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)

Src protein is essential for the regulation of bone turnover primarily via bone resorption because it is required in osteoclast differentiation and function. We followed temporal changes of Src protein abundance in marrow stromal cells induced to mineralize by dexamethasone (DEX), growth in cold temperature, or both. Given the tyrosine kinase function of Src and its numerous substrates, profiles of phosphotyrosine-containing proteins were followed as well. On day 11 of stimulation, specific alkaline phosphatase (ALP) activity at 30 degrees C decreased under DEX relative to 37 degrees C cultures, in accord with increased cell counts. Mineralization per well under DEX increased by 25% at 37 degrees C, whereas at 30 degrees C it increased by more than threefold regardless of the DEX stimulation. At 30 degrees C, on a per cell basis mineralization increased 2.5 and 3 times with and without DEX, respectively. Cultures at 37 degrees C showed a general drop per cell of many phosphotyrosine-containing proteins on day 3 relative to days 1 and 2 in both DEX-stimulated and nonstimulated cultures; several proteins did recover (recuperate) thereafter. On days 1 and 2, the phosphotyrosine signal was higher in several proteins under DEX stimulation; this trend became inverted after day 3. The changes in abundance per cell of Src protein (pp60src) followed a similar trend, and in addition a truncated Src molecule, p54/52src, was detected as a putative cleavage product presumably representing its carboxy terminus. The pp60src was most abundant, relative to its truncated product, in day 7 nonstimulated cultures, whereas under DEX stimulation the truncated species pp54/52src showed the highest relative abundance on days 7. At 30 degrees C, DEX stimulation accentuated the increase in Src protein on day 3, showed no change on day 7, and returned to increase Src protein on day 10. Potassium ionophorvalinomycin, considered to select against mineralizing osteoprogenitors at 30 degrees C, showed on day 10 in the absence of DEX a relative increase in truncated Src protein compared to both DEX-stimulated and nonstimulated cultures in the absence of valinomycin. On day 7 of DEX stimulation, the presence of valinomycin resulted in low p54/52src. Among phosphotyrosine-containing proteins, a 32-34 kDa band, as yet unidentified, showed the most concordant changes with mineralization induction. P32-34 decreased by DEX on days 2 and 8 and increased by low temperature alone or combined with DEX on day 3. On day 7, p32-34 did not change under DEX, but valinomycin selected cells with less phoshpotyrosine-containing p32-34. Taken together, high Src abundance at the start of osteogenic induction followed by a decrease 1 week later is probably related to energy metabolism-dependent induction of mineralization. This is in temporal accord with the increase in Src truncation and fluctuation in mitochondrial membrane potential (which affects mineralization). The reported binding of amino-terminal Src oligopeptide to p32 ADP/ATP carrier in the mitochondrial inner membrane raises the question of its possible involvement in mitochondria-regulated mineralization.
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PMID:Src protein and tyrosine-phosphorylated protein profiles in marrow stroma during osteogenic stimulation. 958 70

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell types and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphorylation in colonocyte differentiation, by using human colonic Caco-2 cells as a model and expression of the brush border enzymes alkaline phosphatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation markers. We studied three tyrosine kinase inhibitors with different modes of action and specificities, viz., genistein, erbstatin analog (EA), and tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanadate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5-75 mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stimulation at 75 mg/ml by 158.6+/- 17.5% and 228.6+/-37.1% of control values, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25 mM) similarly stimulated AKP and DPDD by 138. 6+/-6.6% and 131.8+/-1.5% of control values (n=12, P<0.001). Unexpectedly, EA (0.1-10 mM) had the opposite effect, inhibiting AKP- and DPDD-specific activity significantly at 10 mM with a maximal 14.8+/-6.4% and 26.5+/-2.5% of control values (n=12, each P<0.001). Sodium orthovanadate had a discordant effect on these two differentiation markers. Orthovanadate dose-dependently increased AKP to a maximal 188.5+/-16.1% of basal activity at 1.5 mM but decreased DPDD activity at 1.5 mM to 47.2+/-3.8% (n=9, P<0.001 each). The effects of each agent were preserved when proliferation was blocked with mitomycin C, suggesting that the modulation of phenotype by these agents was independent of any effects of proliferation. The tyrosine phosphorylation of several phosphoprotein bands was affected differently by these agents. In particular, the tyrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility that at least two independent enzymes or pathways regulating tyrosine phosphorylation modulate intestinal epithelial differentiation. Furthermore, tyrosine phosphorylation of the 70-kDa to 71-kDa phosphoprotein may be important in the intracellular signaling by which intestinal epithelial cell differentiation is controlled.
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PMID:Effects of modulation of tyrosine phosphorylation on brush border enzyme activity in human Caco-2 intestinal epithelial cells. 958 12

A number of factors have been shown to influence osteoblastic proliferation, including fluoride. Recent observations suggest that heterotrimeric G proteins are probably involved in the mitogenic response induced by this agent, further suggesting a role of guanosine 5'-triphosphate (GTP)-binding protein-coupled receptors (GPCR) in the regulation of osteoblastic cell growth. We therefore explored what mitogenic factors known to activate GPCR can influence the replication of mouse osteoblast-like MC3T3-E1 cells. Among several candidates, epinephrine was found to be a potent mitogen for these cells, and its effect on the growth and differentiation of these cells was further investigated. Deoxyribonucleic acid (DNA) synthesis was dose dependently enhanced by this catecholamine in the concentration range of 1 nmol/L-10 micromol/L. Stimulation of DNA synthesis by catecholamines was in the order of epinephrine > norepinephrine >> isoproterenol, indicating that alpha adrenergic receptors mediated this cellular response. Further analysis with specific adrenergic receptor agonists and antagonists suggested that the mitogenic response induced by epinephrine in MC3T3-E1 cells is mediated by alpha1 adrenergic receptors. In addition to its effect on cell replication, epinephrine also enhanced alkaline phosphatase (ALP) activity in these cells but had little effect on collagen synthesis and osteocalcin production. As for the mitogenic response, the change in ALP activity was found to be mediated by alpha1 adrenergic receptors. Both effects of epinephrine on cell replication and ALP activity were markedly reduced by pretreatment of the cells with pertussis toxin (PTX), suggesting a role of Gi proteins. These effects were also completely blocked by pretreatment of the cells with 50 micromol/L genistein, a nonselective inhibitor of tyrosine kinase. In conclusion, the results indicate that epinephrine enhances replication and ALP activity of MC3T3-E1 osteoblast-like cells via alpha1 adrenergic receptors coupled to Gi proteins. The signaling mechanism probably involves a tyrosine phosphorylation mechanism. These observations suggest that PTX-sensitive G proteins are potent mediators of cell proliferation and ALP activity of osteoblast-like cells in response to factors acting through G protein-coupled receptors.
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PMID:Catecholamines stimulate the proliferation and alkaline phosphatase activity of MC3T3-E1 osteoblast-like cells. 973 41

Overexpression of bovine growth hormone (bGH) in transgenic (PEPCK-bGH) mice induces resistance to insulin, which is compensated by a major increase in insulin levels. In these animals, hepatic insulin receptors (InsRs) are downregulated while tyrosine kinase activity of wheat germ agglutinin (WGA)-purified InsRs towards exogenous substrates is unexpectedly increased. By normalizing insulinemia, we attempted to determine whether the alterations detected in the early steps of insulin signal transduction are due to exposure to chronically high GH levels or are secondary to hyperinsulinemia. Transgenic PEPCK-bGH animals were treated with a single intraperitoneal administration of streptozotocin (STZ) or were deprived of food for 48 h, to normalize insulin levels. Both fasting and STZ treatment were effective in reducing insulin blood levels to control values or below, while GH levels remained unchanged (STZ treatment) or increased (fasted animals). In the liver of untreated transgenic mice, the number of InsRs as determined by 125I-insulin binding was significantly diminished (65+/-5% and 60+/-6% of normal values in microsomes and solubilized membranes respectively;P<0.01 vs control mice). In treated transgenic mice, the number of InsRs increased to values similar to or slightly higher than those found in normal control mice (STZ-treated: 139+/-26% and 126+/-8%; fasted: 128+/-5% (P<0.05) and 102+/-1.5%, for microsomes and solubilized membranes respectively). Neither treatment altered InsR affinity. InsR concentration in liver as determined by immunoblotting using an antibody against the beta-subunit of the insulin receptor was found to be reduced in transgenic mice (69+/-3% of normal values,P<0.001) and was normalized after both STZ treatment (105+/-4%) and fasting (109+/-4%). Insulin-stimulated autophosphorylation activity of InsRs in transgenic mice was increased (154+/-13%,P<0.01 compared with the control group), essentially normalized by STZ treatment (96+/-14%), and reduced by fasting, to below the values measured in normal control mice (56+/-15%,P<0.05). The potential influence of basal serine/threonine (Ser/Thr) phosphorylation of the InsR beta-subunit on the regulation of the InsRs from transgenic mice was also investigated. The autophosphorylation activity of WGA-purified InsRs from all groups of mice studied was essentially unchanged after dephosphorylation with alkaline phosphatase or mild trypsinization. Consequently, our results suggest that the observed changes in InsR number and autophosphorylation activity in the liver of bGH transgenic mice are directly related to changes in insulin blood levels, and that Ser/Thr phosphorylation is apparently not involved in the regulation of the InsR autophosphorylation activity in this model of insulin resistance.
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PMID:Role of hyperinsulinemia on hepatic insulin receptor concentration and autophosphorylation in the presence of high growth hormone levels in transgenic mice overexpressing growth hormone gene. 979 37

Short chain fatty acids such as sodium butyrate are concentrated in the colonic lumen and may protect against colon carcinogenesis by maintaining colonocytic differentiation, but the mechanisms by which they act are not fully understood. It has recently been suggested that short chain fatty acids modulate cellular tyrosine kinase activity in addition to altering chromatin structure via regulation of histone acetylation and DNA methylation. Therefore, the authors evaluated the influence of tyrosine kinase inhibition on the effects of 10 mM butyrate on human Caco-2 intestinal epithelial differentiation, using alkaline phosphatase and dipeptidyl dipeptidase specific activity as markers of differentiation, and two tyrosine kinase inhibitors, of different mechanisms of action and different effects on Caco-2 brush border enzyme specific activity, to block tyrosine kinase activity. As expected, butyrate stimulated both alkaline phosphatase and dipeptidyl dipeptidase specific activity. The tyrosine kinase inhibitors prevented, and indeed one inhibitor reversed the effects of butyrate on alkaline phosphatase specific activity. However, tyrosine kinase inhibition did not prevent butyrate stimulation of dipeptidyl dipeptidase specific activity. Different pathways are likely to regulate the effects of butyrate on expression of these two brush border enzymes. Butyrate stimulation of alkaline phosphatase, but not dipeptidyl dipeptidase, may involve tyrosine phosphorylation signaling.
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PMID:Tyrosine kinase inhibitors reverse butyrate stimulation of human Caco-2 intestinal epithelial cell alkaline phosphatase but not butyrate promotion of dipeptidyl dipeptidase. 1019 53

Leucocyte alkaline phosphatase (LAP) is a marker of post-mitotic granulocytes and its activity is reduced or absent in chronic myelogenous leukaemia (CML) granulocytes as a consequence of LAP messenger RNA (mRNA) deficiency. We provide evidence that along the granulocytic maturation in normal marrow, the acquisition of LAP surface expression, identified by the monoclonal antibody 1B12.1, was restricted to CD11bbright/CD16bright positive cells. Moreover, in normal granulocytes, exposure to granulocyte colony-stimulating factor (G-CSF) in vitro and in vivo increased the cell surface expression of LAP. Although G-CSF was able to induce the LAP surface expression in CML granulocytes, the inhibition of p210 tyrosine kinase activity by genistein or CGP75148B failed to restore LAP mRNA expression and LAP protein synthesis. In conclusion, the acquisition of LAP protein on the cell surface of granulocytes follows CD16 antigen expression and can be considered as the last marker of terminally differentiated neutrophils. G-CSF is a potent regulator of the LAP mRNA expression and protein synthesis in normal and CML-derived neutrophils. The lack of direct activity of p210 tyrosine kinase on LAP mRNA expression in CML neutrophils supports the notion that the LAP defect in this disease could be related to a precocious and uncontrolled release of white blood cells from the bone marrow into the blood stream.
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PMID:Leucocyte alkaline phosphatase identifies terminally differentiated normal neutrophils and its lack in chronic myelogenous leukaemia is not dependent on p210 tyrosine kinase activity. 1023 80

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kinase. Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a significant decrease in enzyme activity. Dephosphorylation by treatment with alkaline phosphatase completely restored GPI-PLD activity. In addition, incubation of GPI-PLD with trypsin, which results in the generation of distinct peptide fragments, resulted in complete dephosphorylation of radiolabeled GPI-PLD. The site of phosphorylation by PKA was assigned to Thr-286. Tyrosine phosphorylation was only observed in a proteolytically processed fragment of GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD activity.
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PMID:In vitro phosphorylation of purified glycosylphosphatidylinositol-specific phospholipase D. 1038 65

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.
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PMID:Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats. 1039 Nov 42

Retinal pigment epithelial cells (RPE) and the choriocapillaris are on opposite sides of Bruch's membrane and control transport in and out of the retina. In age-related macular degeneration (AMD), they may also be responsible for deposition of material in and on Bruch's membrane and the formation or regression of choroidal neovascularization (CNV). Indocyanine green (ICG) angiography can be used to visualize the choroidal vasculature and CNV. Filling of the choriocapillaris with ICG was delayed in subjects older than 50 years of age, and areas of hypofluorescence were observed in maculas of AMD subjects, often associated with CNV. Laser Doppler flowmetry of the choriocapillaris in the macula demonstrated that choroidal blood flow and volume are reduced in subjects older than 46 years of age and further decreased in subjects with AMD. The human choriocapillaris can be histologically studied in two dimensions by incubating the tissue for alkaline phosphatase activity, flat-embedding it in transparent polymer and sectioning it. Using this technique, choriocapillaris dropout was found to be associated with deposition of material in Bruch's membrane in diabetic subjects. When RPE are removed from Bruch's membrane, the choriocapillaris degenerates; the regeneration of choriocapillaris can be blocked by Genistein, a tyrosine kinase inhibitor. Finally, RPE cells may produce substances that both stimulate the formation and regression of CNV in animal models. These studies suggest that there may be a reduction in choriocapillaris flow in AMD, and this loss of choriocapillaris can be associated with the Bruch's membrane deposits that are hallmarks of AMD. Furthermore, RPE may stimulate the formation and regression of CNV and RPE loss can result in loss of choriocapillaris.
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PMID:Changes in choriocapillaris and retinal pigment epithelium in age-related macular degeneration. 1056 59

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.
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PMID:Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts. 1058 92


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