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)

Diacylglycerols (DAG) play an important role in metabolism, signal transduction and protein kinase activation. Naturally occurring DAGs usually contain a saturated chain in the 1-position and an unsaturated chain in the 2-position. We have investigated the physical behavior of 1-stearoyl-2-oleoyl-sn-glycerol (sn-SODG) both in the dry and hydrated states by means of differential scanning calorimetry, X-ray diffraction, and NMR. In the dry state the saturated stearate and unsaturated oleate chains have difficulties in packing. As a result marked polymorphism occurs as the chains try to find a suitable packing. Eight phases were found in the dry state: alpha (transition temperature = 16.4 degrees C; delta H = 6.8 kcal/mol); beta 4 (20.7 degrees C; 13.8); beta 3 (21.5 degrees C; 13.8); beta 2 (22.2 degrees C; 14.4); beta 1 (23.1 degrees C; 12.3); beta' (25.7 degrees C; 11.9); gamma 1 (-2.9 degrees C; 0.5), and gamma 2 (-5.9 degrees C; 1.2), all of relatively low stability compared to 1,2 distearoyl-sn-glycerol (beta', 77.2 degrees C; 30.6). gamma 1 and gamma 2 are metastable low temperature phases. beta 1-beta 4 are bilayers (d001 = 3.4, 43.4, 44.7, 46.1 A, respectively) with elements of triclinic parallel chain packing, while beta' is a bilayer (d001 = 47.1 A) with orthorhombic perpendicular chain packing. The metastable alpha phase has hexagonal chain packing and an unusual eight-layer structure (d001 = 174 A). Hydrated sn-SODG contains about one-half of a water molecule per diacylglycerol. Three phases can be distinguished gamma w, alpha w (15.1 degrees C; 6.7) and beta w (19.9 degrees C, 14.3). Both alpha w and beta w are bilayers but alpha w has hexagonal chain packing and beta w is predominantly triclinic parallel packing. Thus, when saturated and unsaturated chains must pack side by side, complex chain conformation, disorder, and instability result giving rise to marked polymorphism. Hydration appears to partly stabilize the interactions.
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PMID:Physical behavior of the mixed chain diacylglycerol, 1-stearoyl-2-oleoyl-sn-glycerol: difficulties in chain packing produced marked polymorphism. 822 44

Cyclic AMP (cAMP)-dependent protein kinase (cAMP-kinase) partially purified from the membrane fractions of rat brains was stimulated by novel phosphonoglycosphingolipids (glycolipids) derived from the skin and nerve fibers of Aplysia kurodai. Among various glycolipids tested, a major glycolipid from the skin, 3-O-MeGal beta 1-->3GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2](2-aminoethylphosphonyl-->6)Glc beta 1-->4Glc beta 1-->1ceramide (SGL-II), was most potent, giving half-maximal activation at 32.2 microM. Activation of cAMP-kinase was maximal with 250 microM SGL-II using kemptide as substrate. The effect of SGL-II was additive on kinase activity at submaximal concentrations of cAMP. The kinase activity activated with SGL-II was inhibited by the addition of protein kinase inhibitor peptide, a specific peptide inhibitor for cAMP-kinase. Its inhibitory pattern was similar to that for the catalytic subunit. Of the various substrates tested, the glycolipid-stimulated cAMP-kinase could phosphorylate microtubule-associated protein 2, synapsin I, and myelin basic protein but not histone H1 and casein. The regulatory subunit strongly inhibited the activity of purified catalytic subunit of cAMP-kinase. This inhibition was reversed by addition of SGL-II, as observed for cAMP. SGL-II was capable of partially dissociating cAMP-kinase, which was observed by gel filtration column chromatography. However, the binding activity of cAMP to the holoenzyme was not inhibited with SGL-II. These results demonstrate that the glycolipids can directly activate cAMP-kinase in a manner similar, but not identical, to that of cAMP.
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PMID:Glycolipids isolated from Aplysia kurodai can activate cyclic adenosine 3',5'-monophosphate-dependent protein kinase from rat brain. 826 47

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.
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PMID:Enhancement of recombinant gamma-aminobutyric acid type A receptor currents by chronic activation of cAMP-dependent protein kinase. 826 57

Amyloid beta-protein (A beta) is the major protein of cerebrovascular and plaque amyloid in Alzheimer's disease (AD). Extensive evidence has demonstrated abnormal protein phosphorylation in this disease. We investigated the effect of synthetic A beta with the amino-acid sequence corresponding to cerebrovascular A beta and plaque A beta on the activities of casein kinase I (CK I) and casein kinase II (CK II). These enzymes were purified from bovine brain and casein was used as a substrate. A beta was found to stimulate markedly CK I- and CK II-mediated phosphorylation of casein in a concentration-dependent manner. The effect of plaque A beta was considerably higher than that of cerebrovascular A beta. Heparin, which is known to be a specific inhibitor of CK II, completely inhibited A beta-stimulated CK II activity. A beta itself was not a substrate for casein kinases. These findings were confirmed using other substrates for CK I and CK II. The experiments with synthetic CK II-substrate peptide (Leu-Glu-Leu-Ser-Asp-Asp-Asp-Asp-Glu) and the phosphorylation of erythrocyte membrane proteins by intrinsic membrane-bound CK I in erythrocytes showed marked stimulation in activities of casein kinases in the presence of A beta 1-40 or blocked A beta. We propose that A beta, by stimulating casein kinases, may contribute to abnormal protein phosphorylation in AD, in particular to increased phosphorylation of microtubule-associated proteins, leading to the neurofibrillary tangles formation and neurodegeneration in this disease. Interaction of A beta with protein kinases, thus, may characterize the beginning of the disease.
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PMID:Amyloid beta-protein stimulates casein kinase I and casein kinase II activities. 828 80

Tumor cell interaction with endothelial cells is a crucial step leading to organ-selective metastasis. Adhesion of murine B16 amelanotic melanoma cells (B16a) to murine microvascular endothelial cells (CD3) was enhanced, in a dose- and time-dependent manner, by pretreating CD3 cells with 12(S)-hydroperoxyeicosatetraenoic acid [i.e., 12(S)-HETE], a 12-lipoxygenase metabolite of arachidonic acid. The metabolic precursor of 12(S)-HETE, 12-HPETE (12-hydroperoxyeicosatetraenoic acid) also enhanced B16a cell adhesion to CD3 monolayers, whereas other lipoxygenase products, i.e., 5(S), 11(S), and 15(S)-HETEs were ineffective. 12(S)-HETE-enhanced tumor cell adhesion was blocked by treating endothelial cells with antibodies against the alpha v beta 3 complex or against individual subunits but not with antibodies against alpha 5 beta 1. In contrast, neither of these two integrins appeared to be involved in tumor cell adhesion to unstimulated endothelium. Flow cytometric analysis, immunofluorescent labeling, and image analysis indicated that 12(S)-HETE induced a time- and dose-dependent increase in the surface expression of alpha v beta 3 but not alpha 5 beta 1 on CD3 cells. The increased surface expression of alpha v beta 3 on endothelial cells did not result from an increased transcription or translation of alpha v beta 3 message as confirmed by quantitative reverse transcription-polymerase chain reaction, Northern blotting, and quantitative Western blotting. Instead, subcellular fractionation studies revealed an increased translocation of alpha v beta 3 integrins from the cytosolic pool to the membrane fractions. Pretreatment of endothelial cells with several cytoskeleton-disrupting agents (i.e., cycloheximide or acrylamide to disrupt intermediate filament vimentin, cytochalasin D to disrupt microfilaments, colchicine or Nocodazole to disrupt microtubules) abolished the 12(S)-HETE-enhanced alpha v beta 3 surface expression as well as tumor cell adhesion to endothelial cells. Also, pretreatment of CD3 cells with protein kinase C inhibitor calphostin C, but not with protein kinase A inhibitor H8, blocked 12(S)-HETE-enhanced alpha v beta 3 surface expression and tumor cell adhesion. Collectively, these results suggest that eicosanoid 12(S)-HETE modulates tumor cell interaction with endothelium via protein kinase C- and cytoskeleton-dependent up-regulation of the surface expression of alpha v beta 3 integrin.
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PMID:Activation of microvascular endothelium by eicosanoid 12(S)-hydroxyeicosatetraenoic acid leads to enhanced tumor cell adhesion via up-regulation of surface expression of alpha v beta 3 integrin: a posttranscriptional, protein kinase C- and cytoskeleton-dependent process. 831 70

AP-1 is a transcriptional activator composed of homo- and heterodimers of Jun and Fos proteins. It is involved in activation of genes, such as collagenase, stromelysin, IL-2 and TGF beta 1, by tumour promoters, growth factors and cytokines. AP-1 activity is also elevated in response to transforming oncogenes and is required for cell proliferation. AP-1 activity is subject to complex regulation both transcriptionally and post-transcriptionally. Transcriptional control of jun and fos gene expression determines the amount and composition of the AP-1 complex. The jun and fos genes are regulated both positively and negatively and are highly inducible in response to extracellular stimuli. Post translational control is also important. Both cJun and cFos are subject to regulated phosphorylation. In the case of cJun, phosphorylation of sites near the DNA-binding domain inhibits DNA-binding, while dephosphorylation reverses this inhibition. Phosphorylation of cJun on sites within the N-terminal activation domain increases its ability to activate transcription. The protein kinase phosphorylating these sites is stimulated by cytokines and growth factors. Another mechanism modulating AP-1 activity is transcriptional interference by members of the nuclear receptor family and is relevant for the pathophysiology of rheumatoid arthritis (RA). In RA, chronic inflammation leads to increased AP-1 activity in T cells,macrophages and synoviocytes as a response to secretion of cytokines such as IL-1 and TNF alpha. While the IL-2 gene plays a major role in T cell activation, another AP-1 target gene encodes an enzyme, collagenase, responsible for destruction of bone and tendon.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Various modes of gene regulation by nuclear receptors for steroid and thyroid hormones. 831 34

Two forms (I and II) of phosphoinositide-specific phospholipase C (PLC) were purified from the cytosol of bovine iris sphincter by sequential chromatography on DEAE-Sepharose, EAH-Sepharose, heparin-Sepharose, Sephacryl S-200 gel filtration and Mono Q HR columns. The final step resulted in specific activities of PLC-I and PLC-II of 4.3 and 5.9 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein, which represented up to 295-fold purification compared with that of the starting supernatant. The purified enzymes were further investigated for the presence of isoenzymes and characterized for molecular mass, substrate specificity, pH, Ca2+ requirements and kinetic parameters. Using monoclonal antibodies, PLC-I was identified as PLC-delta 1. The apparent molecular mass of PLC-I as determined by SDS/PAGE and gel filtration was 85 kDa. PLC-II contained an apparently invisible protein band that reacted with the antibody against PLC-gamma 1, and a major 109 kDa protein band that was not recognized by any of the PLC monoclonal antibodies. Further purification of PLC-II by size-exclusion h.p.l.c. resulted in elution of the enzyme activity as a single peak which corresponded to 109 kDa position. Again, this PLC activity was not recognized by any of the PLC monoclonal antibodies. However, the 109 kDa protein activity was recognized by a polyclonal antibody raised against a rat PLC-gamma 1 fragment (amino acids 1272-1287), thus suggesting that this protein is a proteolytic product of PLC-gamma 1. PLC-delta 1 and PLC-gamma 1 were identified in the supernatant fraction and PLC-beta 1 in the membrane fraction of the iris sphincter. Although immunologically different, the catalytic properties of PLC-I and PLC-II were quite similar. The Vmax and Km values for phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis were three to five times greater than those for PI hydrolysis. Both forms preferred PIP and PIP2 over PI and both were inactive against phosphatidylcholine. With PIP2 as substrate, the optimal pH values for PLC-I and PLC-II were 6.5 and 7.5 respectively. Unlike PIP2, PI hydrolysis by both forms was dependent on the presence of free Ca2+. The maximal hydrolysis of PI and PIP2 by both forms occurred at 200 and 5 microM Ca2+ respectively. Incubation of the purified enzymes with the catalytic subunit of protein kinase A (PKA) and [gamma-32P]ATP resulted in increased phosphorylation of PLC-I and PLC-II, but it had no inhibitory effect on their enzyme activities.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Purification and characterization of phosphoinositide-specific phospholipase C from bovine iris sphincter smooth muscle. 838 Sep 92

UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 1-6-N-acetylglucosaminyltransferase (i.e. core 2 GlcNAc-T) of the O-linked oligosaccharide pathway is developmentally regulated in human T cells, and changes in its activity have been associated with malignancies and the Wiskott-Aldrich immunodeficiency syndrome. Chinese hamster ovary (CHO) cells normally express low levels of core 2 GlcNAc-T activity (8-12 pmol/mg/h) which can be accurately measured with a two-step assay employing purified bovine beta 1-4Gal-T and high specific activity UDP-[3H]Gal to radiolabel the core 2 reaction product. CHO cells treated with 2 mM sodium butyrate for 24 h exhibited a 16-fold increase in core 2 GlcNAc-T activity, whereas several other differentiating agents including dimethyl sulfoxide, retinoic acid, phorbol ester, and cholera toxin had no effect on activity. The addition of butyrate, cholera toxin, or dimethyl sulfoxide to CHO cells slowed cell proliferation and induced changes in cell morphology characteristic of cell differentiation. Induction of core 2 GlcNAc-T by butyrate was blocked by actinomycin D and cycloheximide. Butyrate treatment also elevated cytosolic cAMP levels with a time course which paralleled, but preceded, induction of core 2 GlcNAc-T activity by approximately 8 h. The protein kinase inhibitors H-7 and H-8 blocked butyrate-dependent induction of enzyme activity, whereas the inactive analogue H1004 had no effect. Core 2 GlcNAc-T showed a change in Km for UDP-GlcNAc, from 0.50 mM in untreated cells to 4.54 mM in butyrate + cholera toxin treated CHO cells, but no changes in Km for the synthetic acceptor, Gal beta 1-3GalNAc alpha-para-nitrophenyl. Despite the 9-fold increase in Km for sugar nucleotide, Vmax/Km was 8.8-fold greater in treated compared with untreated cells. These observations suggest that in CHO cells induction of core 2 GlcNAc-T by butyrate treatment requires de novo gene transcription/translation, activation of protein kinase(s), and is associated with changes in the kinetic properties of the enzyme.
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PMID:Regulation of UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1-6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) in Chinese hamster ovary cells. 838 71

Previous work has shown that the GABAA-receptor (GABAA-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit/subtypes of the GABAA-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the beta 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the beta 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315-1317) that both PKA and PKC phosphorylate the beta-subunit of the GABAA-R.
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PMID:Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: analysis of the substrate domain. 838 79

The effect of calcium-phospholipid-dependent protein kinase (PKC) on GABAA receptor function was examined in Xenopus oocytes expressing recombinant human GABAA receptor using two-electrode voltage-clamp measurements. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, inhibited GABA-gated chloride currents by approximately 72% in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. Phorbol 12-monomyristate (PMM), a negative control analogue of PMA, did not alter GABAA receptor responses. To investigate whether activation of PKC could alter the modulatory responses of the receptor complex the effect of PMA on benzodiazepine and barbiturate potentiation of GABA responses was assessed. In oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs, diazepam (300 nM) potentiated GABA responses by approximately 160%. Following PMA (5-25 nM) treatment, diazepam potentiation was significantly increased to 333%. No effect of the inactive phorbol ester PMM (25 nM) was observed on diazepam potentiation of GABA responses. PMA enhancement of diazepam potentiation of GABA responses was also observed in oocytes expressing alpha 1 beta 1 gamma 2S subunit cDNAs, indicating that the unique PKC site present in the gamma 2L subunit is not required for observing the PMA effect. PMA (5-25 nM) also enhanced pentobarbital potentiation of GABA responses. In oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs, pentobarbital (25 microM) potentiated GABA receptor responses by approximately 97%. Following treatment with PMA (5-25 nM), pentobarbital potentiation of GABA responses increased to approximately 156%. The present results suggest that protein phosphorylation may alter the coupling between the allosteric modulatory sites within the GABAA receptor complex.
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PMID:Activation of calcium-phospholipid-dependent protein kinase enhances benzodiazepine and barbiturate potentiation of the GABAA receptor. 838 29


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