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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incubation of a highly purified bovine spleen protein tyrosine kinase with [gamma-32P]ATP and Mg2+ resulted in a gradual radioactive labeling of the protein kinase (50 kDa) with no change in the protein kinase activity toward angiotensin II. On the other hand, treatment of the protein tyrosine kinase with an immobilized alkaline phosphatase caused essentially complete loss in the kinase activity, which could be restored by incubation of the enzyme with ATP and Mg2+. By using the alkaline phosphatase-treated kinase, time courses of the protein phosphorylation and the enzyme activation were demonstrated to correlate closely. These results indicate that this protein tyrosine kinase relies on autophosphorylation for activity and that the purified enzyme usually exists in a fully phosphorylated state. The radioactive labeling of the purified kinase during incubation with [gamma-32P]ATP resulted from a phosphate exchange reaction: the exchange of [gamma-32P]phosphate of ATP with the protein bound phosphate as previously suggested (Kong, S.K., and Wang, J.H. (1987) J. Biol. Chem. 262, 2597-2603). It could be shown that the autophosphorylation of phosphatase-treated tyrosine kinase was strongly inhibited by the substrate angiotensin II, whereas the exchange reaction carried out with untreated tyrosine kinase was not. Autophosphorylation is suggested to be an intermolecular reaction since its initial rate is proportional to the square of the protein concentration.
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PMID:Autophosphorylation and autoactivation of spleen protein tyrosine kinase. 317 May 53

The regulation of kinase activity associated with insulin receptor by phosphorylation and dephosphorylation has been examined using partially purified receptor immobilized on insulin-agarose. The immobilized receptor preparation exhibits predominately tyrosine but also serine and threonine kinase activities toward insulin receptor beta subunit and exogenous histone. Phosphorylation of the insulin receptor preparation with increasing concentrations of unlabeled ATP, followed by washing to remove the unreacted ATP, results in a progressive activation of the receptor kinase activity when assayed in the presence of histone and [gamma-32P]ATP. A maximal 4-fold activation is achieved by prior incubation of receptor with concentrations of ATP approaching 1 mM. High pressure liquid chromatographic analysis of tryptic hydrolysates of the 32P-labeled insulin receptor beta subunit reveals three domains of phosphorylation (designated peaks 1, 2, and 3). Phosphotyrosine and phosphoserine residues are present in these three domains while peak 2 contains phosphothreonine as well. Thus, at least seven sites are available for phosphorylation on the beta subunit of the insulin receptor. Incubation of the phosphorylated insulin receptor with alkaline phosphatase at 15 degrees C results in the selective dephosphorylation of the phosphotyrosine residues on the beta subunit of the receptor while the phosphoserine and phosphothreonine contents are not affected. The dephosphorylation of the receptor is accompanied by a marked 65% inhibition of the receptor kinase activity. Almost 90% of the decrease in [32P]phosphate content of the receptor after alkaline phosphatase treatment is accounted for by a decrease in phosphotyrosine content in peak 2, while very small decreases are observed in peaks 1 and 3, respectively. These results demonstrate that the extent of phosphorylation of tyrosine residues in receptor domain 2 closely parallels the receptor kinase activity state, suggesting phosphorylation of this domain may play a key role in regulating the insulin receptor tyrosine kinase.
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PMID:Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity. 653 76

Leukocyte alkaline phosphatase (LAP) is synergistically induced by the combination of all-trans retinoic acid (ATRA) and granulocyte-colony-stimulating factor (G-CSF) in acute promyelocytic leukemia (APL) cells (Gianni' M. et al., Blood 83: 1909-1921, 1994). The role of cAMP and tyrosine kinases in the induction of LAP was investigated. In the APL cell line NB4, adenosine-3': 5'-monophosphothioate, cyclic, Rp isomer, a reversible inhibitor of cAMP-dependent protein kinase (PKA), has no effect on the induction of LAP enzymatic activity and mRNA triggered by ATRA+G-CSF, in conditions where this compound completely blocks the upregulation of LAP transcript caused by the combination of the PKA agonist, dibutyryl-cAMP (db-cAMP), and ATRA. Challenge of NB4 cells with G-CSF, dbcAMP and ATRA causes a much higher induction of LAP relative to that observed in the presence of ATRA+G-CSF or ATRA+dbcAMP. Treatment of NB4 with ATRA and G-CSF results in increases in the tyrosine phosphorylation of several proteins. In the presence of the cytokine and the retinoid, tyrosine kinase inhibitors decrease LAP enzymatic activity and mRNA.
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PMID:Tyrosine kinases but not cAMP-dependent protein kinase mediate the induction of leukocyte alkaline phosphatase by granulocyte-colony-stimulating factor and retinoic acid in acute promyelocytic leukemia cells. 753 55

The technique of two-dimensional gel electrophoresis was used for analysis of tyrosine phosphorylated polypeptide substrates after epidermal growth factor (EGF)-induced stimulation of receptor tyrosine kinase activity in a brush border fraction of human placental syncytiotrofoblast cells. After incubation with [gamma 32P]ATP, followed by autoradiography of the gels, 35 phosphorylated components were detected, of which 8 were strongly tyrosine phosphorylated by EGF. Using a more sensitive assay with phosphotyrosine-specific antibody, an additional 12 polypeptide components were found to be strongly tyrosine phosphorylated by EGF. A number of the phosphorylated substrates could be aligned with components in a protein catalog of the human brush border membrane fraction that was characterized by glycoprotein staining, Triton X-114 fractionation, immunoreaction with specific antibodies, and comigration with 35S-labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well-recognized substrates (calpactin II, ezrin, EGF receptor) included beta-tubulin and serum albumin, while other cytoskeletal proteins and alkaline phosphatase were excluded as substrates. A notable feature of the catalog was that a number of glycoproteins were present in both the membrane and cytoskeletal fraction, suggesting involvement in membrane/cytoskeletal interactions. The data demonstrate the feasibility of using two-dimensional gel electrophoresis in a global way to identify target substrates for tyrosine kinase activity. In addition they suggest that many of these are located in the vicinity of tyrosine kinase at the membrane/cytoskeletal border at a location which is probably involved, at the molecular level, in morphological changes of the plasma membrane associated with cell proliferation.
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PMID:Application of two-dimensional gel analysis to identification and characterization of tyrosine phosphorylated substrates for growth factor receptors. 768 72

The inhibitory glycine receptor (GlyR) is composed of polypeptide subunits that contain intracellular consensus sequences for phosphorylation by protein kinase C (PKC). During whole-cell recording from rat hippocampal neurones, we observed a time-dependent increase of the glycine-induced membrane current. After 22 min the amplitude was 260 + 13% of the initial control response. PKC was involved in the modulation of hippocampal glycine receptors, since the observed effect was more prominent when the phorbol ester PMA, an activator of PKC, was included in the patch pipette. The action of PMA was mimicked by applying the '5-HT2 receptor agonist, alpha-methyl-serotonin, to the cells. The time-dependent increase in glycine responses was reduced by either tamoxifen, an inhibitor of PKC, or by alkaline phosphatase. Protein kinase A and tyrosine kinase were not involved as modulatory drugs of these kinases had no effect. These results provide direct evidence for the regulation of GlyR function by PKC in rat hippocampal neurones.
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PMID:Modulation of hippocampal glycine receptor channels by protein kinase C. 775 15

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple immunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in 32Pi incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.
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PMID:Epidermal growth factor inhibits gap junctional communication and stimulates serine-phosphorylation of connexin43 in WB cells by a protein kinase C-independent mechanism. 808 76

The ligand-stimulated tyrosine kinase activity of the normal human epidermal growth factor (EGF) receptor and a truncated EGF receptor lacking 164 carboxy-terminal (C-terminal) amino acids was examined in intact cells and after Triton X-100 extraction into Triton-soluble and -insoluble (cytoskeletal) preparations. Detergent extraction of the intact and truncated receptors appeared complete using 0.3% Triton as demonstrated by anti-EGF receptor immunoblots, tyrosine kinase assays, and marker enzyme (alkaline phosphatase) solubilization. Higher Triton concentrations yielded no additional EGF receptor extraction and began to inhibit EGF-stimulated kinase activity toward angiotensin II (AII). Furthermore, the tyrosine kinase activity of the truncated EGF receptor exhibited increased sensitivity to Triton extraction, suggesting a lower affinity or a more labile association of this receptor with the cytoskeleton. However, both EGF receptor forms had altered catalytic activity when associated with the cytoskeletal fraction, as evidenced by the increased phosphorylation of the exogenous substrates: AII, src-peptide, and [Val5]AII. Kinetic analyses of both receptor types revealed that the cytoskeletal fractions obtained using 0.3% Triton contain EGF receptor activity that exhibits a Michaelis-Menten constant (Km) for AII that is 2- to 3-fold more favorable than that calculated for the soluble receptor forms. EGF treatment of intact cells containing either the intact or truncated receptor revealed similar phosphorylated proteins in the soluble fraction of both cell types, although there was evidence for the enhanced phosphorylation of certain proteins (e.g. 115 and 50 kilodalton proteins) in cells containing the truncated receptor. There was also a greater number of tyrosine-phosphorylated proteins in the Triton-insoluble fraction of cells containing the truncated receptor, suggesting an altered specificity of this receptor toward selected cytoskeletal proteins. This work indicates that EGF receptor-cytoskeletal interaction may be an important consideration in the control of receptor-kinase activity and has examined the detergent sensitivity of this association. These studies also suggest that the C-terminal domain of the EGF receptor may affect cytoskeletal interaction in addition to influencing the receptor's catalytic capacity.
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PMID:Evidence for the potentiation of epidermal growth factor receptor tyrosine kinase activity by association with the detergent-insoluble cellular cytoskeleton: analysis of intact and carboxy-terminally truncated receptors. 824 11

Fetal liver kinase-1 (Flk-1) is a transmembrane tyrosine kinase that was identified in endothelial cells and populations of cells enriched in hematopoietic progenitors. To characterize the interaction of Flk-1 with potential ligands the receptor extracellular domain was genetically fused to an alkaline phosphatase (AP) tag. A soluble ligand for Flk-1 was identified in the supernatants of numerous mesenchymal cell lines by co-immunoprecipitation with the Flk1-AP fusion protein. This polypeptide was shown by N-terminal sequencing to be vascular endothelial growth factor (VEGF). Receptor-AP fusion proteins can thus be used to identify soluble ligands as well as transmembrane ligands, and this approach is therefore likely to be widely applicable to many types of orphan receptor. The Flk1-AP soluble receptor was also found to bind to cell surfaces, showing two apparent classes of binding site with different affinities. This interaction could be reconstructed by introducing a VEGF expression plasmid into cells. These results indicate that VEGF presented at the cell surface can bind to the Flk-1 receptor, and could mediate a direct cell-cell interaction. The Flk1-AP fusion protein was also found to bind heparin, implying that ligand binding by the Flk-1 receptor may involve a three way interaction between the Flk-1 receptor, VEGF, and heparin-like cell surface proteoglycans.
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PMID:Interactions between the Flk-1 receptor, vascular endothelial growth factor, and cell surface proteoglycan identified with a soluble receptor reagent. 852 58

A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C- gamma/glutathione S-transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-gamma, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a.
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PMID:Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity. 861 95

The Fgr protein-tyrosine kinase, p55(c-fgr), is specifically expressed and functions in cells of myelomonocytic lineages. We examined levels of expression and enzymatic activity of p55(c-fgr) peripheral blood neutrophils of patients with myelodysplastic syndromes (MDS) and chronic myelogenous leukemia (CML) by comparison with those of normal individuals. While neutrophils of eight normal subjects gave uniform results, the specific enzymatic activity of p55(c-fgr), a ratio of the total kinase activity versus the protein level was reduced in seven out of eight patients with MDS and all of five patients with CML. The specific kinase activity of p55(c-fgr) correlated significantly with the activity of neutrophil alkaline phosphatase (NAP) which has been considered to be a marker of neutrophil maturity (r=0.568, P<0.01). The reduced activity of this tyrosine kinase was considered to be a biological parameter for immaturity and to reflect dysfunction of neutrophils of patients with MDS and with CML.
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PMID:Activity of Fgr protein-tyrosine kinase is reduced in neutrophils of patients with myelodysplastic syndromes and chronic myelogenous leukemia. 863 16


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