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)

Src-homology region 2 (SH2) domains, by binding to tyrosine-phosphorylated sequences, mediate specific protein-protein interactions important in diverse signal transduction pathways. Previous studies have shown that activated forms of the Abl tyrosine kinase, including P210BCR/ABL of human chronic myelogenous leukemia, require the SH2 domain for the transformation of fibroblasts. To determine whether SH2 is also required for Bcr/Abl to transform hematopoietic cells, we have studied two SH2 domain mutations in P210BCR/ABL: a point mutation in the conserved FLVRES motif (P210/R1033K), which interferes with phosphotyrosine-binding by SH2, and a complete deletion of SH2 (P210/delta SH2). Despite a negative effect on intrinsic Abl kinase activity, both P210 SH2 mutants were still able to transform the hematopoietic factor-dependent cell lines Ba/F3 and FDC-P1 to growth factor independence. Unexpectedly, both mutants showed greater transforming activity than wild-type P210 in a quantitative transformation assay, probably as a consequence of increased stability of the SH2 mutant proteins in vivo. Cells transformed by both P210 SH2 mutants were leukemogenic in synaptic mice and P210/r1053K mice exhibited a distinct disease phenotype, reminiscent of that induced by v-Abl. These results demonstrate that while the Abl SH2 domain is essential for BCR/ABL transformation of fibroblasts, it is dispensable for the transformation of hematopoietic factor-dependent cell lines.
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PMID:The SH2 domain of P210BCR/ABL is not required for the transformation of hematopoietic factor-dependent cells. 757 59

The proliferation of normal hematopoietic cells is strictly factor dependent, while leukemic cell lines and primary leukemic cells are frequently factor independent. Although autocrine growth stimulation of human leukemias is occasionally observed in vitro, it is possible that mutations of signal-transduction or cell-cycle control genes may also be important in the development of factor independence. We have previously shown that the proto-oncogene Raf-1, a 70-kd serine/threonine protein kinase, is rapidly phosphorylated and activated by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and Steel factor and is likely to be an important intermediate in mitogenic signal transduction pathways in hematopoietic cells. In an effort to better understand the possible role of abnormal signal transduction in the development of factor independence, we compared the state of phosphorylation and associated kinase activity of Raf-1 between a series of factor-dependent human and murine-myeloid normal cells or cell lines and a series of factor-independent myeloid cell lines. In factor-dependent myeloid cells (normal neutrophils; monocytes; and the cell lines MO7, 32Dc13, and FDC-P1), Raf-1 phosphorylation and associated kinase activity was strictly regulated by the supply of growth factor. In contrast, each of eight factor-independent leukemic cell lines examined, HL-60, KG-1, K562, U937, JOSK-S, JOSK-M, JOSK-K, and JOSK-I, expressed hyperphosphorylated Raf-1 with increased Raf-1 associated kinase activity in the absence of growth factor addition. To further explore the relationship of Raf-1 to factor-independent growth, factor-independent sublines were derived from two factor-dependent cell lines, MO7 and FDC-P1, by culture in CSF-deprived medium. Also, several factor-independent sublines were derived by transfection of a cDNA encoding p210BCR/ABL into three different cell lines: MO7, 32Dc13, and FDC-P1. In each case, the new sublines expressed constitutively hyperphosphorylated and activated Raf-1. The correlation of hyperphosphorylation of Raf-1 with factor independence was also observed with primary acute myeloblastic leukemia cells. The rate of "spontaneous" proliferation of primary acute myeloblastic leukemia (AML) cells in vitro correlated with the extent of Raf-1 phosphorylation. These results suggest that the evolution of myeloid leukemic cells to factor independence is associated with phosphorylation and activation of Raf-1, implicating Raf-1 and signal transduction pathways which activate RAf-1 in this process.
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PMID:Factor independence of human myeloid leukemia cell lines is associated with increased phosphorylation of the proto-oncogene Raf-1. 792 78

It has been described that interleukin 3 (IL3) activates JAK2, which in turn stimulates STAT5 activation. We found, however, that IL3 induces tyrosine-phosphorylation of Tyk2 as well as JAK2 in IL3-dependent mouse cell lines, FDC-P2 and Ba/F3. Furthermore, we found that IL3 induces activation of not only STAT5 but also STAT1 and STAT3. Taken together with other observations, these results indicate that IL3, erythropoietin and thrombopoietin share a common JAK-STAT signaling pathway.
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PMID:Interleukin 3 activates not only JAK2 and STAT5, but also Tyk2, STAT1, and STAT3. 863 39

The development of an efficient immune response depends on the capacity of antigen-specific lymphocytes to migrate into secondary lymphoid organs. The first step in the process of lymphocyte extravasation involves lymphocyte binding to the vascular endothelium. Although several adhesion receptors have been implicated in the migration of lymphocytes to inflamed tissue, their role in the extravasation of these cells to normal lymphoid organs is not yet clearly established. The involvement of adhesion molecules in lymphocyte entrance to secondary lymphoid organs can be better assessed in an in vitro system using endothelial cells in culture. Here we report on the isolation and culture of a homogeneous population of adherent cells of endothelial origin derived from human tonsils (TEC) and on adhesion studies performed with these cells. Beginning from primary cultures of human tonsils, we isolated a population of cells that we show by FACScan analysis to present the intracellular endothelial cell marker Von Willebrand factor and LVAP-2, a surface molecule present in venules from lymphoid organs. The cells are negative for FDC, IDC and macrophage markers. They express ICAM-1, VCAM-1 and CD40 both constitutively and in inducible forms and are induced by IFN-gamma to express major histocompatibility complex class II antigens. As opposed to endothelial cells from human umbilical cord (HUVEC), they do not need to be activated by cytokines to bind lymphoid cells via VLA-4. The mAb HP2/1 directed to the integrin VLA-4 blocks adhesion of Ramos and Daudi cells to tumor necrosis factor alpha (TNF-alpha)-treated HUVEC and to untreated TEC but not of tonsil-derived MNC. On the other hand, an anti-VCAM-1 antibody that blocks adhesion of Ramos and Daudi cells to TNF-alpha-treated HUVEC, does not block adhesion of these cells to TEC, suggesting the presence on the tonsillar endothelial cells of a ligand for VLA-4 different from VCAM-1. We show here that this ligand is not fibronectin.
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PMID:Lymphocyte adhesion to endothelium derived from human lymphoid tissue. 873 20

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.
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PMID:The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation. 921 89

Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form. Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity. The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration. Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested. Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR- and GHR-ECDs. The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished. Both analogues exhibited antagonistic activity in the latter cells. To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization.
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PMID:Novel recombinant analogues of bovine placental lactogen. G133K and G133R provide a tool to understand the difference between the action of prolactin and growth hormone receptors. 963 58

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (hbetac). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hbetac. We report here a comprehensive screen of the entire hbetac molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hbetac that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbetac mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbetac activation, dissociate hbetac tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbetac.
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PMID:Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphorylation. 973 Oct 57

The human growth hormone (hGH)-receptor interaction was used to study the relationship between hormone-receptor affinity and bioactivity. hGH has two nonequivalent sites, called site 1 and site 2, that bind two molecules of receptor in a sequential fashion. We produced both site 1 and site 2 high-affinity hGH variants either by combining alanine mutants previously found to improve affinity at site 1 or by random mutagenesis of residues in site 2 followed by phage display and receptor binding selections. The two high-affinity variants, as well as one which combined them, were used in cell proliferation assays with FDC-P1 cells expressing the hGH receptor. Interestingly, none of these variants produced a change in the EC50 for cell proliferation or the levels of JAK2 tyrosine kinase phosphorylation. Next we studied the effect of a reduction in site 1 affinity on cell proliferation. A systematic series of hGH mutants were produced in which affinity for site 1 was reduced from 5- to 500-fold. Surprisingly, the EC50 for cell proliferation was unaffected until affinity was reduced about 30-fold from wild-type hGH. Thus, native hGH-receptor affinity is much higher than it needs to be for maximal JAK2 phosphorylation or cell proliferation. These studies begin to define basic functional tolerances for receptor activation that need to be considered in the design of hGH mimics.
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PMID:Growth hormone binding affinity for its receptor surpasses the requirements for cellular activity. 989 Aug 85

Activation of the SRC family of protein tyrosine kinases is an important component of intracellular signaling in hematopoiesis, but their critical substrates are less well understood. In this report, we describe the cloning and functional characterization of murine SKAP55R (mSKAP55R), an SRC family kinase substrate. Expression of mSKAP55R was examined by Northern blot. Phosphorylation of mSKAP55R was examined by transient transfection of COS cells. For overexpression studies, mSKAP55R was cloned into a bicistronic murine stem cell virus-based retrovirus. Transduced cells (FDC-P1 cell line and murine bone marrow) were FACS isolated by expression of the selectable marker green fluorescent protein.mSKAP55R showed 90% amino acid identity to the recently published human SKAP55R. mSKAP55R contained a central pleckstrin homology domain, a C-terminal SH3 domain, and a putative SRC kinase consensus substrate DEIY(260). mSKAP55R was expressed in all hematopoietic lineages, with relative mRNA levels greatest in cells of the myeloid and erythroid lineages. Induced myeloid differentiation of M1 and HL-60 cell lines was associated with an eight-fold increase in mSKAP55R mRNA. Transient expression of mSKAP55R in COS cells demonstrated that tyrosine 260 was the predominant site of phosphorylation by FYN kinase. Furthermore, this phosphotyrosine was essential for coimmunoprecipitation of FYN with mSKAP55R. Enforced expression of mSKAP55R inhibited in vitro growth of the myeloid FDC-P1 cell line and primary hematopoietic progenitors. In contrast, a tyrosine 260 mutant mSKAP55R had no effect on in vitro growth. These studies implicate mSKAP55R in the processes of myeloid differentiation and growth arrest.
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PMID:Adaptor protein SKAP55R is associated with myeloid differentiation and growth arrest. 1106 73

STI571 (formerly CGP57148) and AG957 are small molecule inhibitors of the protein tyrosine kinase (PTK) p145(abl) and its oncogenic derivative p210(bcr-abl). AG490 is an inhibitor of the PTK Janus kinase 2 (JAK2). No direct comparison of these inhibitors has previously been reported, so this study compared their effects on factor-dependent FDC-P1, 32D, and MO7e cells and their p210(bcr-abl)-expressing factor-independent derivatives. STI571 was a more potent inhibitor of (3)H-thymidine incorporation in p210(bcr-abl)-expressing cells than was AG957, and it showed superior discrimination between inhibitory effects on parental cell lines and effects on their p210(bcr-abl)-expressing derivatives. Assays performed with and without growth factor demonstrated that STI571 but not AG957 reversed the p210(bcr-abl)-driven factor independence of cell lines. p210(bcr-abl)-expressing cells were less sensitive to AG490 than to AG957 or STI571. However, for p210(bcr-abl)-expressing clones from all 3 cell lines, synergistic inhibition was demonstrated between STI571 and concentrations of AG490 with no independent inhibitory effect. Inhibition of nucleic acid synthesis with AG957 treatment was associated with reduced cell numbers, reduced viability, and small pyknotic apoptotic cells. At concentrations of STI571 that reversed the p210(bcr-abl) factor-independent phenotype, STI571 treatment and growth factor deprivation together were sufficient to induce apoptosis. This study concludes that, for the cell lines studied, (1) STI571 is a more potent and more selective inhibitor of a p210(bcr-abl)-dependent phenotype than AG957; (2) AG490 synergizes with STI571 to enhance its inhibitory effect on p210(bcr-abl)-driven proliferation; and (3) the combination of p210(bcr-abl)-tyrosine kinase inhibition and growth factor signal withdrawal can be sufficient to induce apoptotic death of transformed cells. (Blood. 2001;97:2008-2015)
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PMID:Comparison of effects of the tyrosine kinase inhibitors AG957, AG490, and STI571 on BCR-ABL--expressing cells, demonstrating synergy between AG490 and STI571. 1126 65


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