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)

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.
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PMID:Purification and characterization of choline/ethanolamine kinase from rat liver. 215 25

Phosphatidylcholine is apparently essential for mammalian life, since there are no known inherited diseases in the biosynthesis of this lipid. One of its critical roles appears to be in the structure of the eucaryotic membranes. Why phosphatidylcholine is required and why other phospholipids will not substitute are unknown. The major pathway for the biosynthesis of phosphatidylcholine occurs via the CDP-choline pathway. Choline kinase, the initial enzyme in the sequence, has been purified to homogeneity from kidney and liver and also catalyzes the phosphorylation of ethanolamine. Most evidence suggests that the next enzyme in the pathway, CTP:phosphocholine cytidylyltransferase, catalyzes the rate-limiting and regulated step in phosphatidylcholine biosynthesis. This enzyme has also been completely purified from liver. Cytidylyltransferase appears to exist in the cytosol as an inactive reservoir of enzyme and as a membrane-bound form (largely associated with the endoplasmic reticulum), which is activated by the phospholipid environment. There is evidence that the activity of this enzyme and the rate of phosphatidylcholine biosynthesis are regulated by the reversible translocation of the cytidylyltransferase between membranes and cytosol. Three major mechanisms appear to govern the distribution and cellular activity of this enzyme. (i) The enzyme is phosphorylated by cAMP-dependent protein kinase, which results in release of the enzyme into the cytosol. Reactivation of cytidylyltransferase by binding to membranes can occur by the action of protein phosphatase 1 or 2A. (ii) Fatty acids added to cells in culture or in vitro causes the enzyme to bind to membranes, where it is activated. Removal of the fatty acids dissociates the enzyme from the membrane. (iii) Perhaps most importantly, the concentration of phosphatidylcholine in the endoplasmic reticulum feedback regulates the distribution of cytidylyltransferase. A decrease in the level of phosphatidylcholine causes the enzyme to be activated by binding to the membrane, whereas an increase in phosphatidylcholine mediates the release of enzyme into the cytosol. The third enzyme in the CDP-choline pathway, CDP-choline:1,2-diacylglycerol choline-phosphotransferase, has been cloned from yeast but never purified from any source. In liver an alternative pathway for phosphatidylcholine biosynthesis is the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase. This enzyme is membrane bound and has been purified to homogeneity. It catalyzes all three methylation reactions involved in the conversion of phosphatidylethanolamine to phosphatidylcholine.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Boehringer Mannheim Award lecture. Phosphatidylcholine metabolism: masochistic enzymology, metabolic regulation, and lipoprotein assembly. 226 10

The protective effects of protein kinase inhibitors and a calmodulin kinase inhibitor (W-7) against ischemic neuronal damage were examined in the CA1 subfield of the hippocampus. Staurosporine, KT5720, and KT5822 were used as inhibitors of protein kinase C (PKC), cyclic AMP-dependent protein kinase, and cyclic GMP-dependent protein kinase, respectively. All test compounds were injected topically into the CA1 subfield of the hippocampus. In the gerbil ischemia model, staurosporine (0.1-10 ng) administered 30 min before ischemia prevented neuronal damage in a dose-dependent manner. However, KT5720, KT5822, and W-7 were ineffective, even at a dose of 10 ng. In the rat ischemia model, staurosporine (10 ng) also prevented neuronal damage when administered before ischemic insult, although staurosporine administered 10 or 180 min after recirculation was ineffective. These results suggest the involvement of PKC in CA1 pyramidal cell death after ischemia and that the fate of vulnerable CA1 pyramidal cells through PKC-mediated processes could be determined during the early recirculation period.
J Cereb Blood Flow Metab 1990 Sep
PMID:Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat. 238 38

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.
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PMID:Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C. 311 87

We examined alterations in the cyclic AMP generating system and G protein subunits in gerbil hippocampus following 10 min of transient ischemia. In hippocampal slices, basal and isoproterenol- and forskolin-stimulated cyclic AMP accumulations were markedly increased at 6 and 24 h after ischemia. Interestingly, both the inhibition of forskolin-stimulated cyclic AMP and the potentiation of beta-adrenoceptor-stimulated cyclic AMP by a gamma-aminobutyric acidB receptor agonist were attenuated at these time points. Ischemia did not affect the immunolabeling of any of the G protein alpha subunits; only that of beta subunits was significantly decreased, by 28.2%, 4 days after ischemia. In contrast, pertussis toxin-catalyzed [32P]ADP ribosylation declined progressively during the late recirculation period, reaching a significant reduction (25.4%) at 6 h after ischemia. These results suggest that ischemia affects the heterotrimeric conformation (alpha beta gamma) of Gi/Go during the recirculation period, thereby leading to increased cyclic AMP production. Because cyclic AMP-dependent protein kinase A modulates the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-kainate receptor channels, postischemic sensitization of the cyclic AMP generating system may contribute to neuronal degeneration in the hippocampus.
J Cereb Blood Flow Metab 1995 Sep
PMID:Alterations in cyclic AMP generation and G protein subunits following transient ischemia in gerbil hippocampus. 767 81

The effects of cerebral ischemia on calcium/calmodulin-dependent kinase II (CaM kinase II) were investigated using the rat four-vessel occlusion model. In agreement with previous results using rat or gerbil models of cerebral ischemia or a rabbit model of spinal cord ischemia, this report demonstrates that transient forebrain ischemia leads to a reduction in CaM kinase II activity within 5 min of occlusion onset. Loss of activity from the cytosol fractions of homogenates from the neocortex, striatum, and hippocampus correlated with a decrease in the amount of CaM kinase alpha and beta isoforms detected by immunoblotting. In contrast, there was an apparent increase in the amount of CaM kinase alpha and beta in the particulate fractions. The decrease in the amount of CaM kinase isoforms from the cytosol but not the particulate fractions was confirmed by autophosphorylation of CaM kinase II after denaturation and renaturation in situ of the blotted proteins. These results indicate that ischemia causes a rapid inhibition of CaM kinase II activity and a change in the partitioning of the enzyme between the cytosol and particulate fractions. CaM kinase II is a multifunctional protein kinase, and the loss of activity may play a critical role in initiating the changes leading to ischemia-induced cell death. To identify a structural basis for the decrease in enzyme activity, tryptic peptide maps of CaM kinase II phosphorylated in vitro were compared. Phosphopeptide maps of CaM kinase alpha from particulate fractions of control and ischemic samples revealed not only reduced incorporation of phosphate into the protein but also the absence of a limited number of peptides in the ischemic samples. This suggested that certain sites are inaccessible, possibly due to a conformational change, a covalent modification of CaM kinase II, or steric hindrance by an associated molecule. Verifying one of these possibilities should help to elucidate the mechanism of ischemia-induced modulation of CaM kinase II.
J Cereb Blood Flow Metab 1995 May
PMID:Effect of cerebral ischemia on calcium/calmodulin-dependent protein kinase II activity and phosphorylation. 771 3

We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.
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PMID:N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89) inhibits incorporation of choline into phosphatidylcholine via inhibition of choline kinase and has no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. 828 Jan 5

The flow threshold for alterations of the in vitro [3H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [3H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA1 were 18, 34, and 49 ml 100 g-1 min-1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA, and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA1 than in other brain regions, suggesting that the hippocampus CA1 could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA1 and CA3, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions.
J Cereb Blood Flow Metab 1996 May
PMID:Flow threshold for reduction of cyclic AMP binding in the hippocampus CA1 and other brain regions during stroke development in gerbils. 862 51

We examined the effects of FK506, a specific inhibitor of calcineurin, on the binding capacity of cyclic AMP-dependent protein kinase (cAMP-DPK) in gerbils subjected to 2-h cerebral hemispheric ischemia. FK506 (0.1 mg/kg) was infused intravenously at 15 min prior to the induction of ischemia by common carotid artery occlusion. The binding capacity of cAMP-DPK was evaluated by autoradiographic analysis of the cAMP binding, and cerebral blood flow (CBF) was measured by the [14C] iodoantipyrine method. In the sham-operated gerbils. FK506 significantly increased mean arterial blood pressure and tended to decrease CBF, suggesting that FK506 may constrict systemic blood vessels as well as cerebral blood vessels. On the other hand, cAMP binding was not altered by FK506 in the sham-operated gerbils. In the ischemia group of gerbils, FK506 prevented any significant reduction of cAMP binding in the hippocampus CA1 and cerebral cortices on the ischemic side, whereas it exerted no significant influence on the cAMP binding of the nonischemic side. The values of CBF were comparable between the vehicle-treated gerbils and FK506-treated gerbils in the ischemic regions. Preservation of cAMP binding indicates that intracellular signal transduction via cAMP-DPK can be maintained by FK506 despite ischemia, suggesting that this agent may be beneficial for reducing ischemic tissue damage.
J Cereb Blood Flow Metab 1997 Apr
PMID:Calcineurin inhibitor, FK506, prevents reduction in the binding capacity of cyclic AMP-dependent protein kinase in ischemic gerbil brain. 914 23

Choline kinase, the initial enzyme of the CDP-choline pathway, mediates the conversion of choline to phosphorylcholine and is localized in the supernatant fraction of cells. The enzyme also catalyzes the phosphorylation of ethanolamine, functioning as the initial enzyme of the CDP-ethanolamine pathway as well. Yeast choline kinase is encoded by a single structural gene, CKI, which was cloned by the genetic complementation of the choline kinase mutation cki. The deduced sequence comprises 582 amino acid residues with a molecular mass of 66316 Da and bears local sequence similarity to various protein kinases and bacterial antibiotic phosphotransferases. The expression of yeast choline kinase is transcriptionally repressed by myo-inositol and choline in a coordinate manner with other phospholipid-synthesizing enzymes in yeast.
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PMID:Choline kinase from yeast. 937 Mar 17


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