Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)beta receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFbetaR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFbetaR, and TEL/ARG with an IC(50) of approximately 0.5 microM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.
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PMID:ARG tyrosine kinase activity is inhibited by STI571. 1129 Jun 9

Stem cell factor (SCF) is a growth factor that promotes the survival, proliferation, and differentiation of hematopoietic cells. SCF and its receptor, Kit, are normally present in both cell surface and soluble forms. Both forms of Kit can bind SCF. However, the function of soluble Kit is unknown. In order to determine if soluble Kit can modulate SCF activity, we produced a fusion protein, Kit-Fc, comprised of the extracellular domain of murine Kit and the Fc portion of human IgG(1) and investigated its ability to bind 125I-SCF and to inhibit SCF-stimulated hematopoietic colony growth in vitro. Stable cell lines expressing Kit-Fc were generated and Kit-Fc was purified to greater than 95% purity. Scatchard analysis demonstrated that Kit-Fc binds iodinated SCF with high affinity (Kd 570 pM). Kit-Fc also bound to transmembrane SCF displayed on the surface of fibroblasts. The murine mast cell line IC2 was engineered to express murine Kit on the cell surface and was demonstrated to proliferate in the presence of SCF. Kit-Fc completely blocked SCF-stimulated proliferation of IC2-Kit cells, but not IL-3-stimulated growth of IC2-Kit cells, demonstrating the specificity of Kit-Fc. We investigated the ability of Kit-Fc to block SCF-stimulated murine hematopoietic colony growth. Kit-Fc blocked SCF-stimulated erythroid colony growth as effectively as a neutralizing anti-Kit monoclonal antibody, ACK2, but did not block erythropoietin-stimulated erythroid colony growth. Likewise, Kit-Fc blocked SCF-stimulated myeloid colony growth as effectively as ACK2 antibody, but did not block IL-3- or GM-CSF-stimulated myeloid colony growth. These results indicate that a form of soluble Kit binds SCF with high affinity, and can specifically block the ability of SCF to stimulate hematopoietic colony growth, suggesting that one function of soluble Kit may be to modulate SCF bioactivity.
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PMID:Soluble Kit receptor blocks stem cell factor bioactivity in vitro. 1130 Nov 10

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27(Kip1) to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27(Kip1) expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27(Kip1) on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27(Kip1) have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27(Kip1) can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27(Kip1), cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.
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PMID:BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression. 1132 29

The growth factor combination containing early acting cytokines FLT-3 ligand (FL), Stem Cell Factor (SCF) and thrombopoietin (TPO) is able to maintain, for an extended culture period, early stem cells, defined as long-term repopulating NOD/SCID mice (Scid Repopulating Cell-SRC) contained in cord blood (CB). In this culture system, the role of IL-6 and IL-3 has not been clearly established. Using a combination of FL+TPO+SCF with or without IL-6, we were able to form CB CD34+ cells for 30 weeks. The CB CD34+ cells cultured in this system engrafted NOD/SCID mice after 6 weeks of culture; the cells from primary recipients were also able to engraft secondary NOD/SCID mice. When CB CD34+ cells were cultured in the presence of IL-3 in the place of IL-6 we observed an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture. However, more primitive LTC-IC output increased up to week 6 with the growth factor combination containing IL-3 and then decreased and disappeared, while with the growth factor combination with or without IL-6 increased up to week 23. Cells cultured for 4 weeks with the 4-factor combination containing IL-3 engrafted NOD/SCID mice less efficiently. Repopulation of NOD/SCID mice was no longer observed when ex vivo expansion was performed for 6 weeks. This study provides some evidence that no differences could be detected in long-term maintenance and even expansion of human primitive cord blood cells cultured with FL+TPO+SCF in the presence or absence of IL-6. Under the culture conditions employed in this study, the presence of IL-3 reduced the repopulating potential of expanded CB CD34+ cells.
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PMID:Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model. 1138 43

We report a strategy for generating efficient signal transduction with unnatural heterologous receptor combinations. As previously described [Ueda, H., Kawahara, M. et al. (2000) J. Immunol. Methods 241, 159-170], chimeric receptors composed of the V(H)/V(L) domains of anti-hen egg lysozyme antibody HyHEL-10 and N-terminally truncated erythropoietin receptor (EpoR) can be activated by lysozyme. When the cytoplasmic domains of these receptors were substituted with one derived from gp130, IL-3 dependent Ba/F3 cells expressing both V(H)-gp130 and V(L)-gp130 grew dose-dependently when given lysozyme without IL-3. However, cells expressing the heterologous pair of V(H)-gp130 and V(L)-EpoR also showed more efficient and stricter lysozyme-dependent proliferation in the absence of IL-3, indicating this combination is as an efficient and strict signal transducer as wild-type EpoR. The immunoprecipitation data indicated the existence of a preformed V(H)-gp130 and V(L)-EpoR heterodimer in the absence of lysozyme, suggesting the crucial role of a receptor conformational change in signal triggering as well as wild-type EpoR and gp130. Phosphorylation of JAK2, STAT3, and STAT5 was observed upon the addition of lysozyme, suggesting the activation of both EpoR- and gp130-derived signals.
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PMID:A growth signal with an artificially induced erythropoietin receptor-gp130 cytoplasmic domain heterodimer. 1148 Oct 50

Previous studies have indicated a redundancy in the effects of the cytokines, IL-3, IL-5, and nerve growth factor (NGF) on acute priming of human basophils. In the current study, we have examined the effects of these three cytokines on 18-h priming for leukotriene C4 generation, their ability to induce Fc(epsilon)RIbeta mRNA expression, or their ability to sustain basophil viability in culture. We also examine a variety of the signaling steps that accompany activation with these cytokines. In contrast with the ability of IL-3 to alter secretagogue-mediated cytosolic calcium responses following 18-h cultures, 18-h treatment with IL-5 or NGF did not affect C5a-induced leukotriene C4 generation or alter C5a-induced intracellular Ca2+ concentration elevations. IL-3 and IL-5, but not NGF, induced Fc(epsilon)RIbeta mRNA expression and all three improved basophil viability in culture with a ranking of IL-3 > IL-5 > or = NGF. All three cytokines acutely activated the extracellular signal-regulated kinase pathway and the signaling elements that preceded extracellular signal-regulated kinase and cytosolic phospholipase A2 phosphorylation, consistent with their redundant ability to acutely prime basophils. However, only IL-3 and IL-5 induced Janus kinase 2 and STAT5 phosphorylation. This pattern of signal element activation among the three cytokines most closely matched their ability to induce expression of Fc(epsilon)RIbeta mRNA. Induction of the sustained calcium signaling that follows overnight priming with IL-3 appeared to be related to the strength of the early signals activated by these cytokines but the relevant pathway required was not identified. None of the signaling patterns matched the ability of the cytokines to promote basophil survival.
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PMID:Differences in functional consequences and signal transduction induced by IL-3, IL-5, and nerve growth factor in human basophils. 1149 16

The Dbl homology (DH) domain of BCR in P210BCR-ABL (P210/WT) has been thought to have a negative effect on the activation of BCR-ABL because P185BCR-ABL, in which this region is physically deleted, has stronger biochemical and biological activities. To study the role of the DH domain of BCR in the background of P210/WT, the region was replaced with homologous sequences derived from Dbl (P210/Dbl) or CDC24 (P210/CDC24) or with irrelevant sequences from LacZ (P210/LacZ) or luciferase (P210/Luci). Surprisingly, the abilities to transform Rat1 cells or mouse bone marrow cells and induce growth factor independence in interleukin 3-dependent mouse Ba/F3 cells were retained only in P210/Dbl. However, even P210/Dbl could not achieve the wild type level of surviving potential against genotoxins in Rat1 cells and in Ba/F3 cells. Activation of Akt correlated with the biological changes in Rat1 cells but did not correlate with the biological changes in Ba/F3 cells. The DH domain was not tyrosine-phosphorylated in vitro, nor could we find any differences in peptide mapping between in vitro phosphorylated P210/WT and P210/Dbl. Although functions of the DH domain remain to be discovered, we propose that the DH domain makes positive contributions to P210BCR-ABL.
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PMID:The Dbl homology domain of BCR is not a simple spacer in P210BCR-ABL of the Philadelphia chromosome. 1150 48

The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2-3 weeks. A single dose of doxycycline added to the medium (1 microg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60-90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 x 10(6) Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 microg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin- bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.
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PMID:Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette. 1158 26

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.
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PMID:Involvement of Jak2 tyrosine phosphorylation in Bcr-Abl transformation. 1159 27

This report describes 2 patients with a clinical and hematologic diagnosis of chronic myeloid leukemia (CML) in chronic phase who had an acquired t(8;22)(p11;q11). Analysis by fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR) indicated that both patients were negative for the BCR-ABL fusion, but suggested that the BCR gene was disrupted. Further FISH indicated a breakpoint within fibroblast growth factor receptor 1 (FGFR1), the receptor tyrosine kinase that is known to be disrupted in a distinctive myeloproliferative disorder, most commonly by fusion to ZNF198. RT-PCR confirmed the presence in both cases of an in-frame messenger RNA fusion between BCR exon 4 and FGFR1 exon 9. Expression of BCR-FGFR1 in the factor-dependent cell line Ba/F3 resulted in interleukin 3-independent clones that grew at a comparable rate to cells transformed with ZNF198-FGFR1. The growth of transformed cells was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, the farnesyltransferase inhibitors L744832 and manumycin A, the p38 inhibitors SB202190 and SB203580 but not by the MEK inhibitor PD98059. The growth of BaF3/BCR-FGFR1 and BaF3/ZNF198-FGFR1 was not significantly inhibited by treatment with STI571, but was inhibited by SU5402, a compound with inhibitory activity against FGFR1. Inhibition with this compound was associated with decreased phosphorylation of ERK1/2 and BCR-FGFR1 or ZNF198-FGFR1, and was dose dependent with an inhibitory concentration of 50% of approximately 5 microM. As expected, growth of BaF3/BCR-ABL was inhibited by STI571 but not by SU5402. The study demonstrates that the BCR-FGFR1 fusion may occur in patients with apparently typical CML. Patients with constitutively active FGFR1 fusion genes may be amenable to treatment with specific FGFR1 inhibitors.
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PMID:The t(8;22) in chronic myeloid leukemia fuses BCR to FGFR1: transforming activity and specific inhibition of FGFR1 fusion proteins. 1173 86


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