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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitogen-activated protein kinase kinases (MKKs or MEKs) are dual specificity tyrosine/threonine protein kinases that are activated by phosphorylation at two closely spaced serine residues (serines-218 and -222) by the c-mos and raf proto-oncogenes. This double phosphorylation is both necessary and sufficient for MEKs to activate the MAP kinase enzymes in vitro. The specificity or regulation of in vivo signaling to the mammalian MEKs (MEK1 and MEK2) was recently reported also to involve the differential phosphorylation of a proline-rich peptide located between the MEK kinase-subdomains IX and X. Here we report the purification and characterization of an auto-activating protein kinase from bovine brain that phosphorylates serine-298 of the MEK1 and MEK2 proline-rich insert peptides. The auto-activation of the MEK-S298 peptide kinase is the result of an intermolecular phosphorylation event that can be prevented by the peptide substrates. The inactive kinase migrates on gel filtration as a 90 kDa protein, and after activation as a 43 kDa phosphoprotein. Incorporation of 32P[phosphate] into 40-42 kDa proteins on SDS-PAGE parallels the activation of the enzyme, and dephosphorylation by protein phosphatase 2Ac reverses the activation. SDS-PAGE renaturation assays show that the 40 kDa protein has the capacity to autophosphorylate, and exhibits kinase activity towards myelin basic protein after activation. Phosphorylation of purified bovine brain MEK or recombinant MEK1 by the auto-activated kinase does not activate the enzyme, and does not interfere with the in vitro raf-mediated MEK activation. We conclude that still unknown kinases may control the MAP kinase pathway by targeting MEK.
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PMID:Identification and characterization of an auto-activating MEK kinase from bovine brain: phosphorylation of serine-298 in the proline-rich domain of the mammalian MEKs. 941 3

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
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PMID:A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II. 944 23

Protein tyrosine phosphatases were analyzed in oocytes of Ascaris suum. Phosphatases dephosphorylating modified acidic lysozyme were present in high-molecular-weight form (M(r) > 600,000) and as a 50- to 55-kDa protein in the soluble fraction. The low-molecular-weight form of the phosphatase cross-reacted with an antiserum raised against human T-cell protein tyrosine phosphatase and was not distinguishable from the 50- to 55-kDa protein tyrosine phosphatase previously described in the muscular layer of the adult worms (B. Schmid et al. 1996, Molecular and Biochemical Parasitology 77, 183-192). The low-molecular-weight form was also present on immunoblots of high-molecular-weight protein tyrosine phosphatase preparations after denaturing electrophoresis. The same or a similar form of the tyrosine phosphatase was also found in detergent extracts from the pelletal fraction. In addition, another tyrosine phosphatase of 180 kDa molecular mass that dephosphorylated myelin basic protein was also found in extracts from the soluble compartment as well as in detergent extracts from the pelletal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and was resistant to inhibition by vanadate. While the activities of the myelin basic protein-dephosphorylating protein phosphatase remained fairly constant during early development of the oocytes, the activity of the enzyme dephosphorylating modified lysozyme in the pelletal fraction decreased to less than 10% of the initial activity between days 3 and 28 of incubation. Immunocytochemical studies of unfertilized and developing Ascaris eggs revealed association of protein tyrosine kinase and protein tyrosine phosphatase with the egg shell, in addition to their presence in the neighborhood of mitochondria. The amount of enzyme changed with the stage of development. In the larval stage (21 days) protein tyrosine kinase had increased in the chitin layer of the shell and in the nuclei while the relative amount of tyrosine phosphatase decreased in accordance with the biochemical data.
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PMID:Ascaris suum: protein phosphotyrosine phosphatases in oocytes and developing stages. 953 68

Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti-MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA). We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein. The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor. The activation of MAPKs is discussed in relation to the early responses induced by cryptogein.
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PMID:Activation of MAPK homologues by elicitors in tobacco cells. 980 16

The effect of immunosuppressant cyclosporin A (CsA) on inward-rectifying K(+)-channels and biochemical analysis have indicated the presence of cyclophilin in guard cells of Vicia faba. In this study, we identified a full-length cDNA sequence, vcCyP, encoding cyclophilin (CyP), a peptidyl-prolyl cis-trans isomerase of guard cell protoplasts (GCPs) from Vicia faba L. The deduced amino acid sequence revealed that vcCyP contained 171 amino acid residues and exhibited a strong similarity to previously described cytosolic CyP isoforms from other plants. vcCyP had seven extra amino acid residues, which is a characteristic of the cytosolic form of plant CyPs. A complex of recombinant vcCyP and CsA inhibited the phosphatase activity of bovine calcineurin, a type 2B protein phosphatase, with a half-inhibitory concentration of 0.2 microM. Protein phosphatase activity was measured in the cytosolic fraction of GCPs using a 32P-labeled myelin basic protein (32P-MBP) and the activity was increased by a physiological concentration of Ca2+ (1 microM). This Ca(2+)-stimulated phosphatase activity was inhibited by CsA, suggesting the presence of both cytosolic CyP and calcineurin-like protein phosphatase in guard cells. Northern blot analysis showed that the transcription level of vcCyP was much higher in GCPs than in root and leaf tissues of Vicia.
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PMID:Characterization of cytosolic cyclophilin from guard cells of Vicia faba L. 1018 2

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.
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PMID:Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases. 1069 77

Recombinant I(1)(PP2A) and I(2)(PP2A) did not affect the activity of the catalytic subunit of protein phosphatase 1 (PP1(C)) with (32)P-labeled myelin basic protein, histone H1, and phosphorylase when assayed in the absence of divalent cations. However, in the presence of Mn(2+), I(1)(PP2A) and I(2)(PP2A) stimulated PP1(C) activity by 15-20-fold with myelin basic protein and histone H1 but not phosphorylase. Half-maximal stimulation occurred at 2 and 4 nM I(1)(PP2A) and I(2)(PP2A), respectively. Moreover, I(1)(PP2A) and I(2)(PP2A) reduced the Mn(2+) requirement by about 30-fold to 10 microM. In contrast, PP1(C) activity was unaffected by I(1)(PP2A) and I(2)(PP2A) in the presence of Co(3+) (0.1 mM), Mg(2+) (2 mM), Ca(2+) (0.5 mM), and Zn(2+) (0.1 mM). Following gel filtration chromatography on Sephacryl S-200 in the presence of Mn(2+), PP1(C) coeluted with I(1)(PP2A) and I(2)(PP2A) in the void volume. However, when I(1)(PP2A) and I(2)(PP2A) or Mn(2+) were omitted, PP1(C) emerged with a V(e)/V(0) of approximately 1.6. The results demonstrate that I(1)(PP2A) and I(2)(PP2A) associate with and modify the substrate specificity of PP1(C) in the presence of physiological concentrations of Mn(2+). A novel role is suggested for I(1)(PP2A) and I(2)(PP2A) in the reciprocal regulation of two major mammalian serine/threonine phosphatases, PP1 and PP2A.
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PMID:Protein phosphatase 2A inhibitors, I(1)(PP2A) and I(2)(PP2A), associate with and modify the substrate specificity of protein phosphatase 1. 1073 57

Nuclear inhibitor of protein phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of protein phosphatase-1 (PP1(C)). We show here that mutation of these phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1(C). This could be accounted for by an additional inhibitory phosphatase-binding site in the C-terminal region (residues 311-351), with an inhibitory core corresponding to residues 331-337. Following mutation of all three PP1(C)-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1(C). Remarkably, while both NIPP1 domains inhibited the phosphorylase phosphatase activity of PP1(C) independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1(C) with its regulatory subunits.
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PMID:The C-terminus of NIPP1 (nuclear inhibitor of protein phosphatase-1) contains a novel binding site for protein phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding. 1110 70

The genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 comprises many open reading frames (ORFs) which putatively encode eukaryotic-type protein kinase and protein phosphatase. Based on gene disruption analysis, a region of the hypothetical ORF sll1575, which retained a part of the protein kinase motif, was found to be required for normal motility in the original isolate of strain PCC 6803. Sequence determination revealed that in this strain sll1575 was part of a gene (designated spkA) which harbored an entire eukaryotic-type Ser/Thr protein kinase motif. Strain ATCC 27184 and a glucose-tolerant strain derived from the same isolate as the PCC strain had a frameshift mutation dividing spkA into ORFs sll1574 and sll1575. The structural integrity of spkA agreed well with the motility phenotype, determined by colony morphology on agar plates. The spkA gene was expressed in Escherichia coli as a His-tagged protein, which was purified by Ni2+ affinity chromatography. With [gamma-32P]ATP, SpkA was autophosphorylated and transferred the phosphate group to casein, myelin basic protein, and histone. SpkA also phosphorylated several proteins in the membrane fraction of Synechocystis cells. These results suggest that SpkA is a eukaryotic-type Ser/Thr protein kinase and regulates cellular motility via phosphorylation of the membrane proteins in Synechocystis.
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PMID:A eukaryotic-type protein kinase, SpkA, is required for normal motility of the unicellular Cyanobacterium synechocystis sp. strain PCC 6803. 1116 79

Using SDS/polyacrylamide gels that contained myelin basic protein, we identified a 46-kDa protein kinase in tobacco that is transiently activated by cutting. Although the activity of the kinase was rarely detectable in mature leaves, marked activity became apparent within several minutes after isolation of leaf discs and subsided within 30 min. In the presence of cycloheximide (CHX), the kinase activity did not diminish after the isolation over the course of 2 hr, suggesting that protein synthesis was not required for the activation of the kinase. A second cutting of leaf discs between 30 min and 60 min after the isolation failed to activate the kinase, whereas a second cutting given 3 hr after isolation apparently activated the kinase. These results suggest that the 46-kDa protein kinase is desensitized immediately after the first activation, which can be blocked by CHX, but the response ability recovers with time. When protein extracts containing the active kinase were treated with serine/threonine-specific or tyrosine-specific protein phosphatase, the kinase activity was abolished. After immunoprecipitation with antibody against phosphotyrosine, activity of the kinase was recovered in the immunoprecipitate. These results suggest that the active form of the kinase is phosphorylated at both serine/threonine and tyrosine residues. It seems likely that the 46-kDa protein kinase can be activated by dual phosphorylation. The activity of a 46-kDa protein kinase was also detected in leaves of a wide variety of plant species including dicotyledonous and monocotyledonous plants. We propose the name PMSAP (plant multisignal-activated protein) kinase for this kinase because the kinase was also activated by various signals other than cutting.
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PMID:Cutting activates a 46-kilodalton protein kinase in plants. 1160 79


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