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)

Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae protein kinase C was examined. Using pure CTP by synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation. Protein kinase C activity was also dependent on the concentration of CTP synthetase. Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo. Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site. Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro. The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme. This indicated that CTP synthetase was purified in a phosphorylated state. Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity. Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation.
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PMID:Phosphorylation of CTP synthetase from Saccharomyces cerevisiae by protein kinase C. 779 79

Human cell lines express two genetically distinct isoforms of DNA topoisomerase (topo II) II: topo II alpha (p170) and topo II beta (p180). We detected a higher molecular weight form with an apparent molecular mass of about 190 kDa in M phase-arrested HeLa cells (Kimura, K., Saijo, M., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 1173-1176). In this study we confirmed, using anti-topo II alpha and topo II beta monoclonal antibodies, that this higher molecular weight form is topo II beta and consists of doublet bands around 190 kDa. We confirmed that the doublet bands constituted an M phase-specific phenomenon and were not an artifact of the procedure used to accumulate mitotic cells. Digesting the immunoprecipitated materials from mitotic cell extracts with alkaline phosphatase resulted in the disappearance of the doublet bands and the appearance of the 180-kDa band with the concomitant disappearance of 32P label in the region of the doublet bands. Neither heat-inactivated alkaline phosphatase nor phosphodiesterase affected the doublet bands and the 32P label. Topo II beta in interphase cells was also phosphorylated, but the shift in apparent molecular weight was very slight after alkaline phosphatase digestion. Analysis of the labeled phosphoamino acids present in topo II beta from M phase and logarithmically growing cells indicated that phosphorylation occurred mainly on serine and fairly on threonine residues in both topo II beta isoforms. These results indicated that topo II beta is phosphorylated at specific sites in M phase, resulting in the formation of the doublet bands.
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PMID:Identification of the nature of modification that causes the shift of DNA topoisomerase II beta to apparent higher molecular weight forms in the M phase. 792 18

A synthetic tris-sulfotyrosyl dodecapeptide (TRDIY(S)ETDY(S)Y(S)RK-amide), whose primary sequence is identical to the 1142-1153 sequence of the insulin proreceptor, inhibited insulin receptor dephosphorylation in solubilized membranes, and digitonin-permeabilized cells derived from Chinese hamster ovary (CHO) cells expressing high levels of human insulin receptors (CHO/HIRc). It also inhibited the dephosphorylation of a synthetic tyrosine phosphorylated substrate by recombinant PTP-1B, a protein tyrosine phosphatase (PTPase), indicating that it acted via interaction with PTPase(s). A N-stearyl derivative of the peptide caused an approximately 4.5-fold increase in insulin-stimulated receptor autophosphorylaction in intact CHO/HIRc cells. The peptide displayed specificity toward tyrosine-class phosphatases only, as it had no effect on the activities of the serine/threonine phosphatases PP-1 and PP-2A, or alkaline phosphatase. The tyrosine sulfate ester bonds of the peptide were stable when incubated with PTP-1B (1 h, 30 degrees C). These data suggest that the sulfotyrosyl peptide functions as a nonhydrolyzable phosphotyrosyl peptide analogue capable of direct interaction with PTPase catalytic domain.
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PMID:A synthetic tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain inhibits tyrosine dephosphorylation of the insulin receptor in situ. 808 98

The correlation between sequence homology and catalytic importance of a specific amino acid in the E. coli and Liver/Kidney/Bone (L/K/B) human alkaline phosphatase was investigated. For this reason Ala-161 in the E. coli enzyme was substituted with a threonine residue via site-specific mutagenesis. The homologous amino acid in the L/K/B alkaline phosphatase sequence has been shown to cause inactivation of the enzyme. In E. coli alkaline phosphatase the Ala-161-->Thr substitution results in a mutant enzyme with virtually unchanged catalytic properties when compared to the wild-type enzyme. Our results show that Ala-161 in the E. coli alkaline phosphatase does not have an important catalytic role. The results suggest that the three-dimensional topology of the L/K/B alkaline phosphatase may be different from that observed for the enzyme from E. coli.
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PMID:The Ala-161-->Thr substitution in Escherichia coli alkaline phosphatase does not result in loss of enzymatic activity although the homologous mutation in humans causes hypophosphatasia. 832 35

Tau protein was evaluated as a substrate for a proline-directed protein kinase (p34cdc2/p58cyclin A) which recognizes the phosphorylation site motif X-Ser/Thr-Pro-X. The shortest human tau isoform, expressed as a recombinant protein, was phosphorylated to a stoichiometry of 2 mol phosphate/mol tau. Phosphoamino acid analysis revealed phosphorylation of both serine and threonine residues. Phosphorylation of recombinant tau resulted in a decreased ability to induce microtubule assembly but had no effect on the final extent of microtubule formation or on the rate of cold-induced microtubule disassembly. Phosphorylation of tau by the proline-directed protein kinase completely blocked immunoreactivity with antibody SMI33. Phosphorylation did not create the epitopes for the phosphate-dependent antibodies SMI31 or SMI34. Antibody SMI33 recognizes neurofibrillary tangles after treatment with alkaline phosphatase, suggesting that the proline-directed protein kinase may phosphorylate tau at sites that are phosphorylated in Alzheimer's disease.
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PMID:Phosphorylation of tau by proline-directed protein kinase (p34cdc2/p58cyclin A) decreases tau-induced microtubule assembly and antibody SMI33 reactivity. 833 17

Mitotic division in yeast requires the activity of topoisomerase II, a DNA topology modifying enzyme that is able to disentangle sister chromatids after DNA replication. Previous work has shown that topoisomerase II is a phosphoprotein in intact yeast cells. We show here that when dephosphorylated in vitro, topoisomerase II is unable to cleave or decatenate kinetoplast DNA. An efficient kinase activity that modifies topoisomerase II on seven major sites was found to copurify with the enzyme purified from yeast. Characterization of this kinase, analysis of phosphotryptic peptides, and studies with a yeast mutant deficient in casein kinase II, indicate that the copurifying kinase is casein kinase II (CKII). Topoisomerase II itself has no self-phosphorylating activity. Modification of topoisomerase II by the copurifying kinase is sufficient to restore decatenation activity after dephosphorylation by alkaline phosphatase. The CKII target sites have been mapped to multiple serine and threonine residues on 4 tryptic fragments within the C-terminal 350 amino acids of yeast topoisomerase II. These results are consistent with a model in which the C-terminal domain of topoisomerase II is a negative regulatory domain that is neutralized by phosphorylation.
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PMID:Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme. 838 77

Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.
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PMID:Okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells. 862 1

Protein glycosylation has an important influence on a broad range of molecular interactions in eukaryotes, but is comparatively rare in bacteria. Several antigens from Mycobacterium tuberculosis, the causative agent of human tuberculosis, have been identified as glycoproteins on the basis of lectin binding, or by detailed structural analysis. By production of a set of alkaline phosphatase (PhoA) hybrid proteins in a mycobacterial expression system, the peptide region required for glycosylation of the 19 kDa lipoprotein antigen from M.tuberculosis was defined. Mutagenesis of two threonine clusters within this region abolished lectin binding by PhoA hybrids and by the 19 kDa protein itself. Substitution of the threonine residues also resulted in generation of a series of smaller forms of the protein as a result of proteolysis. In a working model to account for these observations, we propose that the role of glycosylation is to regulate cleavage of a proteolytically sensitive linker region close to the acylated N-terminus of the protein.
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PMID:Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kDa antigen from Mycobacterium tuberculosis. 867 Aug 58

Protein phosphatase 2A is a heterotrimeric protein serine/threonine phosphatase consisting of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit. The B subunits determine the substrate specificity of the enzyme. There have been three families of cellular B subunits identified to date: B55, B56 (B'), and PR72/130. We have now cloned five genes encoding human B56 isoforms. Polypeptides encoded by all but one splice variant (B56gamma1) are phosphoproteins, as shown by mobility shift after treatment with alkaline phosphatase and metabolic labeling with [32P]phosphate. All labeled isoforms contain solely phosphoserine. Indirect immunofluorescence microscopy demonstrates distinct patterns of intracellular targeting by different B56 isoforms. Specifically, B56alpha, B56beta, and B56epsilon complexed with the protein phosphatase 2A A and C subunits localize to the cytoplasm, whereas B56delta, B56gamma1, and B56gamma3 are concentrated in the nucleus. Two isoforms (B56beta and B56delta) are highly expressed in adult brain; here we show that mRNA for these isoforms increases severalfold when neuroblastoma cell lines are induced to differentiate by retinoic acid treatment. These studies demonstrate an increasing diversity of regulatory mechanisms to control the activity of this key intracellular protein phosphatase and suggest distinct functions for isoforms targeted to different intracellular locations.
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PMID:The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. 870 17

The transforming growth factor-beta (TGF-beta) is a multifunctional homodimeric protein with an apparent molecular weight of 25 KDa. TGF-beta transduces signals by forming heteromeric complexes of their type-I (T beta R-I) and type-II (T beta R-II) serin/threonine kinase receptors. TGF-beta binds first to T beta R-II receptor, and then the ligand in this complex is recognized by T beta R-I, resulting in formation of a heteromeric receptor complex composed of T beta R-I and T beta R-II. Once received, T beta R-I becomes phosphorylated in the GS domain by the associated constitutively active T beta R-II and transmits the downstream signal. It has been reported that formation of the heteromeric complex is indispensible at least in epithelial cells for growth inhibition and extracellular matrix production induced by TGF-beta. In this study, the functional role of T beta R-II for the TGF-beta-induced signals in osteoblastic cells was investigated by using a dominant negative type of T beta R-II mutant receptors (T beta RIIDNR). ROS 17/2.8 and MG 63 cells were found to express T beta R-I, T beta R-II, and T beta R-III, and their cell growth was inhibited by TGF-beta, whereas alkaline phosphatase activity was stimulated. Cells that were stably transfected with the T beta RIIDNR plasmid showed decreased response to TGF-beta during growth and alkaline phosphatase activity. These results indicate that the intracellular serine/threonine kinase domain of T beta R-II is essential for signal transduction of the TGF-beta-induced alkaline phosphatase activity as well as growth inhibition.
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PMID:[Functional analysis of transforming growth factor-beta type II dominant negative receptor]. 874 21


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