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)

PA28, also referred to as 11S regulator, is a potent activator of the peptidase activities of the proteasome (multicatalytic proteinase complex). Although the role(s) of PA28-20S proteasome complexes in cellular proteolytic processes remain to be defined, these particles have been implicated in antigen processing of major histocompatibility complex (MHC) class I molecules. Our results demonstrate that PA28 is phosphorylated as evidenced by 32P incorporation into a single PA28 species in rabbit reticulocytes. In reticulocytes as well as human erythrocytes, PA28 is normally found in a phosphorylated state as detected by phosphoserine antibody. In human erythrocytes, this antibody recognizes three polypeptides which are also detected by antibody to PA28 on Western blot analysis. Dephosphorylation with alkaline phosphatase treatment completely abolishes the ability of PA28 to activate hydrolysis of Suc-Leu-Leu-Val-Tyr by proteasomes. After exposure to phosphatase, the three polypeptides are no longer recognized by phosphoserine antibody, although binding to PA28 antibody is unaffected. These results suggest that phosphorylation may function in transduction of cytokine and growth factor signals that, in turn, modulate antigen presentation and other processes which involve PA28-20S proteasome complexes.
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PMID:Phosphorylation of the proteasome activator PA28 is required for proteasome activation. 878 Jul 2

The 20 S proteasome processively degrades cell proteins to peptides. Information on the sizes and nature of these products is essential for understanding the proteasome's degradative mechanism, the subsequent steps in protein turnover, and major histocompatibility complex class I antigen presentation. Using proteasomes from Thermoplasma acidophilum and four unfolded polypeptides as substrates (insulin-like growth factor, lactalbumin, casein, and alkaline phosphatase, whose lengths range from 71 to 471 residues), we demonstrate that the number of cuts made in a polypeptide and the time needed to degrade it increase with length. The average size of peptides generated from these four polypeptides was 8 +/- 1 residues, but ranged from 6 to 10 residues, depending on the protein, as determined by two new independent methods. However, the individual peptide products ranged in length from approximately 3 to 30 residues, as demonstrated by mass spectrometry and size-exclusion chromatography. The sizes of individual peptides fit a log-normal distribution. No length was predominant, and more than half were shorter than 10 residues. Peptide abundance decreased with increasing length, and less than 10% exceeded 20 residues. These findings indicate that: 1) the proteasome does not generate peptides according to the "molecular ruler" hypothesis, and 2) other peptidases must function after the proteasome to complete the turnover of cell proteins to amino acids.
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PMID:Range of sizes of peptide products generated during degradation of different proteins by archaeal proteasomes. 944 34

One point mutation which converts glycine-317 to aspartate of tissue-nonspecific alkaline phosphatase (TNSALP) was reported to be associated with lethal hypophosphatasia (Greenberg, C. R., et al. Genomics 17, 215-217, 1993). In order to define the molecular defect of TNSALP underlying the pathogenesis of hypophosphatasia, we have examined the biosynthesis of TNSALP with a Gly317-->Asp substitution. When expressed in COS-1 cells, the mutant did not exhibit alkaline phosphatase activity at all, indicating that the replacement of glycine-317 with aspartate abolishes the catalytic activity of TNSALP. Pulse-chase experiments showed that the newly synthesized mutant failed to acquire Endo H-resistance and to reach the cell surface. Interestingly, this TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate and was rapidly degraded within the cell, though the mutant protein was modified by glycosylphosphatidylinositol (GPI). Lactacystin, an inhibitor of the proteasome, obstructed the degradation of the mutant protein, suggesting the involvement of proteasome as a part of quality control of TNSALP.
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PMID:Intracellular retention and degradation of tissue-nonspecific alkaline phosphatase with a Gly317-->Asp substitution associated with lethal hypophosphatasia. 961 60

The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function. Parathyroid hormone (through activation of protein kinase A) has also been shown to stimulate ubiquitin/proteasome activity in osteoblasts. Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous treatment with cAMP elevating agents inhibited both osteoblastic differentiation based on alkaline phosphatase assay and DNA binding ability of Cbfa1 based on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibitory effect of forskolin on Cbfa1-regulated genes. Northern and Western blot analyses suggested that the inhibition of Cbfa1 by forskolin was mainly at the protein level. Pretreatment with proteasome inhibitors prior to forskolin treatment reversed the effect of forskolin. Furthermore, addition of proteasome inhibitors to forskolin-pretreated samples resulted in recovery of Cbfa1 protein levels and accumulation of polyubiquitinated forms of Cbfa1, indicating a role for the proteasome pathway in the degradation of Cbfa1. These results suggest that suppression of osteoblastic function by the cAMP pathway is through proteolytic degradation of Cbfa1 involving a ubiquitin/proteasome-dependent mechanism.
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PMID:Inhibition of osteoblast-specific transcription factor Cbfa1 by the cAMP pathway in osteoblastic cells. Ubiquitin/proteasome-dependent regulation. 1050 30

We investigated whether the assembly/disassembly of the 26S proteasome is regulated by phosphorylation/dephosphorylation. The regulatory complex disassembled from the 26S proteasome was capable of phosphorylating the p45/Sug1/Rpt6 subunit, suggesting that the protein kinase is activated upon dissociation of the 26S proteasome or that the phosphorylation site of p45 becomes susceptible to the protein kinase. In addition, the p45-phosphorylated regulatory complex was found to be incorporated into the 26S proteasome. When the 26S proteasome was treated with alkaline phosphatase, it was dissociated into the 20S proteasome and the regulatory complex. Furthermore, the p45 subunit and the C3/alpha2 subunit were cross-linked with DTBP, whereas these subunits were not cross-linked by dephosphorylating the 26S proteasome. These results indicate that the 26S proteasome is disassembled into the constituent subcomplexes by dephosphorylation and that it is assembled by phosphorylation of p45 by a protein kinase, which is tightly associated with the regulatory complex. It was also revealed that the p45 subunit is directly associated with the 20S proteasome alpha-subunit C3 in a phosphorylation-dependent manner.
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PMID:Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit. 1114 24

Tissue-non-specific alkaline phosphatase (TNSALP) is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). A TNSALP mutant with an Asn(153)-->Asp (N153D) substitution was reported in a foetus diagnosed with perinatal hypophosphatasia (Mornet, Taillandier, Peyramaure, Kaper, Muller, Brenner, Bussiere, Freisinger, Godard, Merrer et al. (1998) Eur. J. Hum. Genet. 6, 308-314). When expressed ectopically in COS-1 cells, the wild-type TNSALP formed active non-covalently associated dimers, whereas TNSALP (N153D) formed aberrant disulphide-bonded high-molecular-mass aggregates devoid of enzyme activity. Cell-surface biotinylation and digestion with phosphatidylinositol-specific phospholipase C showed that TNSALP (N153D) failed to reach the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. Upon treatment with brefeldin A, TNSALP (N153D) was still co-localized with GM-130, further supporting the finding that this mutant is localized in the cis-Golgi. Consistent with morphological results, pulse-chase experiments showed that newly synthesized TNSALP (N153D) remained endo-beta-N-acetylglucosaminidase H-sensitive throughout the chase. Eventually, after a prolonged chase time, the mutant was found to be partly degraded in a proteasome-dependent manner. Since the mutant TNSALP was significantly labelled with [3H]ethanolamine, a component of GPI, comparable with the wild-type enzyme, it is unlikely that the abortive synthesis of the mutant is due to a defect in GPI-attachment. Interestingly, when asparagine was replaced by glutamine at position 153 (N153D), TNSALP (N153Q) was indistinguishable from the wild-type enzyme in terms of its molecular properties, suggesting the possible importance of amino acids with a polar amide group at position 153. Taken together, these findings indicate that replacing asparagine with aspartic acid at position 153 causes misfolding and incorrect assembly of TNSALP, which results in its retention at the cis-Golgi en route to the cell surface, followed by a delayed degradation, presumably as part of a quality-control process. We postulate that the molecular basis of the perinatal hypophosphatasia associated with TNSALP (N153D) is due to the absence of mature TNSALP at the cell surface.
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PMID:Retention at the cis-Golgi and delayed degradation of tissue-non-specific alkaline phosphatase with an Asn153-->Asp substitution, a cause of perinatal hypophosphatasia. 1180 76

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.
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PMID:Electrophoretic analysis of phosphorylation of the yeast 20S proteasome. 1184 May 41

The catalytic domain of acetylcholinesterase AChE(T) subunits is followed by a C-terminal T peptide which mediates their association with the proline-rich attachment domain (PRAD) of anchoring proteins. Addition of the T peptide induced intracellular degradation and concomitantly reduced to variable degrees the secretion of AChE species differing in their oligomerization capacity and of human alkaline phosphatase. The T peptide forms an amphiphilic alpha-helix, containing a series of conserved aromatic residues. Replacement of two, four or five aromatic residues gradually suppressed degradation and increased secretion. Co-expression with a PRAD- containing protein induced the assembly of PRAD-linked tetramers in the endoplasmic reticulum (ER) and allowed partial secretion of a dimerization- defective mutant; by masking the aromatic side chains, hetero-oligomerization rescued this enzyme from degradation. Degradation was due to ERAD, since it was not blocked by brefeldin A but was sensitive to proteasome inhibitors. Kifunensine reduced degradation, suggesting a cooperativity between the glycosylated catalytic domain and the non-glycosylated T peptide. This system appears particularly well suited to analyze the mechanisms which determine the degradation of correctly folded multidomain proteins in the ER.
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PMID:The C-terminal T peptide of acetylcholinesterase enhances degradation of unassembled active subunits through the ERAD pathway. 1285 69

A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-beta-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu(218)-->Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca(2+) coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.
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PMID:Tissue-nonspecific alkaline phosphatase with an Asp(289)-->Val mutation fails to reach the cell surface and undergoes proteasome-mediated degradation. 1294 72

Fibroblast growth factors (FGFs) play an important regulatory role in skeletal development and bone formation. However, the FGF signaling mechanisms controlling osteoblast function are poorly understood. Here, we identified a role for the Src family members Lyn and Fyn in osteoblast differentiation promoted by constitutive activation of FGF receptor-2 (FGFR2). We show that the overactive FGFR2 S252W mutation induced decreased Src family kinase tyrosine phosphorylation and activity associated with decreased Lyn and Fyn protein expression in human osteoblasts. Pharmacological stimulation of Src family kinases or transfection with Lyn or Fyn vectors repressed alkaline phosphatase (ALP) up-regulation induced by overactive FGFR2. Inhibition of proteasome activity restored normal Lyn and Fyn expression and ALP activity in FGFR2 mutant osteoblasts. Immunoprecipitation studies showed that Lyn, Fyn, and FGFR2 interacted with the ubiquitin ligase c-Cbl and ubiquitin. Transfection with c-Cbl in which the RING finger was disrupted or with c-Cbl with a point mutation that abolishes the binding ability of the Cbl phosphotyrosine-binding domain restored Src kinase activity and Lyn, Fyn, and FGFR2 levels and reduced ALP up-regulation in mutant osteoblasts. Thus, constitutive FGFR2 activation induces c-Cbl-dependent Lyn and Fyn proteasome degradation, resulting in reduced Lyn and Fyn kinase activity, increased ALP expression, and FGFR2 down-regulation. This reveals a common Cbl-mediated negative feedback mechanism controlling Lyn, Fyn, and FGFR2 degradation in response to overactive FGFR2 and indicates a role for Cbl-dependent down-regulation of Lyn and Fyn in osteoblast differentiation induced by constitutive FGFR2 activation.
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PMID:Cbl-mediated degradation of Lyn and Fyn induced by constitutive fibroblast growth factor receptor-2 activation supports osteoblast differentiation. 1519 72


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