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)

The adhesion of Plasmodium falciparum-infected erythrocytes (IRBCs) to human dermal microvascular endothelial cells (HDMECs) under flow conditions is regulated by a Src family kinase- and alkaline phosphatase (AP)-dependent mechanism. In this study, we showed that the target of the phosphatase activity is the ectodomain of CD36 at threonine-92 (Thr92). Mouse fibroblasts (NIH 3T3 cells) transfected with wild-type CD36 or a mutant protein in which Thr92 was substituted by Ala supported the rolling and adhesion of IRBCs. However, while the Src family kinase inhibitors PP1 and PP2 and the specific AP inhibitor levamisole significantly reduced IRBC adhesion to wild-type CD36 transfectants as with HDMECs, the inhibitors had no effect on IRBC adhesion to the mutant cells. Using a phosphospecific antibody directed at a 12-amino-acid peptide spanning Thr92, we demonstrated directly that CD36 was constitutively phosphorylated and could be dephosphorylated by exogenous AP. Endothelial CD36 was likewise constitutively phosphorylated. The phosphospecific antibody inhibited IRBC adhesion to HDMECs that could be reversed by preincubating the antibody with the phosphorylated but not the nonphosphorylated peptide. Pretreatment of HDMECs with AP abrogated the effect of PP1 on IRBC adhesion. Collectively, these results are consistent with a critical role for CD36 dephosphorylation through Src family kinase activation in regulating IRBC adhesion to vascular endothelium.
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PMID:Ectophosphorylation of CD36 regulates cytoadherence of Plasmodium falciparum to microvascular endothelium under flow conditions. 1629 13

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.
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PMID:Rat organic anion transporting protein 1A1 (Oatp1a1): purification and phosphopeptide assignment. 1651 30

Human mesenchymal stem cell (hMSC) differentiation into osteoblasts and the signaling events involved are poorly understood. We recently established that contact with specific extracellular matrix (ECM) proteins, in particular laminin-5, is sufficient to induce an osteogenic phenotype in hMSC through an extracellular signal-related kinase (ERK)-dependent pathway. Activation of ERK 1/2 by laminin-5 induces phosphorylation of the runx2/cbfa-1 transcription factor that controls osteogenic gene expression. We hypothesized that focal adhesion kinase (FAK) mediated signaling pathways supply a link between cell surface integrin-ECM binding and activation of ERK 1/2, and that laminin-5 promotes its osteogenic effects through this pathway. To test this hypothesis, we plated hMSC on a laminin-5 matrix in the presence or absence of FAK-specific small inhibitory RNAs (siRNA), and assayed for phosphorylation of runx2/cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein, osteocalcin, alkaline phosphatase, calcium deposition, and mineral:matrix ratio). We found that siRNA treatment reduced total endogenous FAK protein by approximately 40%, and reduced FAK phosphorylation on Y397 by approximately 33% in cells plated on laminin-5 for 30 min. SiRNA treated cells exhibited a decrease in ERK 1/2 phosphorylation after 1 h, and reduced serine/threonine phosphorylation of Runx2/Cbfa-1 after 8 days. Finally, FAK inhibition blocked osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results establish FAK as an important mediator of laminin-5-induced osteogenic differentiation of hMSC.
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PMID:Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5. 1692 79

Cerebral ischemia and reperfusion induces rapid accumulation of oxidative DNA lesions in the brain, which, if not repaired promptly, may trigger cell death. The base-excision repair (BER) pathway is the main mechanism employed by neurons to repair various types of oxidative DNA damage. Recent studies have suggested that the cellular activity of BER is highly regulated (up- or down-regulated) after ischemic brain injury, and this regulation may contribute to the outcome of cell injury. The mechanism through which cellular BER is regulated in response to neuronal injury is currently poorly understood. In the present study, we have examined BER regulation in the rat model of focal ischemic brain injury induced by 2 hr of middle cerebral artery occlusion and 0-72 hr of reperfusion. As determined using cerebral nuclear extracts, focal ischemia resulted in a marked reduction in BER activities, including the overall BER activity, AP endonuclease activity and DNA polymerase-beta activity, indicating functional impairment of the BER pathway. BER reduction occurred as early as 0.5 hr after the onset of reperfusion. Thereafter, BER activity failed to recover, and there were persistent accumulations of apurinic/apyrimidinic abasic sites and DNA single-strand breaks in ischemic tissues. The reduction in BER during the early reperfusion phase (less than 6 hr) was not accompanied by any alterations in the levels of essential BER enzymes in brain extracts. However, increased serine- and threonine-specific phosphorylation was detected for both AP endonuclease and DNA polymerase-beta after ischemia, with the time course of serine phosphorylation closely correlated to that of changes in BER activity. Furthermore, dephosphorylation of nuclear extracts with alkaline phosphatase largely restored AP endonuclease and DNA polymerase-beta activities. Taking advantage of the neuroprotective effect of mild hypothermia (33 degrees C), which was induced in the brain during the first 2 hr of reperfusion, we found that the post-ischemic suppression of BER activity is a reversible event. Hypothermic treatment diminished the serine-specific phosphorylation of AP endonuclease and DNA polymerase-beta, promoted BER activities, and attenuated the levels of oxidative DNA lesions after ischemia. These results suggest that the functional impairment of the BER pathway after severe focal cerebral ischemia is due to the loss-of-function post-translational modifications of repair enzymes. Further investigations elucidating the precise mechanism underlying the post-translational regulation of BER enzymes may lead to novel therapeutic strategies for cerebral ischemia.
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PMID:Impaired DNA repair via the base-excision repair pathway after focal ischemic brain injury: a protein phosphorylation-dependent mechanism reversed by hypothermic neuroprotection. 1712 26

The hepatocyte growth factor (HGF) signaling pathway was examined in human normal melanocytes and three malignant melanoma cell lines. HGF-induced activation of c-Met, its receptor-tyrosine kinase, was observed in both melanocytes and melanoma cells, whereas phosphatidylinositol 3-kinase (PI3K), a downstream target of c-Met, was not activated in the melanocytes but enhanced in the melanoma cell lines. The electrophoretic mobility of Gab1, the scaffolding adapter protein that couples activated c-Met and PI3K, was slower in the melanocytes than that in the melanoma cells, and the mobility shifted to that of the melanoma cells after treatment with alkaline phosphatase, indicating that Gab1 is highly phosphorylated on serine and threonine in the melanocytes. Introduction of protein kinase C (PKC)-betaII into the melanoma cells, which is expressed in melanocytes but absent in melanoma cells, resulted in serine and threonine phosphorylation of Gab1 and also prevented tyrosine phosphorylation of Gab1 and its association with PI3K. Furthermore, the introduction of PKC-betaII suppressed HGF-induced activation of PI3K, and attenuated the in vitro invasion activity of the melanoma cells. These results indicate that the HGF signaling process from Gab1 to PI3K is negatively regulated by PKC-betaII, and its loss is critical for melanoma cells to gain invasive potential.
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PMID:Protein kinase C-betaII represses hepatocyte growth factor-induced invasion by preventing the association of adapter protein Gab1 and phosphatidylinositol 3-kinase in melanoma cells. 1762 96

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.
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PMID:Chlamydophila pneumoniae PknD exhibits dual amino acid specificity and phosphorylates Cpn0712, a putative type III secretion YscD homolog. 1776 19

A 56-year-old Japanese woman with mandibuloacral dysplasia and type A lipodystrophy is described. Mutation analysis identified a homozygous missense mutation (1585G > A) in exon 9 of the LMNA gene that replaces well-conserved residue alanine at position 529 to threonine (A529T). The woman showed, in addition to the usual clinical manifestations of the disorder, severe progressive skeletal changes: osteoporotic changes with multiple fractures; osteolysis of the right radius; and destructive changes of the vertebrae, leading to compression of the cervical spinal cord and paraplegia. Laboratory findings included markedly reduced bone mineral density; significantly increased urine N-telopeptide of collagen type I, an osteoclast marker; and normal serum bone specific alkaline phosphatase, an osteoblast marker. Regular follow up of adult patients with the disorder is desirable, including skeletal radiography, estimates of bone mineral density, and biochemical markers of bone turnover. Treatment with bisphosphonates to inhibit osteoclast activity is likely to be beneficial.
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PMID:Mandibuloacral dysplasia and a novel LMNA mutation in a woman with severe progressive skeletal changes. 1793 39

The subunits of complex I encoded by the mammalian nuclear genes NDUFS4 (AQDQ protein) and NDUFB11 (ESSS protein) contain serine/threonine consensus phosphorylation sequences (CPS) in their presequence, the first also in the C-terminus. We have studied the impact of PKA mediated phosphorylation on the mitochondrial import of in vitro and in vivo synthesized NDUFS4 protein. The intramitochondrial accumulation of the mature form of in vitro synthesized NDUFS4 protein, but not that of ESSS protein, was promoted by PKA and depressed by alkaline phosphatase (AP). In HeLa cells, control or transfected with the NDUFS4 cDNA construct, the mitochondrial level of mature NDUFS4 protein was promoted by 8-Br-cAMP and depressed by H89. Ser173Ala mutagenesis in the C-terminus CPS abolished the appearance in mitochondria of the mature form of NDUFS4 protein. The promoting effect of PKA on the mitochondrial accumulation of mature NDUFS4 protein appears to be due to inhibition of its retrograde diffusion into the cytosol.
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PMID:cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I. 1829 24

We investigated the response of extracellular phosphatase to heat shock in heterotrophic Chenopodium rubrum L. cell cultures. Surprisingly, in contrast to the generally used acid phosphatase, an extracellular alkaline phosphatase showed the most sensitive response. This phosphatase was characterized as a marker for cellular stimulation by its high correlations with induced changes of extracellular pH: 10microM nigericin (correlation coefficient r=0.91), 100microM salicylic acid (r=0.84), heat shock 5min 37 degrees C (r=0.79), and heat shock after pre-treatment with 5microM fusicoccin (r=0.92) or 0.5% ethanol (r=0.90). Cellular stimulation was estimated with concentrations of acids and bases, yielding similar levels of pH change (0.5 pH) in cell-free supernatant: salicylic acid (200microM), benzoic acid (600microM), HCl (140microM), NaOH (100microM), and KOH (100microM). The Golgi apparatus inhibitor Brefeldin A (200microM) reduced the heat-shock-induced phosphatase (-33%). The pH optimum of heat-shock-induced phosphatase was 3; however, there the proportion of constitutive phosphatase was higher than at pH 8-9.5, indicating different pH dependence of constitutive and induced activity. Thus, heat-shock-induced phosphatase was characterized by alkaline activity with inhibitors (10microM molybdate: -52%, 2.5mM phosphate: -64%, 10microM ZnCl(2): -82%), substrates (2.5mM, tyrosine phosphate: 255pkat g(-1), p-nitrophenyl phosphate: 92pkat g(-1), serine phosphate: 0, threonine phosphate: 0), Hill coefficient (nH=1.4) indicating two binding sites, and the extent of heat-shock stimulation (p-nitrophenyl phosphate: +190%, tyrosine phosphate: +180%). SDS-PAGE showed a correlation of alkaline phosphatase with the heat-shock-induced release of highly N-glycosylated 53kDa protein, detected by peroxidase-labeled concanavalin A affinoblotting after endoglycosidase H treatment. The 53kDa protein showed no in-gel phosphatase activity after SDS-PAGE and regeneration treatment, in contrast to a putative dimer (105kDa).
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PMID:Extracellular alkaline phosphatase is a sensitive marker for cellular stimulation and exocytosis in heterotroph cell cultures of Chenopodium rubrum. 1843 30

This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia coli alkaline phosphatase (EAP) is a well-defined nonspecific phosphated monoesterase and Ser-, Thr- or Tyr-phosphorylated peptides served as substrates for EAP in preliminary experiments. Based on the known catalytic mechanism of EAP, the recombinant site-directed mutant EAP-S102L was generated, whose catalytic activity was blocked, but its binding ability was preserved. For EAP-S102L the catalytic rate constant, k(cat), was reduced by a factor of 1000, while the Michaelis-Menten constant, K(m), remained almost unchanged. Crystallographic analysis of the EAP-S102L/phophorylated peptide complex revealed that EAP-S102L could bind the phosphate group of the phosphorylated peptide but lacked nucleophilic attack potential which was essential for the catalytic ability of EAP. Finally, by combining the fluorescence-labeled EAP-S102L with non-phophorylated peptide chips, kinases could be detected from tumor cell samples. The recombinant EAP-S102L construct is perhaps the first functional binding protein derived from a native enzyme, illustrating how one single mutation tremendously alters protein function.
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PMID:Development of a universal phosphorylated peptide-binding protein for simultaneous assay of kinases. 1934 57


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