Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Perilipin and ADRP, located on the surface of intracellular lipid droplets, are proposed to be involved in adipocyte lipid metabolism. The aim of the present study was to investigate the effect of PKA and PKC activities on the distribution of perilipin and ADRP in primary cultured adrenal cells, and the role of ERK in PMA- and calphostin C-induced steroidogenesis. Immunofluorescence staining indicated that in addition to p160, a capsular protein of steroidogenic lipid droplets, perilipin and ADRP were localized on the lipid droplet surface. Stimuli such as activation of PKA by db cAMP or inhibition of PKC by calphostin C, which increase corticosterone synthesis in various magnitudes, caused detachment of p160 and perilipin, but not ADRP, from the lipid droplet surface. Activation of PKC by PMA induced increase in corticosterone synthesis, however, it did not affect the distribution of perilipin, p160, or ADRP on the lipid droplet surface, suggesting the presence of mechanisms for promoting sterodiogensis other than causing detachment of lipid droplet surface proteins. We further demonstrated that ERK pathway was involved in PMA-induced steroidogenesis, since PD98059, specific inhibitor of MEK, blocked the increases in steroidogenesis and phosphorylation of ERK caused by PMA, but not by cAMP-PKA. These data indicate that p160, perilipin, and ADRP were all located on the lipid droplet surface in rat adrenal cells. On the basis of its non-responsiveness to lipolytic stimulation, ADRP may be a structural protein of the lipid droplet surface, whereas their immediate response to lipolytic stimuli suggest that perilipin and p160 are functional proteins. PKC regulates adrenal steroidogenesis through ERK cascade, whereas PKA pathway does not involve ERK.
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PMID:Immunocytochemical studies on lipid droplet-surface proteins in adrenal cells. 1221 Jul 50

Tumor necrosis factor-alpha (TNF-alpha) stimulates lipolysis in human adipocytes. However, the mechanisms regulating this process are largely unknown. We demonstrate that TNF-alpha increases lipolysis in differentiated human adipocytes by activation of mitogen-activated protein kinase kinase (MEK), extracellular signal-related kinase (ERK), and elevation of intracellular cAMP. TNF-alpha activated ERK and increased lipolysis; these effects were inhibited by two specific MEK inhibitors, PD98059 and U0126. TNF-alpha treatment caused an electrophoretic shift of perilipin from 65 to 67 kDa, consistent with perilipin hyperphosphorylation by activated cAMP-dependent protein kinase A (PKA). Coincubation with TNF-alpha and MEK inhibitors caused perilipin to migrate as a single 65-kDa band. Consistent with the hypothesis that TNF-alpha induces perilipin hyperphosphorylation by activating PKA, TNF-alpha increased intracellular cAMP approximately 1.7-fold, and the increase was abrogated by PD98059. Furthermore, H89, a specific PKA inhibitor, blocked TNF-alpha-induced lipolysis and the electrophoretic shift of perilipin, suggesting a role for PKA in TNF-alpha-induced lipolysis. Finally, TNF-alpha decreased the expression of cyclic-nucleotide phosphodiesterase 3B (PDE3B) by approximately 50%, delineating a mechanism by which TNF-alpha could increase intracellular cAMP. Cotreatment with PD98059 restored PDE3B expression. These studies suggest that in human adipocytes, TNF-alpha stimulates lipolysis through activation of MEK-ERK and subsequent increase in intracellular cAMP.
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PMID:Tumor necrosis factor-alpha stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cAMP. 1235 29

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKA-mediated lipolysis, we have expressed constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect.
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PMID:Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols. 1247 20

The intracellular role of placental protein 17b (PP17b)/TIP47 has been controversial, because it is considered to be a protein required for mannose 6-phosphate receptor transport from endosome to trans-Golgi as well as a neutral lipid droplet-associated protein. The similarity between the amino acid sequences of PP17 variants, adipophilin and perilipins, and between their gene structures indicate that PP17b as well as other alternatively spliced PP17 variants belong to the lipid storage droplet protein family, containing also some differentiation factors. Using a specific antibody, PP17b was detected in lipid droplet fractions and co-localized with neutral lipid droplets stained by Nile red, and fluorescently labelled PP17 antibody in HeLa cells with confocal microscopy. PP17b was also detected in milk, associated to milk lipid globule membranes. Cytostatic agents induced apoptosis and PP17b synthesis in HeLa cells, which was significantly inhibited by protein kinase C (PKC) inhibitor, indicating the involvement of NF-kappa B and AP-1 transcription factors in this process, while protein kinase A (PKA) inhibitor had only a modest inhibitory effect. Cell differentiation induced by dibutyryl cyclic AMP or phorbol myristate acetate also increased PP17b synthesis, demonstrating its strong involvement in cell differentiation. PP17b synthesis was higher in M than in G0/G1 phases in control, apoptotic and differentiated cells. This data shows that PP17b is a neutral lipid droplet-associated protein, and its expression is regulated by PKC- and PKA-dependent pathways.
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PMID:Lipid droplet and milk lipid globule membrane associated placental protein 17b (PP17b) is involved in apoptotic and differentiation processes of human epithelial cervical carcinoma cells. 1263 Dec 76

Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the beta-adrenergic receptor-adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A-phosphorylated HSL and perilipin A.
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PMID:Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. 1281 Jul 3

Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093-1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.
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PMID:Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes. 1283 20

Our previous studies have demonstrated that natriuretic peptides (NPs), peptide hormones with natriuretic, diuretic, and vasodilating properties, exert a potent control on the lipolysis in human adipocytes via the activation of the type A guanylyl cyclase receptor (1, 2). In the current study we investigated the intracellular mechanisms involved in the NP-stimulated lipolytic effect in human preadipocytes and adipocytes. We demonstrate that the atrial NP (ANP)-induced lipolysis in human adipocytes was associated with an enhanced serine phosphorylation of the hormone-sensitive lipase (HSL). Both ANP-mediated lipolysis and HSL phosphorylation were inhibited in the presence of increasing concentrations of the guanylyl cyclase inhibitor LY-83583. ANP did not modulate the activity of the cAMP-dependent protein kinase (PKA). Moreover, H-89, a PKA inhibitor, did not affect the ANP-induced lipolysis. On primary cultures of human preadipocytes, the ANP-mediated lipolytic effect was dependent on the differentiation process. On differentiated human preadipocytes, ANP-mediated lipolysis, associated with an increased phosphorylation of HSL and of perilipin A, was strongly decreased by treatment with the inhibitor of the cGMP-dependent protein kinase I (cGKI), Rp-8-pCPT-cGMPS. Thus, ANP-induced lipolysis in human adipocytes is a cGMP-dependent pathway that induces the phosphorylation of HSL and perilipin A via the activation of cGKI. The present study shows that lipolysis in human adipocytes can be controlled by an independent cGKI-mediated signaling as well as by the classical cAMP/PKA pathway.
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PMID:Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes. 1297 Mar 65

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser433, Ser492, and Ser517). Peri 301-517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.
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PMID:Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis. 1452 48

Perilipin A is the most abundant lipid droplet-associated protein in adipocytes and serves important functions in regulating triacylglycerol levels by reducing rates of basal lipolysis and facilitating hormonally stimulated lipolysis. We have previously shown that the central region of perilipin A targets and anchors it to lipid droplets, at least in part via three moderately hydrophobic sequences that embed the protein into the hydrophobic core of the droplet. The current study examines the roles of the amino and carboxyl termini of perilipin A in facilitating triacylglycerol storage. Amino- and carboxyl-terminal truncation mutations of mouse perilipin A were stably expressed in 3T3-L1 preadipocytes, which lack perilipins. Triacylglycerol content of the cells was quantified as a measure of perilipin function and was compared with that of cells expressing full-length perilipin A or control cells lacking perilipins. The amino-terminal sequence between amino acids 122 and 222, including four 10-11-amino acid sequences predicted to form amphipathic beta-strands and a consensus site for cAMP-dependent protein kinase, and the carboxyl terminus of 112 amino acids that is unique to perilipin A were critical to facilitate triacylglycerol storage. The precocious expression of full-length perilipin A in 3T3-L1 preadipocytes aided more rapid storage of triacylglycerol during adipose differentiation. By contrast, the expression of highly truncated amino- or carboxyl-terminal mutations of perilipin failed to serve a dominant negative function in lowering triacylglycerol storage during adipose differentiation. We conclude that the amino and carboxyl termini are critical to the function of perilipin A in facilitating triacylglycerol storage.
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PMID:The amino and carboxyl termini of perilipin a facilitate the storage of triacylglycerols. 1461 73

HSL (hormone-sensitive lipase) is a key enzyme in the mobilization of fatty acids from acylglycerols in adipocytes as well as non-adipocytes. In adipocytes, catecholamines stimulate lipolysis mainly through PKA (protein kinase A)-mediated phosphorylation of HSL and perilipin, a protein coating the lipid droplet. The anti-lipolytic action of insulin is mediated mainly via lowered cAMP levels, accomplished through activation of phosphodiesterase 3B. Phosphorylation of HSL by PKA occurs at three sites, the serines 563, 659 and 660, both in vitro and in primary rat adipocytes. Phosphorylation of Ser-659 and -660 is required for in vitro activation as well as translocation from the cytosol to the lipid droplet, whereas the role of the third PKA site remains elusive. Adipocytes isolated from homozygous HSL-null mice, generated in our laboratory, exhibit completely blunted catecholamine-induced glycerol release and reduced fatty acid release, suggesting the presence of additional, although not necessarily hormone-activatable, triacylglycerol lipase(s). Basal hyperinsulinaemia, release of exaggerated amounts of insulin during glucose challenges and retarded glucose disposal during insulin tolerance tests suggest that HSL-null mice are insulin resistant. Liver, adipose tissue and skeletal muscle appear all to be sites of impaired insulin sensitivity in HSL-null mice.
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PMID:Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. 1464 Oct 8


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