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)

Rainbow trout were used to investigate the hormonal regulation by glucagon and insulin of hepatic triacylglycerol (TG) lipase activation. Two purified preparations of the trout hepatic TG lipase enzyme, the 110,000-g preparation and the resuspended ammonium sulfate fraction (ASF), were activated up to 58% with (in mM) 0.5 ATP, 0.01 cAMP, 5 MgCl2, and exogenous protein kinase over control levels. ATP or cAMP alone had no effect on activation. Activation of the trout hepatic lipase was reversible; complete inactivation of the ASF was obtained within 3 h in the presence of exogenous phosphorylase phosphatase. Adenosine 3',5'-cyclic monophosphate (cAMP)/ATP-dependent 32P-phosphorylation of trout hepatic lipase was observed within 5 min of incubation with the cAMP/ATP-Mg2+ activation system and 25 microCi [32P]ATP. Hormonal modulation of trout hepatic lipase phosphorylation was studied in isolated hepatocytes. Hepatocytes were incubated with [32P]-monopotassium phosphate for 3 h, then exposed to mammalian glucagon (GLU). Within 5 min, increased lipolysis was accompanied by a 95% increase in phosphorylation of the enzyme. Mammalian insulin (INS) depressed GLU-stimulated phosphorylation by 56% and inhibited GLU-stimulated lipolysis. These results indicate that GLU and INS modulate lipolysis in trout liver by altering phosphorylation of the TG lipase enzyme.
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PMID:Glucagon and insulin regulate lipolysis in trout liver by altering phosphorylation of triacylglycerol lipase. 834 95

Although G-protein- and protein kinase-mediated pathways have been reported to activate phospholipase D (PLD) following cell stimulation, the relation between these activation pathways and the mechanistic details of lipase stimulation remain unknown. We have studied activation of PLD by GTP gamma S (guanosine 5'-O-(thiotriphosphate)), and its potentiation by ATP, in a cell-free system derived from U937 human promonocytic leukocytes. ATP, in the micromolar to millimolar range, significantly augmented GTP gamma S-stimulated PLD activity (2.6-fold) and the combination resulted in a 15-fold increase in PLD activity compared to control. ATP alone did not stimulate PLD activity. Measurement of endogenous cytosolic ATP levels and nucleotide depletion with activated charcoal demonstrated that stimulation of PLD by GTP gamma S proceeds by both ATP-dependent and -independent pathways. Nucleotide specificity data suggested that the ATP-dependent pathway involves kinase activity. The tyrosine phosphatase inhibitor vanadate augmented PLD activity stimulated by GTP gamma S/ATP by 41% (p < 0.01). Conversely, the tyrosine kinase inhibitors genistein and herbimycin A decreased PLD activity stimulated by GTP gamma S/ATP by 58 and 35%, respectively (p < 0.001 for each). Mixing experiments utilizing subcellular fractions from herbimycin A-treated cells suggested that the relevant tyrosine kinase activity is membrane-associated. Despite its role in ATP-induced potentiation, tyrosine kinase activity is neither necessary nor sufficient for activation of PLD in this system. Protein kinase C (PKC) is unlikely to play a role in potentiation by ATP as PKC activity is not stimulated under conditions of maximal PLD activation.
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PMID:ATP-induced potentiation of G-protein-dependent phospholipase D activity in a cell-free system from U937 promonocytic leukocytes. 837 59

The mechanisms responsible for the diminished lipolytic response of adipocytes to catecholamines after litter removal from lactating rats and their modulation by growth hormone have been investigated. Lactation, litter removal and growth-hormone treatment did not alter the ability of noradrenaline to activate protein kinase A (A-kinase), showing that the defect in signal transduction in rats after litter removal is after A-kinase. Litter removal had no effect on hormone-sensitive lipase activity itself, but the proportion of the lipase associated with the fat droplet was decreased; growth-hormone treatment increased hormone-sensitive lipase activity and the proportion associated with the fat droplet. In addition, a number of other adaptations in the beta-adrenergic signal-transduction system occur during the lactation cycle and in response to growth hormone treatment, including changes in receptor number, adenylate cyclase activity and cyclic AMP phosphodiesterase activity, but a defect in the ability of hormone-sensitive lipase to associate with the lipid droplet appears to be the major reason for the diminished response to catecholamines on litter removal.
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PMID:Mechanisms involved in the adaptations of the adipocyte adrenergic signal-transduction system and their modulation by growth hormone during the lactation cycle in the rat. 838 54

In the previous studies we have shown that testosterone increases lipolytic responsiveness to catecholamines in rat white adipocytes, and that is associated with an up-regulation of beta-adrenergic receptor density. However, the postreceptor events involved in the testosterone induced enhancement of beta-adrenergic receptor activated lipolysis in these cells have not been adequately studied, and were therefore investigated in the present study. Male Sprague Dawley rats were divided into three groups: control, castrated, and castrated treated with testosterone. The beta-adrenergic receptor-mediated cAMP accumulation, measured with RIA after isoproterenol (a beta-adrenergic agonist) stimulation was decreased in castrated rats, and reversed by testosterone treatment, suggesting a testosterone effect at or proximal to adenylate cyclase. However, no differences between the groups were found in abundance of G alpha protein messenger RNAs (G alpha s, G alpha i-1, and G alpha i-2) as analyzed by Northern blot and a solution hybridization RNase protection assay, or in G protein mass measured with a quantitative enzyme-linked immunosorbent assay in fat cell membrane preparation. Lipolysis stimulated by N6-monobutyryl-cAMP was reduced in castrated rats and recovered by testosterone treatment, suggesting that components distal to the adenylate cyclase, i.e. protein kinase A (PKA) and/or hormone sensitive lipase (HSL) also are involved in testosterone regulation of lipolysis. In conclusion, these and previous results suggest that the testosterone-induced increase in lipolytic response to catecholamines in rat white adipocytes is mediated through several events including an increased beta-adrenergic receptor density, probably an increased adenylate cyclase activity and an increased protein kinase A/hormone sensitive lipase activity at the postreceptor level with apparent absence of effect on the expression of G-proteins.
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PMID:Postreceptor events involved in the up-regulation of beta-adrenergic receptor mediated lipolysis by testosterone in rat white adipocytes. 838 92

Mobilization of lipids from adipose tissue during prolonged exercise is of key importance for the supply of energy to the working muscle. During exercise lipid mobilization is mainly stimulated by increased catecholamine production leading to acceleration of the beta-adrenoceptor mediated lipolysis rate in fat cells. This causes breakdown of triglycerides in fat cells to glycerol and free fatty acids, which then are delivered to the blood stream. Decreased insulin production, enhanced adipose tissue blood flow and decreased reesterification of free fatty acids in fat cells contribute to the enhancement of lipid mobilization during strenuous and long-term light exercise. Several additional factors modulate the lipolytic response to exercise as well. Endurance training increases the lipolytic action of catecholamine whereas the opposite occurs during ageing. These alterations are at least in part mediated by changes in the function of the final step in lipolysis activation, the protein kinase-hormone sensitive lipase complex. There are also gender and regional differences in the lipolytic response to exercise. Women mobilize more lipids from the subcutaneous abdominal area than men, whereas a low rate of lipid mobilization from the peripheral subcutaneous areas is observed in either sex. In pathophysiological states, which are associated with catabolism such as fasting and insulin dependent diabetes, there is an enhanced lipolytic response to exercise, because of increased beta-adrenoceptor function.
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PMID:Impact of exercise on adipose tissue metabolism in humans. 858 Oct 90

The purpose of the present study was to determine the possible interaction of cyclic AMP (cAMP) and the synthesis of prostacyclin [measured as immunoreactive 6-keto-prostaglandin (PG)F1 alpha] elicited by the beta adrenergic receptor agonist isoproterenol (ISOP), in freshly dissociated rabbit ventricular myocytes. ISOP (10(-13) to 10(-11) M) increased 6-keto-PGF1 alpha synthesis without altering the level of cAMP. Increasing the concentration of ISOP from 10(-10) to 10(-7) M enhanced accumulation of cAMP, which was associated with a decline in 6-keto-PGF1 alpha synthesis. Forskolin (10(-6) M), an activator of adenylyl cyclase, and 3-isobutyl-1-methylxanthine (10(-5) M), an inhibitor of cAMP phosphodiesterase, increased cAMP accumulation and inhibited ISOP-induced 6-keto-PGF1 alpha synthesis. 8-(4-chlorophenylthio) (cpt)-cAMP (10(-7) M) also inhibited ISOP-induced 6-keto-PGF1 alpha production. On the other hand, miconazole (10(-4) M), an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-induced 6-keto-PGF1 alpha synthesis in myocytes. Miconazole also attenuated ISOP-, forskolin- and cpt-cAMP-induced increases in protein kinase A activity. The protein kinase A inhibitor H-89 {N-[2-(p-bromocinnamylamino)ethyl] -5-isoquinolinesulfonamide} attenuated the ISOP (10(-7) M)-induced increase in the activity of this enzyme and minimized the decline in 6-keto-PGF1 alpha synthesis produced by 10(-7) M ISOP and the inhibitory effect of cpt-cAMP and forskolin on 6-keto-PGF1 alpha production. 3-Isobutyl-1-methylxanthine, forskolin and cpt-cAMP did not alter the conversion of exogenous arachidonic acid to 6-keto-PGF1 alpha. These data indicate that beta adrenergic receptor activation promotes prostacyclin synthesis in rabbit ventricular myocytes and that cAMP acts as an inhibitory modulator. This action is mediated via activation of protein kinase A, probably by decreasing the activity of the lipase, involved in beta adrenergic receptor-induced arachidonic acid release.
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PMID:Modulation by cyclic AMP of beta adrenergic receptor-stimulated prostacyclin synthesis in rabbit ventricular myocytes. 876 95

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.
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PMID:Domain-structure analysis of recombinant rat hormone-sensitive lipase. 891 75

Bile-salt-dependent lipase (BSDL) is secreted by the pancreas into the duodenum, where it catalyses the hydrolysis of dietary lipid esters on activation by bile salts. The secretion pathway of BSDL is comparable with that of other digestive enzymes produced by pancreatic acinar cells. However, in contrast with these other enzymes, BSDL is partly associated with endoplasmic reticulum membranes as part of a folding complex, including a Grp94-related protein to which BSDL is transiently linked. The release of BSDL from membranes occurs once its glycosylation is completed [Bruneau and Lombardo (1995) J. Biol. Chem. 270, 13524-13533]. In the present study, investigations concerning the mechanism of association/dissociation of BSDL with membranes of microsomes were performed. For this purpose the role of ATP and that of the possible phosphorylation of BSDL were examined. For the first time, it is shown that human pancreatic BSDL is phosphorylated, probably at a serine residue, during its transport within the acinar cell. The phosphorylation of BSDL is provoked by calphostin C, an inhibitor of protein kinase C. In the presence of 1-(isoquinolinesulphonyl)2-methylpiperazine, a non-specific inhibitor of serine/threonine protein kinase A, C or G, or of calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N', N'-tetra-acetic tetra(acetoxymethyl)ester, the phosphorylation of BSDL elicited by calphostin C is abolished. These data suggested that the phosphorylation of BSDL within human pancreatic microsomes is under the control of a cascade of protein kinases. We have also shown that the phosphorylation of BSDL appears to be involved in the release of the enzyme from microsome membranes. Nevertheless ATP, which modifies the conformation of BSDL, triggers this association, and an unhydrolysable ATP analogue was unable to promote it.
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PMID:Association of bile-salt-dependent lipase with membranes of human pancreatic microsomes is under the control of ATP and phosphorylation. 935 26

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.
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PMID:Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase. 935 44

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triacylglycerols and cholesteryl esters. HSL activity is regulated via phosphorylation-dephosphorylation, with cyclic AMP-dependent protein kinase increasing activity following phosphorylation of a single serine and Ca2+/calmodulin-dependent protein kinase II phosphorylating another serine at a basal site. The current studies used site-directed mutagenesis to show that Ser-563 of rat HSL is phosphorylated by cyclic AMP-dependent protein kinase and that Ser-565 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II. Mutation of Ser-563-->Ala eliminated HSL hydrolytic activity against cholesteryl ester, triacylglycerol, and diacylglycerol substrates to the same extent as mutation of Ser-423-->Ala, the presumed catalytic site. Mutation of Ser-565-->Ala modestly decreased HSL activity. In contrast, mutation of Ser-563-->Asp preserved HSL hydrolytic activity and even increased activity 20% above the control wild-type enzyme. Molecular modeling of the catalytic pocket of HSL suggested the involvement of Val-710. Mutation of Val-710-->Ala resulted in an 85% loss of HSL hydrolytic activity. The results of these studies illustrate the importance of the presence of a hydroxyl group or negative charge at residue 563, either for proper conformation of rat HSL or for proper stabilization of substrate to allow maintenance of hydrolytic activity, as well as the importance of the involvement of additional amino acids in the catalytic pocket of the enzyme.
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PMID:Mutational analysis of structural features of rat hormone-sensitive lipase. 963 39


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