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)

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.
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PMID:Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator. 158 85

Gel retardation and DNAase 1 footprinting experiments have been performed to characterize the promoter sequences of exon 1a and 1b of the human ABL gene. Several Sp1 motifs and CCAAT boxes are found to be protected by nuclear proteins in the 1b promoter but none of the 7 reported Sp1 sites in 1a were found to bind protein. Multiple sets of initiation sites seem to exist in the 1b promoter region which may represent individual initiation sites, distributed over a DNA region of up to 700 bp. Starting with the most distal initiation site, 1a and 1b ABL promoter sequences show a high degree of homology, suggesting that one is derived from the other. However, multiple evolutionary changes in the 1a promoter sequence indicate that type 1a ABL expression may be differently regulated than 1b.
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PMID:Characterization of the human ABL promoter regions. 216 52

The chronic myelogenous leukemia-associated P210 BCR-ABL oncogene protein product has been produced using the baculovirus expression system. High-level expression of the P210 BCR-ABL protein required the removal of GC rich 5' non-coding sequences. P210 BCR-ABL synthesized in insect cells is an active tyrosine protein kinase indistinguishable from P210 BCR-ABL isolated from human cells. Both proteins utilize angiotensin II as a phosphate acceptor in vitro with a Km for ATP of approximately 1.5 microM. P210 BCR-ABL produced in insect cells undergoes autophosphorylation in vitro and in vivo. Gel filtration of P210 BCR-ABL reveals that the protein elutes as a high molecular weight complex of about 800 kD. Approximately 4 to 5 mg of P210 BCR-ABL is produced in one liter of infected insect cells. Following cell disruption and a three-step ion exchange and gel filtration purification procedure, 0.4 mg of soluble P210 BCR-ABL is obtained per liter of suspension culture. An alternative procedure employing detergent extraction and immunoaffinity chromatography gave higher yields and purity from smaller amounts of infected cell extracts. The availability of intact, soluble and enzymatically active P210 BCR-ABL represents a significant advance for studying the biochemical and biophysical properties of the ABL oncogene family of proteins.
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PMID:Baculovirus expression of functional P210 BCR-ABL oncogene product. 249 63

The presence of amidases cleaving the tripeptide VAL.LEU.ARG.pNA, and liberating from human plasma kininogen substance(s) able to contract uterine smooth muscle and to lower blood pressure (uterus contracting and hypotensive activity), has been demonstrated in vascular and muscle tissues from normally perfused and ischaemic areas of dog hearts. Studies were carried out on blood free preparations of coronary arteries and veins and normally perfused and ischaemic ventricle. All the tissues were found to contain both acid optimum (pH 6) and alkaline optimum (pH greater than 9) enzymes forming uterus contracting substance (UCS, bioassayed on isolated uterus of rats in oestrus), the highest levels of both activities being found in arterial tissues and the least in ventricle. Enzyme levels in ischaemic or normally perfused ventricle did not differ significantly. Gel filtration (Sephacryl, S-300) of coronary artery extracts gave one peak each of acid optimum enzyme with a molecular weight of 38,300 +/- 800 daltons and alkaline optimum enzyme with a molecular weight of 92,100 +/- 4000 daltons. Both acid and alkaline enzyme fractions cleaved the tripeptide substrate with pH optima identical to those for UCS formation. The acid optimum activity, both of UCS formation and tripeptide cleavage, was inhibited by pepstatin but not by aprotinin or soybean trypsin inhibitor (SBTI). The alkaline optimum activity was inhibited by aprotinin and SBTI but not pepstatin. Both acid and alkaline optimum enzymes released a hypotensive agent from a plasma protein substrate. The molecular weight and response to inhibitors of the acid optimum enzyme were similar to a cathepsin, and those of the alkaline optimum enzyme were similar to plasma kallikrein.
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PMID:Enzymes in normally perfused and ischaemic dog hearts which release a substance with kinin like activity. 277 62

Thrombopoietin (TPO) is a growth and differentiation factor for megakaryocyte-lineage cells. The receptor for TPO, c-MPL, is a member of the hematopoietic cytokine receptor family and has previously been shown to rapidly activate one or more cytoplasmic tyrosine kinases after ligand binding. In this study, we found that activation of the TPO receptor rapidly induced tyrosine phosphorylation of two members of the Jak tyrosine kinase family, JAK2 and TYK2, but not JAK1 or JAK3, in two different factor-dependent hematopoietic cell lines. The activation of both JAK2 and TYK2 was dose- and time-dependent and was associated with rapid tyrosine phosphorylation of a series of STAT proteins including STAT1, STAT3, and STAT5. Gel-shift assays indicated that one or more of these STATs is likely to participate in the formation of specific DNA-binding complexes. The activation of tyrosine kinases and signal propagation through tyrosine phosphorylation are likely to represent important initial steps in mediating the activities of TPO in myeloid cells.
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PMID:The thrombopoietin receptor c-MPL activates JAK2 and TYK2 tyrosine kinases. 754 16

Ferritin, the major intracellular iron storage protein of eucaryotic cells, is regulated during inflammation and malignancy. We previously reported that transcription of the H subunit of ferritin (ferritin H) is negatively regulated by the adenovirus E1A oncogene in mouse NIH 3T3 fibroblasts (Y. Tsuji, E. Kwak, T. Saika, S. V. Torti, and F. M. Torti, J. Biol. Chem. 268:7270-7275, 1993). To elucidate the mechanism of transcriptional repression of the ferritin H gene by E1A, a series of deletions in the 5' flanking region of the mouse ferritin H gene were constructed, fused to the chloramphenicol acetyltransferase (CAT) gene, and transiently cotransfected into NIH 3T3 cells with an E1A expression plasmid. The results indicate that the E1A-responsive region is located approximately 4.1 kb 5' to the transcription initiation site of the ferritin H gene. Further analyses revealed that a 37-bp region, termed FER-1, is the target of E1A-mediated repression. This region also serves as an enhancer, augmenting ferritin H transcription independently of position and orientation. FER-1 was dissected into two component elements, i.e., a 22-bp dyad symmetry element and a 7-bp AP1-like sequence. Insertion of these DNA sequences into a ferritin H-CAT chimeric gene lacking an E1A-responsive region indicated that (i) the 22-bp dyad symmetry sequence by itself has no enhancer activity, (ii) the AP1-like sequence has moderate enhancer activity which is repressed by E1A, and (iii) the combination of the dyad symmetry element and the AP1-like sequence is required for maximal enhancer activity and repression by E1A. Gel retardation assays and cotransfection experiments with c-fos and c-jun expression vectors suggested that members of the Fos and Jun families bind to the AP1-like element of FER-1 and contribute to its regulation. In addition, gel retardation assays showed that E1A reduces the ability of nuclear proteins to bind to the AP1-like sequence without affecting the levels of nuclear factors that recognize the 22-bp dyad symmetry element. Taken together, these results demonstrate that FER-1 serves as both an enhancer of ferritin H transcription and a target for E1A-mediated repression.
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PMID:FER-1, an enhancer of the ferritin H gene and a target of E1A-mediated transcriptional repression. 765 32

Interleukin-9 (IL-9) is a T-cell-derived multifunctional cytokine that can stimulate the proliferation of a human megakaryocytic leukemia cell line, MO7E. Previous studies suggested that protein tyrosine phosphorylation may be involved in IL-9 signaling pathways. However, tyrosine kinases activated by IL-9 have not been identified. In this report we show that IL-9 induces tyrosine phosphorylation and activation of the JAK family tyrosine kinases including JAK1, JAK3, and Tyk2. The kinetic studies indicate that tyrosine phosphorylation and activation of JAK kinases induced by IL-9 occurred within 1 minute, peaked by 5 to 10 minutes, and persisted at least for 45 minutes. Furthermore, we show that signal transducers and activators of transcription (Stat) 91 or related protein and an 88-kD Stat 91-associated protein are rapidly tyrosine phosphorylated following IL-9 treatment. Gel shift assays confirm that nuclear extracts from MO7E cells stimulated with IL-9 specifically interact with a DNA element termed gamma activated site. These results suggest that actions of IL-9 may, in part, be mediated through JAK kinase-Stat signaling cascades.
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PMID:Tyrosine phosphorylation and activation of JAK family tyrosine kinases by interleukin-9 in MO7E cells. 775 43

A reciprocal translocation between chromosomes 9 and 22 creates the Philadelphia (Ph1) chromosome in chronic myelogenous leukemia. This translocation results in the fusion of the ABL and the BCR genes to form a BCR/ABL fusion gene, the product of which has a greatly increased protein tyrosine kinase activity in comparison with the normal ABL protein. The chromosome 22 translocation breakpoints are concentrated within a 5.8-kilobase region named the major break-point cluster region (Mbcr). Gel mobility shift and DNase I footprinting assays have defined binding sites for three proteins, BIF 1-3 (BCR intron factors 1-3), lying within a 427-base pair fragment of the Mbcr. This 427-base pair fragment functions as a transcriptional silencer with both the BCR as well as a heterologous promoter. The silencing is position- and orientation-independent. The transcriptional effects are greatest in chronic myelogenous leukemia cells, decreased in HeLa and B-cells, and absent in T-lymphocytes. Gel mobility shift assays show a corresponding difference in pattern when the T-lymphocyte nuclear extract is compared with other cell lines. The Mbcr appears to contain a novel group of transcriptional silencers that share a common binding motif with a recently described suppressor in the mouse Adh-1 gene.
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PMID:A novel transcriptional suppressor located within a downstream intron of the BCR gene. 814 70

IL-2-, IL-12-, and IFN-alpha-mediated signaling pathways were analyzed in primary NK cells and in the NK3.3 cell line. Gel mobility shift and immunoprecipitation analyses revealed that in addition to activating STAT3 (signal transducer and activator of transcription-3) and STAT5, IL-2 induced tyrosine and serine phosphorylation of STAT1 alpha, which formed IFN-gamma-activated sequence-binding complexes by itself and with STAT3. Although IL-2 and IFN-alpha activated STAT1 alpha and STAT5, IL-2 predominantly activated STAT5, while IFN-alpha predominantly activated STAT1 alpha. IL-2 induced less STAT1 alpha activation and IFN-alpha induced greater STAT5 activation in NK3.3 cells compared with preactivated primary NK cells. In NK3.3 cells, IL-2 induced comparable formation of c-fos promoter sis-inducible element IFN-gamma-activated sequence-binding complexes containing STAT3 alone with complexes containing STAT3 and STAT1 alpha, while in preactivated primary NK cells, it preferentially induced complexes containing STAT3 and STAT1 alpha. Thus, signaling in NK3.3 cells is not always identical with that in primary NK cells. In contrast to IL-2 and IFN-alpha, IL-12 induced strong tyrosine phosphorylation of STAT4 and variable weak phosphorylation of STAT3. However, supershift analyses using the c-fos promoter sis-inducible element probe showed that IL-12 activated STAT4, STAT1 alpha, and STAT3, and induced complexes containing STAT4 only, STAT4 with STAT1 alpha, STAT3 with STAT1 alpha, or STAT1 alpha only in preactivated primary NK cells. STAT1 alpha activation by IL-12 correlated with increased phosphorylation of serine, but not tyrosine. Finally, IL-2 induced tyrosine phosphorylation of JAK1 and JAK3, while IL-12 induced phosphorylation of JAK2 and TYK2 in both preactivated primary NK and NK3.3 cells. Differential phosphorylation and consequent differential activation of both separate and overlapping STAT proteins by IL-2, IL-12, and IFN-alpha may provide a molecular basis for the similarities and differences in the actions of these cytokines on NK cells.
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PMID:Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-alpha. 868 6

Interleukin-7 (IL-7) stimulates the proliferation of normal and leukemic B and T cell precursors and T lymphocytes. Activation of the JAK/STAT pathway has been implicated in IL-7R signaling. We investigated which STAT complexes are formed upon stimulation of B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells with IL-7. Gel retardation assays with STAT-binding oligonucleotides showed that IL-7 induces the formation of two major STAT complexes in BCP-ALL cells. Supershifts with anti-STAT antibodies identified these as STAT1 and STAT5 complexes. This pattern of STAT activation was seen in all BCP-ALL cases that respond to IL-7 in proliferation assays. IL-7 also induced STAT/DNA binding in BCP-ALL cases that failed to proliferate in response to IL-7, suggesting that the ability of IL-7R to activate the JAK/STAT pathway per se is not sufficient for proliferation induction. To determine the contribution of the cytoplasmic domain of the IL-7 receptor alpha chain (IL-7R alpha) to activation of STAT proteins, transfectants of the murine pro-B cell line BAF3 were made that express chimeric receptors consisting of the extracellular domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) and the transmembrane and intracellular domains of human IL-7R alpha. Activation of the chimeric G-CSF-R/IL-7R alpha with G-CSF resulted in a full proliferative response and induced the phosphorylation of JAK1 but not JAK2. Major STAT complexes activated by G-CSF-R/IL-7R alpha contained STAT1 or STAT5, while some formation of STAT3-containing complexes was also seen. These findings establish that STAT1 and STAT5, and possibly STAT3, are activated upon stimulation of precursor B cells with IL-7. The data further indicate that the IL-7R alpha chains are directly involved in the activation of JAKs and STATs and have a major role in proliferative signaling in precursor B cells.
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PMID:Interleukin-7 signaling in human B cell precursor acute lymphoblastic leukemia cells and murine BAF3 cells involves activation of STAT1 and STAT5 mediated via the interleukin-7 receptor alpha chain. 870 37


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