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)

Fluoride stimulates bone cell proliferation and nodule formation in fetal rat calvarial osteoblastic cells. In addition, fluoride enhances alkaline phosphatase activity, a marker of osteoblastic differentiation, in a dose-dependent manner. The effects of fluoroalumino complex (AlFx) on cell proliferation and differentiation were markedly reduced by tyrosine kinase inhibitor; 1 mM genistein or 1 microg/ml herbimycin. It suggests that tyrosine kinase-mediated mitogenic signaling involves a series of protein-protein interactions between tyrosine-phosphorylated receptors, Shc and Grb2, resulting in an AlFx-induced mitogenic effect. The results indicate that AlFx dose-dependently enhances the tyrosine phosphorylation of the adaptor molecule Shc (p52) and its association with Grb2 in the tyrosine kinase mediated pathway. In addition, AlFx decreases the phosphorylation level of CREB without any change on the amount of CREB protein. Taken together, the results suggest that adaptor proteins, including Shc and Grb2 of the protein tyrosine kinase cascade are implicated in fluoride-induced mitogenic activity of fetal rat calvarial osteoblastic cells. Furthermore, CREB which passes signals from cAMP to transcriptional factor CRE, modulates the fluoroaluminate-induced metabolism of bone cells via a decrease of phosphorylation level.
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PMID:Mechanism of mitogenic effect of fluoride on fetal rat osteoblastic cells: evidence for Shc, Grb2 and P-CREB-dependent pathways. 1095 25

Several transmembrane transporters of organic compounds are regulated by phosphorylation/dephosphorylation mechanisms. The aim of this study was to investigate the possible regulation of the human extraneuronal monoamine transporter, hEMT, by these mechanisms. The experiments were performed using HEK293 cells stably transfected with pcDNA3hEMT (293hEMT). The characteristics of hEMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) were studied by incubating the cells at 37 degrees C for 1 min with 200 nM [3H]MPP+. Uptake of [3H]MPP+ by 293hEMT cells was not affected or only slightly reduced by modulators of protein kinase A, protein kinase C, or protein kinase G. It was not affected by an inhibitor of protein tyrosine kinase and was reduced by mitogen-activated protein kinase inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was independent of extracellular Ca2+ and strongly reduced by Ca2+/calmodulin pathway inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced in the presence of non-selective phosphodiesterase inhibitors (IBMX, caffeine, theophylline). The effect of IBMX was independent of extracellular Ca2+ its IC50 was found to be 82.0 microM (66.2-101.6 microM; n=4), and its inhibitory effect resulted from a significant decrease in the maximal velocity of [3H]MPP+ uptake, with no change in the Michaelis-Menten constant. [3H]MPP+ uptake was reduced by 8-methoxy-methyl-IBMX, a selective inhibitor of the Ca2+/calmodulin-dependent phosphodiesterase (PDE1), but not by zaprinast, a selective inhibitor of PDE5. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced by protein tyrosine phosphatase inhibitors, by an alkaline phosphatase inhibitor and, by contrast. showed an increase in the presence of exogenous alkaline phosphatase. In conclusion, these results suggest that hEMT is regulated by phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state.
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PMID:Regulation of human extraneuronal monoamine transporter (hEMT) expressed in HEK293 cells by intracellular second messenger systems. 1177 2

High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.
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PMID:Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function. 1594 59

Interleukin (IL)-6 has been considered as an osteolytic factor involved in periodontal disease. However, the function of IL-6 in osteoblastic differentiation of periodontal ligament cells is not clear. We examined the effects of IL-6 and its soluble receptor (sIL-6R) on osteoblastic differentiation of periodontal ligament cells. Osteoblastic differentiation was induced by ascorbic acid. Osteoblast markers, including alkaline phosphatase activity and Runx2 gene expression, were examined. The mechanism of action of IL-6 on osteoblastic differentiation was evaluated by insulin-like growth factor (IGF)-I production and specific inhibitors for the IL-6-signaling molecule. IL-6/sIL-6R enhanced alkaline phosphatase activity and Runx2. Alkaline phosphatase activity was reduced by anti-IGF-I antibody. Mitogen-activated protein kinase and Janus protein tyrosine kinase inhibitors diminished alkaline phosphatase induced by IL-6/sIL-6R. We conclude that IL-6/sIL-6R increases ascorbic-acid-induced alkaline phosphatase activity through IGF-I production, implying that IL-6 acts not only as an osteolytic factor, but also as a mediator of osteoblastic differentiation in periodontal ligament cells.
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PMID:IL-6 induces osteoblastic differentiation of periodontal ligament cells. 1880 47

Erythropoietin (EPO) is a pleiotropic growth factor. Of interest for skeletal tissue engineering, the non-hematopoietic capabilities of EPO include its osteogenic and angiogenic potencies. The main aim of this study was to investigate the dose-response relationship and determine the lowest effective dose of EPO that reliably increases the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Additional aims were to elucidate the surface receptors and to investigate the role of the intracellular signaling pathways by blocking the mammalian target of rapamycin (mTOR), Jak-2 protein tyrosine kinase (JAK2), and phosphoinositide 3-kinases (PI3K). The primary outcome measures were two mineralization assays, Arsenazo III and alizarin red, applied after 10, 14, and 21 days. Moreover, alkaline phosphatase activity, cell number, and cell viability were determined after 2 and 7 days. A proportional dose-response relationship was observed. In vivo, the lowest effective dose of 20 IU/ml should be used for further research to accommodate safety concerns about adverse effects. Ex vivo, the most effective dose of 100 IU/ml could facilitate vascularization and bone ingrowth in cell-based scaffolds. The expression of non-hematopoietic receptors EPOR and CD131 was documented, and EPO triggered all three examined intracellular pathways. Future studies of the efficacy of EPO in cell-based tissue engineering can benefit from our findings.
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PMID:The osteogenic effect of erythropoietin on human mesenchymal stromal cells is dose-dependent and involves non-hematopoietic receptors and multiple intracellular signaling pathways. 2405 11


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