cAMP-dependent protein kinase activation mediated by beta 3-adrenergic receptors parallels lipolysis in rat adipocytes.

Catecholamine-induced lipolysis is chiefly mediated through the recently characterized beta 3-adrenergic receptor (AR) in rat adipocytes. Discrepancies between the ability of beta 3-AR agonists to stimulate adenylyl cyclase and the resulting lipolysis were recently reported. cAMP-dependent protein kinase (A-kinase) activation induced by these agonists was compared to lipolysis. Agonist potencies were similar for A-kinase activity ratios and lipolysis. The same A-kinase activity ratio to lipolysis relationship was found for the beta 3-AR agonists tested.     

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.     

Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling

Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Its effects on energy homeostasis, glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and -R2. With the exception of APPL1 and RACK1, no intracellular binding partners of adiponectin receptors are reported and thus signaling pathways downstream of these receptors remain largely unknown. To incorporate adiponectins protective potential in drug development it is essential to understand adiponectin signaling cascades in detail. A yeast two-hybrid approach employing AdipoR1s cytoplasmatic N-terminus led to the identification of the regulatory subunit of protein kinase CK2. We confirmed the interaction in co-immunoprecipitation, ELISA experiments and co-localization analysis in mammalian cells. Furthermore we could localize the interaction site in an N-terminal basic region close to the transmembrane domain. In adiponectin stimulation experiments of C2C12 mouse myotubes and MCF7 cells incorporating CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) we found a modulator role of CK2 in adiponectin signaling. Accordingly we identified the regulatory subunit of protein kinase CK2 as a novel intracellular partner of AdipoR1 and have strong evidence of CK2 as an effector molecule in adiponectin signaling. Since CK2 is involved in signaling cascades of other adipokines and hormones, e.g. leptin and insulin, our findings suggest a possible key function in crosstalk between adiponectin and insulin signaling pathways and could provide further insight into the anti-diabetic effects of adiponectin.     

Nitric oxide production from rat adipocytes is modulated by beta3-adrenergic receptor agonists and is involved in a cyclic AMP-dependent lipolysis in adipocytes.

It is established that the modulation of beta(3)-adrenoceptor function could be associated with impairment of lipolysis in white fat and be responsible for disturbed lipid metabolism. Though two isoforms of nitric oxide synthase (NOS) were reported in adipocytes, the role of nitric oxide (NO) in adipose tissue is still ambiguous. The present work was directed to study the interplay between NO production and beta-adrenoceptor/cyclic AMP (cAMP) pathway on lipid mobilization (glycerol and nonesterified fatty acids, NEFA) in cultures of rat adipocytes isolated from epididymal white adipose tissue. beta-Nonselective (isoprenaline) and beta(3)-selective (BRL-37344) agonists and the postadrenoceptor agents such as dibutyryl-cAMP, forskolin, and 3-isobutyl-1-methylxanthine significantly increased nitrite, glycerol, and NEFA levels with BRL-37344 being the most potent. Conversely, addition of beta-nonselective (propranolol) or beta(3)-selective (bupranolol) antagonist or the adenylyl cyclase inhibitor (SQ 22,536) significantly reduced beta-agonist-induced NO production and lipolysis. For beta-adrenoceptor agonists, antagonists, and their pairs, there was a positive correlation between medium nitrite and glycerol or NEFA with r(2) being 0.90 and 0.84, respectively. The possible relationship between NO and lipolysis was revealed after adipocyte treatment with nonspecific (N(omega)-nitro-l-arginine methyl ester, l-NAME) and specific (aminoguanidine) NOS inhibitors. Both l-NAME and aminoguanidine significantly inhibited the lipolytic effect of BRL-37344. Moreover, NO-donor (S-nitroso-N-acetylpenicillamine) at higher concentration increased basal glycerol and NEFA levels. 8-bromo-cyclic GMP had no effect on adipocyte lipolysis. These data suggest that beta-adrenergic lipolysis, specifically beta(3)-adrenoceptor effect, which is realized via the adenylyl cyclase/cAMP/protein kinase A signaling cascade, involves NO production downstream of beta(3)-adrenoceptor/cAMP pathway.     

Role of beta 1- and beta 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes

To evaluate the physiological functions of₁-,₂-, and₃-adrenoceptors (ARs) in brownadipose tissue, the lipolytic and respiratory effects of variousadrenergic agonists and antagonists were studied in rat brownadipocytes. The -agonists stimulated both lipolysis and respiration(8-10 times above basal levels), with the following order ofpotency (concentration eliciting 50% of maximum response):CL-316243 (₃) > BRL-37344(₃) > isoproterenol (mainly₁/₂) > norepinephrine (NE; mainly₁/₂) > epinephrine (mainly₁/₂) dobutamine (₁)  procaterol (₂). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (₁ antagonist) revealed thatmore than one type of receptor mediates NE action. It is concluded fromour results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through ₁-ARs,2) NE, at higher levels, stimulateslipolysis and respiration via both₁- and₃-ARs,3)₂-ARs play only a minor role,and 4)₃-ARs may represent thephysiological receptors for the high NE concentrations in the synapticcleft, where the high-affinity₁-ARs are presumablydesensitized. It is also suggested that lipolysis represents theflux-generating step regulating mitochondrial respiration.

     

Effect of endothelin-1 on lipolysis in rat adipocytes

Objective : To explore the role of endothelin‐1 (ET‐1) on lipid metabolism, we examined the effect of ET‐1 on lipolysis in rat adipocytes. Research Methods and Procedure : Adipocytes isolated from male Sprague‐Dawley rats, weighing 400 to 450 grams, were incubated in Krebs‐Ringer buffer with or without 10^?7 M ET‐1 for various times or with various concentrations of ET‐1 for 4 hours; then glycerol release into the incubation medium was measured. In addition, selective ET_AR and ET_BR blockers were used to identify the ET receptor subtype involved. We also explored the involvement of cyclic adenosine monophosphate (cAMP) in ET‐1‐stimulated lipolysis using an adenylyl cyclase inhibitor and by measuring changes in intracellular cAMP levels in response to ET‐1 treatment. To further explore the underlying mechanism of ET‐1 action, we examined the involvement of the extracellular signal‐regulated kinase (ERK)‐mediated pathways. Results : Our results showed that ET‐1 caused lipolysis in rat adipocytes in a time‐ and dose‐dependent manner. BQ610, a selective ET_AR blocker, blocked this effect. The adenylyl cyclase inhibitor, 2′, 5′‐dideoxyadenosine, had no effect on ET‐1‐stimulated lipolysis. ET‐1 did not induce an increase in intracellular cAMP levels. In addition, ET‐1‐induced lipolysis was blocked by inhibition of ERK activation using PD98059. Coincubation of cells with ET‐1 and insulin suppressed ET‐1‐stimulated lipolysis. Discussion : These findings show that ET‐1 stimulates lipolysis in rat adipocytes through the ET_AR and activation of the ERK pathway. The underlying mechanism is cAMP‐independent. However, this non‐conventional lipolytic effect of ET‐1 is inhibited by the anti‐lipolytic effect of insulin.     

Protein phosphatase 6 down-regulates TAK1 kinase activation in the IL-1 signaling pathway

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.     

A novel protein kinase A-independent, beta-arrestin-1-dependent signaling pathway for p38 mitogen-activated protein kinase activation by beta2-adrenergic receptors

A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by beta(2)-adrenergic receptors (beta(2)-ARs). However, the detailed mechanism of beta(2)-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by beta(2)-ARs in human embryonic kidney 293A cells. The beta(2)-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of beta-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of beta-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by beta-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47(phox) knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a beta-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for beta(2)-AR-mediated early activation of p38 MAPK.     

Protein kinase Cepsilon: the mitochondria-mediated signaling pathway

Mitochondria, which are the cellular energy plants, also act as the integration center of cellular signaling pathways. Apoptosis is a well-known pathway in which mitochondria are involved. Protein kinase Cepsilon has been classified as a novel type of protein kinase C and is involved in many cellular events regulating mitochondrial function. Much evidence has accumulated regarding the relationships between mitochondria-mediated apoptosis and protein kinase Cepsilon. Therefore, by focusing on these relationships, in particular the anti-apoptotic effects of protein kinase Cepsilon on mitochondrial function, we highlight the importance and significance of protein kinase Cepsilon in cell survival and death.     

Beta-adrenergic receptor subtypes mediating lipolysis in porcine adipocytes. Studies with BRL-37344, a putative beta3-adrenergic agonist

The beta3-selective adrenergic receptor ligand BRL 37344 (BRL) was used to differentiate the presence and functional role of beta-adrenergic receptor (betaAR) subtypes in pig tissues. BRL did not stimulate adenylyl cyclase in membrane preparations or increase lipolysis from pig adipocytes. In contrast to some species, BRL appears to be a poor agonist for the pig betaAR and is not a useful betaAR ligand. Based on displacement of [3H]dihydroalprenolol binding, BRL exhibited a 100-fold selectivity for pig betaAR subtypes in adipose and skeletal muscle membranes. The high affinity site was proposed to be the beta2AR. When used as an antagonist, BRL blockade of the high affinity site did not interfere with isoproterenol-stimulated lipolysis but did inhibit adenylyl cyclase activation. Results indicate that the high affinity betaAR (betaAR) is not linked to lipolysis, possibly due to intracellular compartmentalization. Therefore, betaAR subtypes may have function-specific effects.     

Prolonged treatment with prostaglandin E1 increases the rate of lipolysis in rat adipocytes

Prolonged treatment of adipocytes with certain inhibitors of lipolysis, including N(6)-phenylisopropyl adenosine (PIA) and prostaglandin E(1) (PGE(1)) leads to down-regulation of G(i). Prolonged treatment with PIA increases the rate of lipolysis, and we have reported that tumor necrosis factor-alpha (TNF alpha) stimulates lipolysis by down-regulating G(i). To determine the relationship between G(i) concentration and lipolysis, we have investigated the effect of two other acute inhibitors of lipolysis; PGE(1), which down-regulates G(i), and nicotinic acid (NA), which does not down-regulate G(i). Rat adipocytes were incubated with PIA (300 nM), PGE(1) (3 microM) or nicotinic acid (1 mM) for 24 h. The rate of lipolysis (glycerol release) was increased approximately 2 to 3-fold in PIA-treated cells, and in PGE(1)-treated cells. Conversely, the rate of lipolysis was not altered by the prolonged nicotinic acid treatment. These findings support the hypothesis that the rate of lipolysis in adipocytes is determined, at least partly, by the cellular concentration of G(i) proteins.     

Protein kinase C-dependent antilipolysis by insulin in rat adipocytes

Recently, we have shown that protein kinase C (PKC) activated by phorbol 12-myristate 13-acetate (PMA) attenuates the beta1-adrenergic receptor (beta1-AR)-mediated lipolysis in rat adipocytes. Stimulation of cells by insulin, angiotensin II, and alpha1-AR agonist is known to cause activation of PKC. In this study, we found that lipolysis induced by the beta1-AR agonist dobutamine is decreased and is no longer inhibited by PMA in adipocytes that have been treated with 20 nM insulin for 30 min followed by washing out insulin. Such effects on lipolysis were not found after pretreatment with angiotensin II and alpha1-AR agonists. The rate of lipolysis in the insulin-treated cells was normalized by the PKCalpha- and beta-specific inhibitor G? 6976 and PKCbeta-specific inhibitor LY 333531. In the insulin-treated cells, wortmannin increased lipolysis and recovered the lipolysis-attenuating effect of PMA. Western blot analysis revealed that insulin slightly increases membrane-bound PKCalpha, betaI, and delta, and wortmannin decreases PKCbetaI, betaII, and delta in the membrane fraction. These results indicate that stimulation of insulin receptor induces a sustained activation of PKC-dependent antilipolysis in rat adipocytes.     

The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.     

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.     

Inositol hexakisphosphate kinase-1 interacts with perilipin1 to modulate lipolysis

Lipolysis leads to the breakdown of stored triglycerides (TAG) to release free fatty acids (FFA) and glycerol which is utilized by energy expenditure pathways to generate energy. Therefore, a decrease in lipolysis augments fat accumulation in adipocytes which promotes weight gain. Conversely, if lipolysis is not complemented by energy expenditure, it leads to FFA induced insulin resistance and type-2 diabetes. Thus, lipolysis is under stringent physiological regulation, although the precise mechanism of the regulation is not known. Deletion of inositol hexakisphosphate kinase-1 (IP6K1), the major inositol pyrophosphate biosynthetic enzyme, protects mice from high fat diet (HFD) induced obesity and insulin resistance. IP6K1-KO mice are lean due to enhanced energy expenditure. Therefore, IP6K1 is a target in obesity and type-2 diabetes. However, the mechanism/s by which IP6K1 regulates adipose tissue lipid metabolism is yet to be understood. Here, we demonstrate that IP6K1-KO mice display enhanced basal lipolysis. IP6K1 modulates lipolysis via its interaction with the lipolytic regulator protein perilipin1 (PLIN1). Furthermore, phosphorylation of IP6K1 at a PKC/PKA motif modulates its interaction with PLIN1 and lipolysis. Thus, IP6K1 is a novel regulator of PLIN1 mediated lipolysis.     

Homocysteine suppresses lipolysis in adipocytes by activating the AMPK pathway

Hyperhomocysteinemia (HHcy) is an independent risk factor for coronary artery disease. Emerging evidence suggests that HHcy is also associated with adipocyte tissue dysfunction. One of the principal functions of adipose tissue is to provide energy substrate via lipolysis. In the present study, we investigated the effects of homocysteine (Hcy) on lipolysis in adipocytes. We found that Hcy inhibited release of glycerol and fatty acids, two typical indicators of the lipolytic response, in primary adipocytes and fully differentiated 3T3-L1 adipocytes in a dose-dependent manner under both basal and isoproterenol-stimulated conditions. In differentiated 3T3-L1 adipocytes, decreased glycerol and free fatty acid (FFA) release was associated with elevation of intracellular TG content. Further studies showed that Hcy-mediated antilipolytic responses were independent of the cyclic AMP-PKA and MEK-ERK1/2 pathways. However, Hcy increased phosphorylation levels of AMP-activated protein kinase (AMPK) and its downstream enzyme acetyl-CoA carboxylase. Compound C, an AMPK inhibitor, abolished Hcy-induced reduction of glycerol and FFA release under both basal and isoproterenol-stimulated conditions. Furthermore, AMPKα1 siRNA reversed Hcy-inhibited glycerol release. Supplementation of exogenous Hcy in the diet for 2 wk lowered circulating glycerol and FFA levels. Moreover, Hcy supplementation was associated with elevated leptin levels and reduced adiponectin levels in plasma. These results show that Hcy inhibits lipolysis through a pathway that involves AMPK activation.     

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.     

The mammalian formin FHOD1 interacts with the ERK MAP kinase pathway

Formin homology 2 domain containing protein (FHOD1), a mammalian formin, regulates cytoskeletal architecture, enhances cell migration, and induces gene expression from the serum response element. In this study, we describe co-precipitation of FHOD1 with components of the ERK MAP kinase pathway while co-precipitation of FHOD1 with p38 MAP kinase and JNK was not observed. In addition, FHOD1 co-localized to lamellipodia with Raf-1 and to stress fibers with MEK. FHOD1-induced gene expression from the serum response element was dependent on ERK MAP kinase activation, and the native skeletal actin promoter were activated by FHOD1 through the SRF site. However, FHOD1-induced stress-fiber formation and gene expression from the skeletal actin promoter was independent of ERK activation. These novel data demonstrate that FHOD1-ERK MAP kinase interaction regulates key aspects of FHOD1 biology.     

TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes

Tumor necrosis factor-alpha (TNF-alpha) increases adipocyte lipolysis after 6-12 h of incubation. TNF-alpha has been demonstrated to activate mitogen-activated protein (MAP) kinases including extracellular signal-related kinase (ERK) and N-terminal-c-Jun-kinase (JNK) in different cell types. To determine if the MAP kinases have a role in TNF-alpha-induced lipolysis, 3T3-L1 adipocytes were treated with the cytokine (10 ng/ml), in the presence or absence of PD98059 or U0126 (100 micromoles), specific inhibitors of ERK activity. We demonstrated that U0126 or PD98059 blocked TNF-alpha-induced ERK activity and decreased TNF-alpha-induced lipolysis by 65 or 76% respectively. The peroxisome-proliferator-activated receptor gamma (PPARgamma) agonists, rosiglitazone (ros), and 15-deoxy-Delta-(12,14)- prostaglandin J(2) (PGJ2) have been demonstrated to block TNF-alpha-induced lipolysis. Pretreatment of adipocytes with these agents almost totally blocked TNF-alpha-induced ERK activation and reduced lipolysis by greater than 90%. TNF-alpha also stimulated JNK activity, which was not affected by PD98059 or PPARgamma agonist treatment. The expression of perilipin, previously proposed to contribute to the mechanism of lipolysis, is diminished in response to TNF-alpha treatment. Pretreatment of adipocytes with PD98059 or ros significantly blocked the TNF-alpha-induced reduction of perilipin A protein level as determined by Western analysis. These data suggest that activation of the ERK pathway is an early event in the mechanism of TNF-alpha-induced lipolysis.     

Protein kinase A regulates AKAP250 (gravin) scaffold binding to the beta2-adrenergic receptor

A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the beta(2)-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor-scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold-receptor interactions. Gravin constructs lacking the AKAP domain displayed no binding to the receptor. Metabolic labeling studies in vivo demonstrate agonist-stimulated phosphorylation of gravin and enhanced gravin-receptor association. Analysis of the AKAP domain revealed two canonical PKA sites phosphorylated in response to elevated cAMP, blocked by PKA inhibitor, and essential for scaffold-receptor association and for resensitization of the receptor. The AKAP appears to provide the catalytic PKA activity responsible for phosphorylation of the scaffold in response to agonist activation of the receptor as well as for the association of the scaffold with the receptor, a step critical to receptor resensitization.