efficient transcription in vivo. Six additional protein-binding regions were identified more to the 5'. Of these, one is found in an inverted repeat in the 3' coding and splice donor region of BCR exon 1.

ABR, an active BCR-related gene

The human BCR gene on chromosome 22 is specifically involved in the Philadelphia translocation, t(9;22), a chromosomal rearrangement present in the leukemic cells of patients with chronic myeloid leukemia or acute lymphoblastic leukemia. In most cases, the breakpoints on chromosome 22 are found within a 5.8 kb region of DNA designated the major breakpoint cluster region (Mbcr) of the BCR gene. Hybridization experiments have indicated that the human genome contains BCR gene-related sequences. Here we report the molecular cloning of one of these loci, for which we propose the name ABR. In contrast with the other BCR-related genes studied to date, ABR represents a functionally active gene and contains exons very similar to those found within the Mbcr. Unlike the BCR gene, the ABR gene exhibits great genomic variability caused by two different variable tandem repeat regions located in two introns. All other BCR gene-related sequences isolated so far and the BCR gene itself are located on chromosome 22. In contrast, the ABR gene is located on chromosome 17p.

A deletion/insertion polymorphism in the human BCR gene on chromosome 22.

A 2.3 kb single-copy genomic EcoRI-BamHI fragment was isolated from the putative first intron of the human BCR gene and subcloned into the Bluescribe vector pBS M13+ (pA-EB2.3). Multiple enzymes resolve a two-allele deletion/insertion type RFLP. A 1 kb difference between the alleles is seen in BamHI, Bg1II and HindIII digests. The deleted/inserted material includes EcoRI and TaqI sites; thus, using these enzymes the fragment lengths corresponding to the alleles do not differ by 1 kb. The BCR gene is within chromosome band 22q11. A family was studied in which the parents represented both homozygotes. The child was heterozygous as expected.