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PubMed1997

BCR-ABL gene variants.

The BCR-ABL hybrid gene, the main product of the t(9;22)(q34;q11) translocation, is found in the leukaemic clone of at least 95% of CML patients. The fusion protein encoded by BCR-ABL varies in size, depending on the breakpoint in the BCR gene. Three breakpoint cluster regions have been characterized to date: major (M-bcr), minor (m-bcr) and micro (mu-bcr). The overwhelming majority of CML patients have a p210 BCR-ABL gene (M-bcr), whose mRNA transcripts have a b3a2 and/or a b2a2 junction. There is apparently no significant difference between patients with a 5' or a 3' M-bcr breakpoint, except maybe for a slight predominance of b3a2-expressing cases among those with increased platelet counts (ET-like syndrome). The smallest of the fusion proteins, p190BCR-ABL, (m-bcr breakpoint) is principally associated with Ph-positive ALL. Rare cases of CML are due to a p190-type of BCR-ABL gene and, in these, the disease tends to have a prominent monocytic component, resembling CMML. CML resulting from a p230 BCR-ABL gene (mu-bcr breakpoint) is also rare, and has been associated with the CNL variant and/or with marked thrombocytosis. Exceptional CML cases have been described with BCR breakpoints outside the three defined cluster regions, or with unusual breakpoints in ABL resulting in BCR-ABL transcripts with b2a3 or b3a3 junctions, or with aberrant fusion transcripts containing variable lengths of intronic sequence inserts. The reciprocal ABL-BCR gene found in the derivative 9q+ chromosome of the t(9;22) is transcriptionally active in nearly two-thirds of CML patients but has not been shown so far to have a functional role in CML. 'Ph-negative CML' comprises cases of typical CML in whom the BCR-ABL gene can be detected by molecular methods and others who are genuinely BCR-ABL negative and usually have an atypical disease phenotype.

Animals↗

PubMed2001

The significance of bcr-abl molecular detection in chronic myeloid leukemia patients "late," 18 months or more after transplantation.

The bcr-abl chimeric messenger RNA is frequently detected in chronic myeloid leukemia (CML) patients after bone marrow transplantation. It was previously reported that the relapse risk of bcr-abl detection 6 to 12 months after transplantation was greater than 40%. This risk decreased as the time between transplantation and detection increased. To further define the relapse risk associated with bcr-abl molecular detection in "late" CML survivors, 379 consecutive CML patients alive at 18 months after transplantation or later were studied. Ninety of 379 patients (24%) had at least one positive bcr-abl test 18 months after transplantation or later; 13 of 90 bcr-abl-positive patients (14%) and 3 of 289 bcr-abl-negative patients (1.0%) relapsed. The median time from bcr-abl detection to relapse was 916 days (range, 251-2654 days). The hazard ratio of relapse associated with bcr-abl detection was 19.2 (P <.0001). The stage of disease, chronic graft-versus-host disease, and the donor type did not alter the association between bcr-abl and relapse. Quantification of bcr-abl was performed on 344 samples from 85 bcr-abl-positive patients by means of a real-time quantitative reverse transcriptase-polymerase chain reaction assay. The median bcr-abl change of patients who relapsed was significantly greater than those that remained in remission (P =.002). The median bcr-abl level at relapse was 40 443 bcr-abl copies per microg RNA (range, 960-299 552). Of 73 bcr-abl-positive patients who failed to relapse, 69% had only one positive test at a median of 24 copies bcr-abl per microg RNA. The detection of bcr-abl is common following transplantation. The prognostic significance of a qualitative bcr-abl can be refined by quantitative assays and thus may target patients who would benefit from early intervention.

Adolescent↗

PubMed2001

Expression of a truncated first exon BCR sequence in chronic myelogenous leukemia cells blocks cell growth and induces cell death.

We have shown that a deletion mutant form of Bcr [Bcr(64-413)] is a strong inhibitor of the tyrosine kinase of Bcr-Abl in vitro and also inhibits its oncogenic growth effects (Liu et al., Cancer Res., 56: 5120-5124, 1996). To determine the effects of this Bcr-Abl kinase inhibitor on chronic myelogenous leukemia (CML) cells, we cloned BCR(64-413) into a recombinant, replication-defective adenovirus to express useful quantities of Bcr(64-413) in a wide variety of cells in culture. Infection of Cos1 cells with plaque-purified virus at a multiplicity of infection of 20-40 induced high expression of Bcr(64-413) as detected by Western blotting. Infection of hematopoietic cells at modest multiplicities of infection (20-40) required special conditions involving shifting cycling cells to a nongrowing condition involving serum starvation and cell crowding. Under these conditions, both Bcr-Abl-positive and -negative hematopoietic cells can be efficiently infected by adenovirus, as demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of cells infected by beta-galactosidase (beta-GAL) adenovirus. We found that expression of Bcr(64-413) in Bcr-Abl-positive K562 and BV-173 cells, but not Bcr-Abl-negative SMS-SB cells, increased cell-cell clumping and inhibited cell growth. In contrast to the effects of the Bcr(64-413) adenovirus, the beta-GAL adenovirus, despite infecting both types of cells, did not block growth or increase cell-cell clumping of Bcr-Abl-positive and -negative hematopoietic cells. Expression of Bcr(64-413) protein in primary cultures of cells from CML patients with active disease interfered with cell growth, induced apoptosis (as measured by annexin staining), and increased cell-cell clumping, whereas the beta-GAL adenovirus and mock-infected cells lacked these effects. In contrast, normal marrow cells did not exhibit these effects on infection with Bcr(64-413) adenovirus. We conclude from these findings that Bcr(64-413) interferes with the oncogenic effects of Bcr-Abl and therefore has the potential for use in therapy of CML.

Adenoviridae↗

PubMed2002

Molecular characterization and sensitivity of STI-571 (imatinib mesylate, Gleevec)-resistant, Bcr-Abl-positive, human acute leukemia cells to SRC kinase inhibitor PD180970 and 17-allylamino-17-demethoxygeldanamycin.

Using human acute leukemia HL-60/Bcr-Abl (with ectopic expression of p185 Bcr-Abl) and K562 cells (with endogenous expression of p210 Bcr-Abl) subjected to a continuous selection pressure of up to 1.0 micro M Gleevec (imatinib mesylate, STI-571), we have isolated Gleevec-resistant K562 R (+Bcr-Abl), K562 R (-Bcr-Abl), and HL-60/Bcr-Abl R cells, which display disparate level and activity of Bcr-Abl tyrosine kinase (TK). As compared with their sensitive counterparts, Gleevec-resistant cell types were >/=5-fold resistant to Gleevec-induced apoptosis. Bcr-Abl protein levels were significantly increased in HL-60/Bcr-Abl R and K562 R (+Bcr-Abl) cells, but K562 R (-Bcr-Abl) cells showed a marked decline in the mRNA and protein levels and activity of Bcr-Abl. Bcr-Abl TK level and activity corresponded to the signal transducers and activators of transcription-5 DNA binding activity and up-regulation of heat shock protein 70 levels. The decline in Bcr-Abl expression and TK activity in K562 R (-Bcr-Abl) cells was associated with reduced AKT kinase and signal transducers and activators of transcription-5 DNA binding activities and increased sensitivity to the death ligand Apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and 1-beta-D-arabinofuranosylcytosine-induced apoptosis. All Gleevec-resistant cell types were sensitive to 17-allylamino-17-demethoxygeldanamycin (17-AAG)- and PD180970 (a SRC and Bcr-Abl TK inhibitor)-induced apoptosis. Treatment with 17-AAG or PD180970 also induced apoptosis of CD34+ leukemic cells from three patients with chronic myeloid leukemia in blast crisis who had progressive leukemia while receiving Gleevec therapy. Taken together, these findings indicate that in addition to overexpression or mutations in Bcr-Abl, resistance to Gleevec may also develop due to a loss of Bcr-Abl expression. These findings also support the rationale to test the in vivo efficacy of 17-AAG and PD180970 against STI-571-resistant Bcr-Abl-positive acute leukemias.

Benzamides↗

PubMed2000

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7.

There is currently no satisfactory model allowing analysis of dose-effect relationships of BCR-ABL proteins in human hematopoietic cells. To study comparatively the proliferative, differentiative and anti-apoptotic actions of different levels of BCR-ABL proteins in the context of the same cellular background, we have introduced the BCR-ABL gene into the GM-CSF-dependent pluripotent human cell line UT-7. Individual clones expressing BCR-ABL were analyzed by Western blots. After normalization to equivalent levels of endogenous ABL protein, 14 clones always grown in GM-CSF were found to express low but variable levels of BCR-ABL whereas two clones selected in the absence of GM-CSF expressed very high levels of BCR-ABL. All low-level BCR-ABL expressing clones exhibited a behavior similar to that of the GM-CSF-dependent parental cells as they ceased to proliferate upon growth factor deprivation and showed a strong proliferative response upon GM-CSF addition. One out of 14 clones showed progressive GM-CSF independence during culture over several weeks and was found to have a significant increase of BCR-ABL expression at that time. The resistance of this clone (E8-2) to different apoptotic stimuli was found to be increased as compared to its low BCR-ABL-expressing counterpart (E8-1) and similar to that observed in clones with very high levels of BCR-ABL (UT-7/9 and UT-7/11) which were totally resistant to apoptotic stimuli. When injected into nude mice, parental UT-7 cells and clones with low-level of BCR-ABL were not tumorigenic over 10 weeks of observation whereas UT-7 clones with high levels of BCR-ABL (UT-7/9, UT-7/11 and UT-7/E8-2) induced aggressive tumors in 2-4 weeks with a significant correlation between the amount of BCR-ABL protein and the rate of tumor growth. In conclusion, the establishment of an in vitro and in vivo CML model using UT-7 cells suggests for the first time in human cells, that the fully transformed phenotype induced by BCR-ABL requires high levels of BCR-ABL expression. These findings suggest that variable levels of BCR-ABL in primary patient cells could also be responsible for the different phenotypic features seen in chronic and acute phases of CML, such as the differentiation ability induced by growth factors.

Animals↗

PubMed1995

Structural and signaling requirements for BCR-ABL-mediated transformation and inhibition of apoptosis.

BCR-ABL is a deregulated tyrosine kinase expressed in Philadelphia chromosome-positive human leukemias. Prolongation of hematopoietic cell survival by inhibition of apoptosis has been proposed to be an integral component of BCR-ABL-induced chronic myelogenous leukemia. BCR-ABL elicits transformation of both fibroblast and hematopoietic cells and blocks apoptosis following cytokine deprivation in various factor-dependent cells. To elucidate the mechanisms whereby BCR-ABL induces transformation and blocks apoptosis in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. Single amino acid substitutions in the GRB2 binding site (Y177F), Src homology 2 domain (R552L), or an autophosphorylation site in the tyrosine kinase domain (Y793F) do not diminish the antiapoptotic and transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. A BCR-ABL molecule containing all three mutations (Y177F/R552L/Y793F) exhibits a severe decrease in transforming and antiapoptotic activities compared with the wild-type BCR-ABL protein in 32D myeloid progenitor cells. Ras is activated, the SHC adapter protein is tyrosine phosphorylated and binds GRB2, and myc mRNA levels are increased following expression of all kinase active BCR-ABL proteins with the exception of the Y177F/R552L/Y793F BCR-ABL mutant in 32D cells. We propose that BCR-ABL uses multiple pathways to activate Ras in hematopoietic cells and that this activation is necessary for the transforming and antiapoptotic activities of BCR-ABL. However, Ras activation is not sufficient for BCR-ABL-mediated transformation. A BCR-ABL deletion mutant (delta 176-427) that activates Ras and blocks apoptosis but has severely impaired transforming ability in 32D cells has been identified. These data suggest that BCR-ABL requires additional signaling components to elicit tumorigenic growth which are distinct from those required to block apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed1993

The ABL-BCR fusion gene is expressed in chronic myeloid leukemia.

Although the BCR-ABL hybrid gene on chromosome 22q-plays a pivotal role in the pathogenesis of chronic myeloid leukemia (CML), little is known of the reciprocal chimeric gene, ABL-BCR, formed on chromosome 9q+. By reverse transcription/polymerase chain reaction amplification (RT/PCR) we have detected ABL-BCR mRNA in cells from 31 of 44 BCR-ABL positive CML patients and 3 of 5 CML cell lines. Of the 34 positive samples, 31 had classical t(9;22) (q34;q11) translocations; in 3 samples there was no Philadelphia (Ph) and/or 9q+ chromosomes. ABL-BCR expression consisted of ABL(Ib)-BCR mRNA in 26 patients and of both ABL(Ib)-BCR and ABL(Ia)-BCR mRNA species in 6 patients. The ABL-BCR transcripts encoded one or, more rarely, both of the two potential junctions, designated ABL-b3 and ABL-b4, which differed in size by 75 bp. In 2 patients, the BCR exon b3 was not present in either the BCR-ABL or the corresponding ABL-BCR transcript, whereas in 5 patients exon b3 was present in both transcripts. Direct sequencing of PCR fragments representing the full-length coding sequence of ABL-BCR cDNAs type Ib-b3, Ia-b3, Ib-b4, and Ia-b4 showed an open reading frame predicted to encode fusion proteins of 370 to 414 amino-acids. If an ABL-BCR gene product is produced in CML cells, it may be relevant as a mechanism for deregulating the GTPase activating protein (GAP) function of BCR.

Base Sequence↗

PubMed2003

Bcr-Abl regulates protein kinase Ciota (PKCiota) transcription via an Elk1 site in the PKCiota promoter.

The protein kinase C (PKC) family of serine/threonine kinases plays an important role in numerous cancer signaling pathways, including those downstream of the bcr-abl oncogene. We demonstrated previously that atypical PKCiota is required for Bcr-Abl-mediated resistance of human K562 chronic myelogenous leukemia (CML) cells to Taxol-induced apoptosis. Here, we report that the pattern of PKC isozyme expression characteristic of CML cells is regulated by Bcr-Abl. When Bcr-Abl was expressed in Bcr-Abl-negative HL-60 promyelocytic leukemia cells, expression of the PKCbetaI, PKCbetaII, and PKCiota genes was induced, whereas expression of the PKCdelta gene was reduced to levels similar to those found in CML cells. Given the importance of PKCiota in Bcr-Abl-mediated transformation, we characterized the mechanism by which Bcr-Abl regulates PKCiota expression. A 1200-bp PKCiota promoter construct isolated from genomic DNA was highly active in Bcr-Abl-positive K562 cells and was activated when Bcr-Abl-negative cells were transfected with Bcr-Abl. Bcr-Abl-mediated induction of the PKCiota promoter was dependent upon MEK1/2 activity, but not phosphatidylinositol 3-kinase or p38 MAPK activity. Mutational analysis of the PKCiota promoter revealed a region between 97 and 114 bp upstream of the transcriptional start site that is responsible for Bcr-Abl-mediated regulation. Mutation of a consensus Elk1-binding site within this region abolished Bcr-Abl-mediated regulation. We conclude that Bcr-Abl regulates PKCiota expression through the MEK-dependent activation of an Elk1 element within the proximal PKCiota promoter. Our results indicate that Bcr-Abl-mediated transformation involves transcriptional activation of the PKCiota gene, which in turn is required for Bcr-Abl-mediated chemoresistance.

Cell Transformation, Neoplastic↗

PubMed2005

Disruption of the inhibitor of apoptosis protein survivin sensitizes Bcr-abl-positive cells to STI571-induced apoptosis.

The Bcr-abl oncogene induces hematopoietic cell transformation and protects cells from apoptosis; however, the mechanisms whereby Bcr-abl blocks apoptosis are poorly defined. We examined whether the inhibitor of apoptosis protein (IAP) family, in particular survivin, are regulated by Bcr-abl. Overexpression of Bcr-abl in Mo7e or BaF3 hematopoietic cells elevated survivin mRNA and protein concomitant with a 4-fold increase in survivin promoter activity. The region of the survivin promoter responding to Bcr-abl was narrowed down to a 116 bp fragment between nucleotides -1,194 and -1,078. The IAP family member IAP-like protein-2 was also up-regulated by Bcr-abl. Disruption of Bcr-abl in Bcr-abl-transduced BaF3 cells by small interfering RNA resulted in 3- to 4-fold reduction in survivin protein confirming the link between Bcr-abl and survivin. Survivin disruption in Bcr-abl-transduced Mo7e cells, or in K562 cells that endogenously express Bcr-abl, by transfection with dominant-negative or antisense survivin constructs promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, which was accompanied by caspase-dependent cleavage of Bcr-abl, mitochondrial membrane potential disruption, and enhanced mitochondrial cytochrome c release. Although ectopic survivin protected K562 cells from apoptosis induced by STI571, it did not protect cells from apoptosis induced either by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of TRAIL plus Hemin. Our results identify a new signal pathway downstream of Bcr-abl, in addition to the Bcl-2 family involved in the antiapoptotic effects of Bcr-abl, and suggest that anti-survivin therapy may have utility in patients with chronic myelogenous leukemia.

Animals↗

PubMed2000

The c-Fes protein-tyrosine kinase suppresses cytokine-independent outgrowth of myeloid leukemia cells induced by Bcr-Abl.

The c-Fes protein-tyrosine kinase exhibits strong expression in myeloid hematopoietic cells. Previous studies have shown that Fes induces differentiation in the chronic myelogenous leukemia-derived cell line K-562, suggesting that the Fes signal for differentiation is dominant to the Bcr-Abl signal for transformation in these cells. In addition, Fes has been shown to associate with and phosphorylate Bcr on NH2-terminal sequences retained within Bcr-Abl. To determine whether Fes interacts directly with Bcr-Abl, kinase-inactive Bcr-Abl was coexpressed with Fes in 293T cells, and phosphorylation was assessed by anti-phosphotyrosine immunoblotting. Bcr-Abl was strongly phosphorylated by Fes under these conditions, suggestive of direct interaction. Similarly, tyrosine phosphorylation of kinase-inactive Fes was observed after coexpression with active Bcr-Abl. To test for the interaction of Fes with Bcr-Abl under physiological conditions, wild-type and kinase-defective Fes were stably expressed in the cytokine-dependent myeloid leukemia cell line, DAGM. Expression of either form of Fes alone did not affect the proliferation or interleukin 3 dependence of these cells. The DAGM/Fes cells were then infected with Bcr-Abl retroviruses, and their rates of cytokine-independent outgrowth were compared. Fes dramatically suppressed Bcr-Abl-induced DAGM cell outgrowth relative to a cell line expressing beta-galactosidase as a negative control. This effect required Fes tyrosine kinase activity, because the kinase-inactive form of Fes did not affect Bcr-Abl-induced cell outgrowth. The phosphotyrosine content of both wild-type and kinase-inactive Fes was strongly enhanced after coexpression with Bcr-Abl in DAGM cells, similar to the 293T result. Phosphorylation of wild-type Fes correlated with stimulation of Fes tyrosine kinase activity in the presence of Bcr-Abl. These results show that Fes and Bcr-Abl interact in myeloid cells, leading to Fes activation and suppression of Bcr-Abl-induced conversion to cytokine independence.

Cell Division↗

PubMed2006

Functional and structural requirements for the internalization of distinct BCR-ligand complexes.

Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag-BCR complexes. Previous studies using anti-BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft-mediated signaling are dispensable for BCR-mediated internalization of Ag-specific BCR. Therefore, we investigated the relationship between BCR signaling and endocytosis by defining the role of early kinase signaling in the BCR-mediated internalization of a model Ag (haptenated protein). The results demonstrate that Src kinases and Syk-mediated BCR signaling are not essential for BCR-mediated Ag internalization. Moreover, by comparing Ag and Ab, it was determined that while both localize to clathrin-coated pits, the internalization of Ab-BCR complexes is more susceptible to inhibition of signaling and highly sensitive to disruption of lipid rafts and the actin cytoskeleton compared to Ag-BCR complexes. Thus, these results demonstrate that the nature of the ligand ultimately determines the functional requirements and relative contribution of lipid rafts and other membrane structures to the internalization of BCR-ligand complexes.

Animals↗

PubMed1998

Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/threonine kinase.

Bcr is a novel serine/threonine protein kinase that is believed to require two cysteine pairs for activity (Maru and Witte, Cell, 67, 459, 1991). Tyrosine phosphorylated Bcr has dramatically reduced kinase activity, and tyrosine 360 of Bcr, which is one of the sites of phosphorylation by the Bcr-Abl oncoprotein, is required for transkinase activity (Liu et al., Mol. Cell Biol., 16, 998, 1996). Results presented here indicate that Bcr tyrosine 328 is also phosphorylated within Bcr-Abl expressing cells and is required for Bcr's serine/threonine kinase activity. Bcr Y328F, like Bcr Y360F, had defective transkinase activity but can autophosphorylate. However, the Y328F/Y360F double mutant of Bcr is defective in both trans- and autokinase activities. Taken together with the kinase inhibitory effects of tyrosine phosphorylation of Bcr by Bcr-Abl, our studies with tyrosine to phenylalanine Bcr mutants indicate that the hydroxyl residues of tyrosines 328 and 360 play crucial roles in Bcr's kinase activity.

Amino Acid Sequence↗

PubMed1996

A coiled-coil tetramerization domain of BCR-ABL is essential for the interactions of SH2-containing signal transduction molecules.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (ph1)-positive leukemia. We have previously shown SH2-containing phosphotyrosine phosphatase SHP-2 forms stable complexes with BCR-ABL and Grb2 in BCR-ABL transformed cells (T., Tauchi, et al. J. Biol. Chem. 269, 15381, 1994). To elucidate the structural requirement of BCR-ABL for the interactions with SH2-containing signaling molecules, we examined a series of BCR-ABL mutants which include the Grb2 binding site deleted BCR-ABL (1-63 BCR/ABL), the tetramerization domain deleted BCR-ABL (64-509 BCR/ABL), and the SH2 domain deleted BCR-ABL (BCR/ABL delta SH2). These BCR-ABL mutants were previously shown to reduce the transforming activity in fibroblasts. We found that the tetramerization domain deleted BCR-ABL did not induce the tyrosine phosphorylation of SHP-2 and the interactions of BCR-ABL, SHP-2, and Grb2. In vitro kinase assays have also shown the tetramerization domain deleted BCR-ABL mutant did not phosphorylate GST-SHP-2 in vitro. SHP-2 was co-immunoprecipitated with P13Kinase in BCR/ABL p210 transformed cells, however this interaction was not observed in the tetramerization domain deleted BCR-ABL mutant. Therefore the tetramerization domain of BCR-ABL is essential for interactions of these downstream molecules.

Fusion Proteins, bcr-abl↗

PubMed2001

Involvement of Jak2 tyrosine phosphorylation in Bcr-Abl transformation.

We have previously reported that the Jak2 tyrosine kinase but not Jak1 is tyrosine phosphorylated in the absence of IL-3 in Bcr-Abl positive M3.16 cells, which are rendered IL-3 independent by BCR-ABL gene expression. We have explored the involvement of Jak2 tyrosine phosphorylation in Bcr-Abl oncogenic effects. Our results indicate that Jak2 became tyrosine-phosphorylated in a number of cell lines expressing Bcr-Abl, when maintained in medium lacking IL-3, whereas Bcr-Abl negative cells lacked Jak2 tyrosine phosphorylation. Jak2 was poorly tyrosine-phosphorylated in cells expressing the SH2 deletion mutant of Bcr-Abl compared to either wild-type Bcr-Abl or its SH3 deletion mutant. Moreover, tyrosine phosphorylation of Jak2 by Bcr-Abl was inhibited by the Abl tyrosine kinase inhibitor, STI 571, in a dose-dependent manner. This inhibition of Bcr-Abl kinase by the drug did not interfere with the ability of Jak2 and Bcr-Abl to form a complex. Studies with deletion mutants of Bcr-Abl indicated that the C-terminal domain of Abl within Bcr-Abl was involved in complex formation with Jak2. Similarly, GST-Abl pull-down assays confirmed the strong binding to Jak2 by the C-terminus of Abl. Jak2 peptide substrate studies indicated that the Bcr-Abl and Abl tyrosine kinases specifically phosphorylated Y1007 of Jak2 but only poorly phosphorylated Y1008. Phosphorylation of Y1007 of Jak2 is known to be critical for its tyrosine kinase activation. Tyrosine residue 1007 of Jak2 was phosphorylated in 32Dp210 cells as measured by Western blotting with a phosphotyrosine 1007 sequence-specific antibody. A kinase-inactive Jak2 mutant blocked the colony forming ability of K562 cells. Tumor formation of K562 cells in nude mice was similarly inhibited by this kinase-inactive Jak2 mutant. This inhibition was independent of Stat5 tyrosine phosphorylation. Furthermore, tyrosine-phosphorylated Jak2 was detected in blood cells from CML patients in blast crisis but not in a normal marrow sample. In summary, these findings provide strong evidence that the Jak2 tyrosine kinase is a critical factor in Bcr-Abl malignant transformation.

Animals↗

PubMed1997

A coiled-coil tetramerization domain of BCR-ABL is essential for the interactions of SH2-containing signal transduction molecules.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemia. We have previously shown SH2-containing phosphotyrosine phosphatase SHP-2 forms stable complexes with BCR-ABL and Grb2 in BCR-ABL-transformed cells (Tauchi, T., Feng, G. S., Shen, R., Song, H. Y., Donner, D., Pawson, T., and Broxmeyer, H. E. (1994) J. Biol. Chem. 269, 15381-15387). To elucidate the structural requirement of BCR-ABL for the interactions with SH2-containing signaling molecules, we examined a series of BCR-ABL mutants which include the Grb2 binding site-deleted BCR-ABL (1-63 BCR/ABL), the tetramerization domain-deleted BCR-ABL (64-509 BCR/ABL), and the SH2 domain-deleted BCR-ABL (BCR/ABL deltaSH2). These BCR-ABL mutants were previously shown to reduce the transforming activity in fibroblasts. We found that the tetramerization domain-deleted BCR-ABL did not induce the tyrosine phosphorylation of SHP-2 and the interactions of BCR-ABL, SHP-2, and Grb2. In vitro kinase assays have also shown that the tetramerization domain-deleted BCR-ABL mutant did not phosphorylate GST-SHP-2 in vitro. SHP-2 was co-immunoprecipitated with phosphatidylinositol 3-kinase in BCR/ABL p210-transformed cells; however, this interaction was not observed in the tetramerization domain-deleted BCR-ABL mutant. Therefore the tetramerization domain of BCR-ABL is essential for interactions of these downstream molecules.

Cell Line↗

PubMed2001

p160 Bcr mediates platelet-derived growth factor activation of extracellular signal-regulated kinase in vascular smooth muscle cells.

BACKGROUND: 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). METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Animals↗

PubMed1989

Characterization of bcr gene products in hematopoietic cells.

The bcr gene plays a critical role in the pathogenesis of two human leukemias associated with the Philadelphia chromosome: chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). In both instances a chimeric gene, formed between bcr and the abl protooncogene, results in expression of fused bcr-abl proteins with tyrosine kinase activity. There is controversy regarding the normal gene products of bcr. We investigated this problem by several techniques and found proteins of 190/185, 155, 135, 125, 108, 83 and 47 kDa in several human cell lines by immunoprecipitation with two distinct site-directed anti-bcr antibodies termed anti-bcr(738-753) and anti-bcr(898-911). The 190/185, 155, 125 and 108 kDa proteins were consistently detected by anti-bcr(898-911) antibodies by immunoblotting. Antibodies pre-reacted with excess bcr peptide did not detect these proteins. These proteins were labeled with [32P]orthophosphate in vivo and in vitro by [gamma 32P]ATP in immune complex kinase assays performed with anti-bcr antibodies indicating that these proteins are phosphorproteins. Following labeling in kinase assays, phosphoamino acid analyses detected both phosphoserine and phosphothreonine. In structural studies using one dimensional peptide maps derived by partial V8 protease treatment, the 185, 155, 135, 125 and 108 kDa proteins shared several peptide fragments but contained unique fragments as well. Similarly 2-dimensional maps of proteins labeled in the kinase assay exhaustively digested with trypsin, revealed homology between the 155, 135, 125, 108, and 83 kDa proteins. bcr proteins sedimented in glycerol gradients as putative complexes detected in the cytoplasm of the cell. A 47 kDa protein as well as the recently identified Ph-P53 protein appeared to be associated with bcr proteins based on their co-sedimentation in glycerol gradients and co-immunoprecipitation with several different anti-bcr antibodies. None of the proteins exhibited a precursor-product relationship in pulse-chase experiments conducted with [35S]methionine. We conclude that human cells express several different bcr gene products ranging in size from 190 to 83 kDa, and that these proteins can form specific intracellular cytoplasmic complexes with other cellular proteins.

Adenosine Triphosphate↗

PubMed2003

The interaction of the Bcr-Abl tyrosine kinase with the Src kinase Hck is mediated by multiple binding domains.

Bcr-Abl is found in more than 95% of cases with CML. The mechanism of Bcr-Abl-induced transformation is not fully understood. Bcr-Abl is a constitutively active tyrosine kinase with transforming capacity for hematopoietic cells. We demonstrated recently that the Src kinase Hck interacts directly with Bcr-Abl by a kinase-independent mechanism. Moreover, the inhibition of the Hck kinase seems to block some of the transforming effects of Bcr-Abl. To identify the binding domains mediating this interaction of Hck with Bcr-Abl, we co-expressed different plasmid and baculovirus vectors containing mutants or single domains of Bcr-Abl and/or Hck in COS7 and Sf9 cells. At least four independent binding regions for Hck were identified in Bcr-Abl, one in Bcr, one in the region comprising the SH3 and SH2 domain of Abl, one in the SH1 domain of Abl, and one in the C-terminal domain of Abl. In the Hck kinase, deletion of the SH2 and/or the SH3 region abolished binding to Bcr-Abl. In contrast, deletion of the Hck SH1 domain enhanced binding of Hck to Abl and Bcr-Abl. In conclusion, the results indicate that the interaction of Bcr-Abl with Hck is mediated by a novel, complex mechanism that involves multiple domains of Bcr-Abl and the SH2 and SH3 domains of Hck.

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