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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When erythrocytes are incubated with 32Pi, incorporation of label into phosphoproteins is a gradual process, increasing for at least 2 hours. Membrane phospholipids also are labeled. Exogenous protein kinase substrates are unlabeled in these incubations. This suggests that labeling by 32Pi occurs into polypeptides inside the erythrocytes. When erythrocytes are incubated with [gamma-32P]ATP and active protein kinase, membrane polypeptides are not labeled. Only exogenously added protein kinase substrates and the regulatory subunit of protein kinase (and its contaminants) are labeled. This suggests that labeling from [gamma-32P]ATP and active protein kinase occurs in the compartment outside the erythrocytes. Apyrase (EC 3.6.1.5) eliminates such labeling, demonstrating that it was occurring in the compartment external to the erythrocytes. However, in incubations of cells with 32Pi, apyrase has no effect on the incorporation into membrane polypeptides and phospholipids, demonstrating that this labeling occurs on the inside of the membrane. Thus, additions of apyrase to intact particles incubated with protein kinase substrates and 32Pi provides a method for identifying internally exposed polypeptides in the plasma membranes of a variety of systems.
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PMID:Defining erythrocyte internal labeling by phosphorylation. 658 27

The phosphorylation of surface proteins by ecto-protein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ecto-protein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [gamma-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32Pi. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled Pi. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-alpha-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca(2+)-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ecto-protein kinase and surface protein phosphorylation in PC12 cells: interactions with nerve growth factor. 841 43

Tyrosine as well as serine/threonine protein kinase inhibitors have potentially two sites of interaction with their targets: the protein-substrate binding site and the ATP binding site. The latter could be modelized by measuring the capacity of protein kinase inhibitors to inhibit ATPase activities. In order to do so, we assess a novel, highly sensitive HPLC method based on hydrophilic separation of [gamma-32P]ATP and [32P]Pi. The novel assay is presented. Furthermore, the potency of 13 protein kinase inhibitors was tested on two types of ATPase, namely: apyrase and partially purified liver mitochondria F1-ATPase. The method described for the assay of ATPase can be used with almost any type of enzyme catalyzing this activity. Only cibacron blue and suramin show interesting capacities in inhibiting these ATPase activities pointing out that several widely used protein kinase inhibitors are at least somewhat specific in that they do not inhibit these two ATPases.
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PMID:Studies of the potency of protein kinase inhibitors on ATPase activities. 843 62

The ecto-ATPase from chicken gizzard (smooth muscle) was solubilized, and the 66-kDa cell membrane ecto-ATPase protein was purified. The protein was then subjected to both enzymatic and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced. Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the cDNA encoding the ecto-ATPase. Two overlapping partial clones were sequenced, yielding the complete coding region and a long 3'-untranslated sequence. The deduced amino acid sequence is in agreement with the N-terminal and peptide sequences obtained from the purified protein. The chicken muscle ecto-ATPase is a slightly basic (predicted pI = 7.93) 494-amino acid protein (54.4 kDa), containing a single transmembrane domain at each end of the protein. The majority of the protein is predicted to be extracellular, making it a Type Ia plasma membrane protein. There are four putative N-glycosylation sites, a single potential cAMP/cGMP-dependent protein kinase phosphorylation site, as well as a single putative tyrosine kinase phosphorylation site. Analysis of the sequence using the BLAST programs demonstrated homology with other ecto-ATPases and ecto-apyrases, including those from the parasitic protozoan Toxoplasma gondii, potato tubers, and garden pea, as well as a guanosine diphosphohydrolase from yeast. However, the most striking homology observed was to the human and mouse lymphoid cell activation antigen 39 (CD39), a molecule now known to have apyrase activity. The chicken ecto-ATPase showed considerable amino acid sequence homology with CD39 over the entire length of the sequence, excluding about 30-40 amino acids at the extreme ends of the protein (which include the two membrane-spanning helices). The sequence homology between the gizzard ecto-ATPase and CD39 was confirmed by Western blots demonstrating immunocross-reactivity between mono- and polyclonal antibodies raised against the chicken ecto-ATPase and two commercially available monoclonal antibodies against the human CD39 protein. The results suggest that the muscle ecto-ATPase may be involved in cell adhesion, since the highly homologous CD39 protein is involved in homotypic adhesion of activated B lymphocytes.
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PMID:Complementary DNA cloning and sequencing of the chicken muscle ecto-ATPase. Homology with the lymphoid cell activation antigen CD39. 899 5

In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.
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PMID:GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor. 958 May 66

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.
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PMID:Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1. 1086 13

A cDNA encoding a pea nuclear apyrase was previously cloned. Overexpressions of a full-length and a truncated cDNA have been successfully expressed in Escherichia coli BL21(DE3). The resulting fusion proteins, apyrase and the C-terminus (residues 315-453) of apyrase, were used for calmodulin (CaM) binding and phosphorylation studies. Fusion protein apyrase but not the C-terminus of apyrase can be recognized by polyclonal antibody pc480. This suggested that the motif recognized by pc480 was located in the N-terminal region of apyrase. The recombinant apyrase protein also showed an activity 70 times higher than that of endogenous apyrase using ATP as a substrate. The recombinant apyrase has a preference for ATP more than other nucleoside triphosphate substrates. CaM can bind to recombinant apyrase, but not to the C-terminus of apyrase. This implies that the CaM-binding domain must be in the first 315 amino acids of the N-terminal region of apyrase. We found that one segment from residue 293 to 308 was a good candidate for the CaM-binding domain. This segment 293 FNKCKNTIRKALKLNY 308 has a basic amphiphilic-helical structure, which shows the predominance of basic residues on one side and hydrophobic residues on the other when displayed on a helical wheel plot. Using the gel mobility shift binding assay, this synthetic peptide was shown to bind to CaM, indicating that it is the CaM-binding domain. Both recombinant apyrase and the C-terminus of apyrase can be phosphorylated by a recombinant human protein kinase CKII. Phosphorylation does not affect CaM binding to recombinant apyrase. However, CaM does inhibit CKII phosphorylation of recombinant apyrase and this inhibition can be blocked by 5 mM EGTA.
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PMID:Regulation of a recombinant pea nuclear apyrase by calmodulin and casein kinase II. 1112 82

Gliosis is characterized by hypertrophic and hyperplastic responses of astrocytes to brain injury. To determine whether injury of astrocytes produced by an in vitro model of brain trauma activates extracellular signal-regulated protein kinase (ERK), a key regulator of cellular proliferation and differentiation, astrocytes cultured on deformable SILASTIC membranes were subjected to rapid, reversible strain (stretch)-induced injury. Activation of ERK was observed 1 min after injury, was maximal from 10 to 30 min, and remained elevated for 3 hr. Activation of ERK was dependent on the rate and magnitude of injury; maximum ERK activation was observed after a 20-60 msec, 7.5 mm membrane displacement. ERK activation was blocked by inhibiting MEK, the upstream activator of ERK. Activation of ERK was reduced when calcium influx was diminished. When extracellular ATP was hydrolyzed by apyrase or ATP/P2 receptors were blocked, injury-induced ERK activation was significantly reduced. P2 receptor antagonist studies indicated a role for P2X2 and P2Y1, but not P2X1, P2X3, or P2X7, receptors in injury-induced ERK activation. These findings demonstrate for the first time that ATP released by mechanical injury is one of the signals that triggers ERK activation and suggest a role for extracellular ATP, P2 purinergic receptors, and calcium-dependent ERK signaling in the astrocytic response to brain trauma.
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PMID:Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors. 1265 94

The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2beta isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2alpha isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2alpha or NTPDase2beta cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2alpha was predominantly membrane-bound, whereas NTPDase2beta had combined cell surface and intracellular localization. alpha and beta isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5'-triphosphates and nucleoside-5'-diphosphates. NTPDase2beta exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid, and differential regulation by protein kinases. NTPDase2beta was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2alpha activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties.
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PMID:C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation. 1536 80

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.
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PMID:ATP and purinergic receptor-dependent membrane traffic in bladder umbrella cells. 1611 Mar 27


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