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)

We investigated the effects of insulin, insulin-like growth factor-I (IGF-I), and phorbol 12-myristate 13-acetate (PMA) on neutral cholesteryl esterase activity in cultured rat vascular smooth muscle cells. Insulin and IGF-I at concentrations between 10(-9) mol/L and 10(-6) mol/L significantly decreased neutral cholesteryl esterase activity in growth-arrested vascular smooth muscle cells in a dose-dependent manner but with no influences on the intracellular concentration of 3',5'-adenosine monophosphate (cyclic AMP). Treatment of cells with KT5720 (10(-7) mol/L to 10(-5) mol/L), a specific inhibitor of cyclic AMP-dependent protein kinase, significantly decreased neutral cholesteryl esterase activity in a dose-dependent manner. Incubation of cells for 6 to 12 hours with PMA (10(-9) mol/L to 10(-6) mol/L), an activator of protein kinase C, significantly increased neutral cholesteryl esterase activity in a dose-dependent manner. However, down-regulation of protein kinase C activity by long-term incubation (18 to 48 hours) with PMA resulted in a significant decrease in neutral cholesteryl esterase activity. Treatment of cells with UCN-01 (10(-7) mol/L to 10(-5) mol/L), a specific protein kinase C inhibitor, decreased the enzyme activity in a dose-dependent manner and completely blocked the activation of the enzyme by PMA. When insulin or IGF-I at a concentration of 10(-6) mol/L was present in the medium containing CL 277,082--an inhibitor of acyl coenzyme A:cholesterol acyltransferase--cellular cholesteryl ester content of the cells significantly increased. In contrast, after the treatment with PMA at a concentration of 10(-6) mol/L in the presence of CL 277,082, the net cholesteryl ester content of the cells significantly declined. These data suggest that both insulin and IGF-I may increase cholesteryl ester accumulation in arterial smooth muscle cells by decreasing arterial cholesteryl ester hydrolysis. The data also suggest that neutral cholesteryl esterase is activated not only by cyclic AMP-dependent protein kinase but also by protein kinase C. Thus growth factors may exert their antilipolytic or lipolytic actions specifically by modulating neutral cholesteryl esterase activity in vascular smooth muscle cells. Neutral cholesteryl esterase of vascular smooth muscle cells may be regulated by recholesteryl esterase of vascular smooth muscle cells may be regulated by reversible phosphorylation, with the phosphorylated form being the active form.
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PMID:Effects of insulin, insulin-like growth factor-I, and phorbol esters on neutral cholesteryl esterase activity in cultured rat vascular smooth muscle cells. 766 69

The mechanism for antiatherogenic effects of 17 beta-estradiol (E2) was investigated in J774 A.1 cells incubated with beta-VLDL. E2 at physiological concentrations (0.25 and 2.5 nM) inhibited an accumulation of cellular cholesteryl esters and enhanced their hydrolysis in foam cells. These phenomena were preceded by activation of neutral cholesterol esterase through an increase in cyclic AMP-dependent protein kinase activity. 17 alpha-estradiol, progesterone, and testosterone lacked such stimulatory effects on neutral cholesteryl esterase.
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PMID:The regulation of cholesteryl ester metabolism by 17 beta-estradiol in macrophages. Activation of neutral cholesterol esterase. 769 21

Hormone sensitive lipase (HSL) is an enzyme of relatively broad specificity, having the ability to hydrolyze tri-, di- and mono-acylglycerols as well as cholesterol esters and small water-soluble substrates. This broad specificity allows HSL to perform a variety of functions in several tissues. A key feature of HSL is its ability to be activated via phosphorylation by cyclic AMP-dependent protein kinase. In addition it is phosphorylated at a second site by several kinases, notably AMP-activated protein kinase. Phosphorylation of this site apparently plays a role in rendering the enzyme hormone-insensitive, in that prior phosphorylation at site 2 prevents phosphorylation and activation at site 1 by cyclic AMP-dependent protein kinase. Investigation of the protein phosphatases responsible for dephosphorylation of these sites has indicated that phosphatase 2A plays a predominant role but also that protein phosphatase 2C is a significant phosphatase targeted against both phosphorylation sites. Evidence indicates that HSL has at least three functional domains which contain (a) the phosphorylation sites which control activity, (b) the active site responsible for the catalytic activity and (c) a lipid binding site responsible for anchoring the lipase at the water-lipid interface. Using limited proteolytic studies we have found that it is possible to cleave HSL into several fragments including a stable domain of M(r) approximately 17.6 kDa which contains the active site serine residue. Digestion under similar conditions also generates a stable domain of M(r) approximately 11.5 kDa containing both phosphorylation sites. Furthermore, under appropriate conditions it is possible to digest HSL and retain activity against water-soluble substrates but with the concomitant loss of activity against triacylglycerol, implying that a lipid binding domain is lost during this procedure. HSL is responsible for the neutral cholesterol esterase activity in macrophages and it may play a role in the accumulation of cholesterol esters which occur during the development of foam cells. HSL activity is reduced in macrophage foam cells, at least partly due to increased activity of a cytosolic HSL inhibitor protein. A finding unexplained for many years has been that, although lipolysis can be stimulated 50-100-fold in adipocytes by lipolytic hormones, HSL can apparently only be activated 2-3-fold via phosphorylation in vitro by cyclic AMP-dependent protein kinase. One possibility to explain this discrepancy is that an additional anchoring protein is missing from the in vitro system and indirect evidence is now accumulating for such a protein.
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PMID:The multifunctional role of hormone-sensitive lipase in lipid metabolism. 794 81

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl2 and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca2+/calmodulin; this latter effect was blocked by the chelator ethylene-glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca2+, Ca2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.
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PMID:Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation. 813 99

Intracellular effector systems which utilize PKA and PKC can be pharmacologically activated by forskolin and phorbol 12-myristate 13-acetate (PMA) and appear to be important for regulation of steroidogenesis by cells of the corpus luteum. In this study the effect of pharmacologic activation of PKA (forskolin) or PKC (PMA) on the activity of adenylate cyclase, cholesterol esterase, P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5, delta 4 isomerase (3 beta HSD) was determined. Basal adenylate cyclase activity (as measured by intracellular and secreted cAMP) was extremely low in both large and small luteal cells. Forskolin stimulated adenylate cyclase activity in both large and small luteal cells but progesterone production was increased only in small cells. PMA inhibited progesterone production by large and forskolin-stimulated small cells without altering adenylate cyclase activity. Basal cholesterol esterase activity was greater in small than in large cells and was stimulated by forskolin only in small cells. PMA did not significantly alter cholesterol esterase activity in either cell type. Activity of P450scc or 3 beta HSD was measured by conversion of hydroxylated cholesterol derivatives (P450scc) or pregnenolone (3 beta HSD) to progesterone. Although basal progesterone production was 47 times greater in large than small cells, there was only 5.1 (P450scc) and 6.4 (3 beta HSD) times greater enzyme activity in large than in small luteal cells. Activation of PKA and/or PKC did not alter the activity of P450scc or 3 beta HSD in either cell type.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Steroidogenic enzyme activity after acute activation of protein kinase (PK) A and PKC in ovine small and large luteal cells. 814 91

Macrophages contain a neutral cholesteryl ester hydrolase that can be activated by cAMP-dependent protein kinase. Immunological studies strongly suggest that hormone-sensitive lipase (HSL) is probably responsible for the cholesteryl ester hydrolase activity in macrophages; however, due to the very low level of expression in macrophages, it has been difficult to determine whether the macrophage cholesteryl ester hydrolase and adipose HSL are, in fact, products of the same gene. We have used the sensitive polymerase chain reaction (PCR) technique to demonstrate expression of HSL mRNA in resident and thioglycollate-elicited mouse peritoneal macrophages, as well as in the P388D1 mouse macrophage cell line. PCR was performed using oligonucleotide primer sequences present on adjacent exons of the mouse HSL gene to allow discrimination between products derived from HSL mRNA or genomic DNA sequences; specificity of the PCR was demonstrated by the absence of a product in liver, which does not express HSL mRNA. Northern blot analysis of poly (A)+ RNA from peritoneal macrophages with a mouse adipose HSL cDNA probe demonstrated a low abundance of mRNA of 3.2 kb, identical in size to HSL mRNA in adipose tissue. These findings, together with the results of previous studies demonstrating similarities between HSL and macrophage neutral cholesteryl ester hydrolase, strongly support the conclusion that both are products of a single gene. The development of a PCR assay for HSL mRNA may allow further study of the regulation of neutral cholesteryl ester hydrolase expression in macrophages and foam cells, and its potential role in atherogenesis.
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PMID:Expression of hormone-sensitive lipase mRNA in macrophages. 826 20

This study was designed to evaluate the effects of aging on steroidogenesis and intracellular cholesterol processing in rat Leydig cells. Maximum gonadotropin-induced testosterone secretion was significantly reduced in Leydig cells from 18 to 27-month-old rats compared to 2 to 5-month-old rats. The decreased production of testosterone in older groups persisted after incubation with cAMP analogs or other non-specific stimulatory agents. This age-related loss in testosterone response was not due to changes in gonadotropin receptor concentration, cAMP concentration, protein kinase A activation or the activity of key steroidogenic enzymes. The content of cellular cholesteryl esters doubled as rats aged from 5 to 18 months, and this high cholesteryl esters level remained constant through 27 months. The ability of hCG to mobilize (hydrolyze) stored cholesteryl ester for testosterone production was significantly reduced (65-75%) in cells from the older rats. This change could be accounted for by the decline in activity of neutral cholesteryl esterase in Leydig cells from 18-month-old rats. In contrast, the activity of a non-specific lysosomal acidic cholesteryl esterase did not change with age. The activity of HMG CoA reductase, the rate limiting enzyme in cholesterol biosynthesis decreased about 70% between 5 and 18 months and fell slightly further as the rats aged to 27 months. Also, [14C]acetate or [3H]H2O incorporation into cellular sterols showed a similar decline. Cyanoketone plus hCG stimulated pregnenolone production was reduced about 70-80% in old as compared to young cells. Leydig cells from young rats responded to hCG with increased accumulation of mitochondrial cholesterol in the presence and absence of steroidogenic inhibitors. On the other hand, old cells responded poorly to hCG and mitochondrial cholesterol levels were little affected by hCG plus cycloheximide or aminoglutethimide. Together, these data indicate that alterations in the intracellular processing and metabolism of cholesteryl esters occur in Leydig cells of aging rats, and we suggest they may be responsible for the observed age-related changes in testosterone production.
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PMID:Age-related decline in the steroidogenic capacity of isolated rat Leydig cells: a defect in cholesterol mobilization and processing. 839 38

Diethylumbelliferyl phosphate (DEUP) is an organophosphate cholesteryl ester hydrolase inhibitor that blocks steroidogenesis mainly by preventing cholesterol transport into the mitochondria of steroidogenic cells. In the present study, we show that DEUP blocks the cAMP-stimulated mitochondrial accumulation of the 30-kDa mitochondrial proteins (recently named steroidogenic acute regulatory StAR proteins) that are believed to be the cycloheximide-sensitive factors induced by trophic hormones and cAMP. Inhibition of mitochondrial StAR accumulation by DEUP is dose dependent and closely parallels inhibition of progesterone synthesis. Stimulated lactate production, another cAMP-dependent process in MA-10 cells, is also inhibited by DEUP. Inhibition of protein kinase A (PKA) action would explain the inhibition of these two unrelated processes. However, the cytosolic PKA activity of DEUP-treated MA-10 10 cells was normal. Moreover, the activity of purified PKA was unaffected by DEUP. The inhibition of StAR synthesis was not caused by a direct effect of DEUP on the labile proteins since DEUP-treated cells required more than 24 h to recover steroidogenic capacity after DEUP treatment. Further evidence that the synthesis of StAR was not directly affected was obtained using the constitutively active R2C cells. Progesterone synthesis by these cells also involves StAR, but neither StAR synthesis or steroid synthesis is sensitive to DEUP. Lactate formation in dibutyryl-cAMP-stimulated R2C cells is, however, sensitive to inhibition by DEUP. These data can be best explained by DEUP acting on a long-lived factor involved in the cAMP/PKA response pathway, but not involved in constitutive steroidogenesis.
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PMID:Diethylumbelliferyl phosphate inhibits steroidogenesis by interfering with a long-lived factor acting between protein kinase A activation and induction of the steroidogenic acute regulatory protein (StAR). 853 19

For several reasons it seems reasonable to suspect that perilipins participate in lipid hydrolysis. First, they are located at the lipid droplet surface, the presumed site of HSL and cholesteryl esterase action. Secondly, they are polyphosphorylated by PKA in concert with lipid hydrolysis. Finally, these proteins appear to be expressed primarily, if not solely, in adipocytes and steroidogenic cells, cells in which lipid hydrolysis is stimulated by cyclic AMP and mediated by HSL or cholesteryl esterase(s), whereas other cells that contain abundant neutral lipid depositions contain no perilipin [13]. Interestingly, these closely related hydrolases share no homology with other mammalian lipases [3]. Although such attributes provide a link between perilipin and lipid hydrolysis, we have no evidence that perilipins participate directly in, or are necessary for, lipid catabolism. The basis for the strong affinity between the perilipins and neutral lipids is unknown. Clearly, lipids and perilipins are tightly linked, as evidenced by selective targeting of epitope-tagged perilipin to lipid droplets and by the paradoxical appearance of lipid droplets in pre-adipocytes transfected with a sense perilipin A construct. Indeed, in differentiating adipocytes the earliest lipid depositions are associated with perilipins, and restriction of perilipin synthesis with anti-sense constructs may impede lipid formation and deposition. It remains to be determined if, in the normal course of events, perilipins are directed toward lipid depositions or if lipids are transported to perilipin foci. Whatever the temporal sequence, the result is that neutral lipids are encased in perilipin-bounded droplets.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Perilipin: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells. 856 27

Premenopausal women are at a lower risk of coronary heart disease relative to age matched men. However, the underlying mechanisms are not clearly understood. This article studies the effects of 17 beta-estradiol (17 beta-E2) at physiological concentrations on the cholesterylester metabolism in macrophages (J774 A.1 cells) with a particular focus on neutral cholesterol esterase (N-CEase). Cells were incubated with beta-VLDL, [1-14C]oleic acid and 17 beta-E2 (0.25 and 2.5 nM). 17 beta-E2 dose-dependently reduced cholesteryl-[1-14C]oleate (14C-CO) at 36 h and 48 h relative to the control. It also stimulated hydrolysis of 14C-CO in foam cells on 36 h and 48 h incubation. In addition, 17 beta-E2 markedly increased N-CEase activity at 24 h and 36 h. This increase preceded the enhanced hydrolysis of cholesterylester, 17 alpha-E2 (inactive isomer), estrone and estriol had no stimulatory action on N-CEase, whereas progesterone and testosterone inhibited it. 17 beta-E2-treatment (24 h) increased the activity of cyclic AMP-dependent protein kinase (A-kinase). DEAE-cellulose column chromatography revealed that an isoform (type II) of A-kinase appeared in 17 beta-E2-treated cells in addition to type I of A-kinase found in the control cells. These results suggest that inhibition of cholesterylester accumulation in macrophages by 17 beta-E2 is mediated by an enhancement of N-CEase activity possibly through an increase in A-kinase.
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PMID:Inhibition of cholesterylester accumulation by 17 beta-estradiol in macrophages through activation of neutral cholesterol esterase. 867 86


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