A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 M cyclic AMP (cAMP), 1 mM calcium and 1 M calmodulin (Ca²⁺/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 M phorbol 12,13-dibutyrate (Ca²⁺/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca²⁺/calmodulin, respectively, but was unaffected in the presence of Ca²⁺/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 M okadaic acid and 0.01 M microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: Evidence against a cAMP- dependent effect

In intact guinea pig ventricles, acetylcholine (ACH) has been shown to attenuate the positive inotropic effects of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, by reducing protein phosphorylation without altering cAMP levels. In the present study, we tested the hypothesis that the cAMP-independent inhibitory action of ACH is also evident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PLB) were determined in unlabeled and ³²P-labeled guinea pig ventricular cardiomyocytes, respectively. IBMX increased PKA activity ratio and phosphorylation of PLB in a dose-dependent manner. When cardiomyocytes were incubated simultaneously with IBMX (0-1 mM) and ACH (2 M), ACH attenuated PLB phosphorylation stimulated by low concentration (10-100 M) but not by high concentrations (> 200 M) of IBMX. EC₅₀ value for IBMX-induced phosphorylation of PLB was 32 ± 6 M and increased nearly 3-fold after addition of ACH while PKA activity ratio remained unchanged. The rank order of cyclic nucleotide derivatives to phosphorylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibutyryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAMP. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosphorylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of ACH on PLB phosphorylation cannot be explained by a reduction in IBMX-stimulated cAMP levels but may involve the activation of protein phosphatases.     

Effect of malathion on nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes

Summary The effect of malathion, an organophosphorus insecticide, on DNA and RNA synthesis was investigated by measuring the rate of incoporation of ³H thymidine and ³H uridine, respectively, into human lymphocytes stimulated by phytohemagglutinin (PHA). Increasing concentrations of malathion, from 10 to 70 g/ml, were added to human lymphocyte cultures at different times in relation to PHA introduction. The lowest applied dose of malathion (10 g/ml) in most cases led to increased incorporation of both ³H thymidine and ³H uridine. Higher concentrations of malathion (30, 50, 70 g/ml) caused a time- and dosedependent decrease of radioisotope incorporation.     

Role of Ca2+-calmodulin dependent phospholamban phosphorylation on the relaxant effect of β-adrenergic agonists

The role of the Ca²⁺-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of -adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]₀ from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca²⁺-calmodulin dependent protein kinase activity. 3×10^–8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol³²P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10^–6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]₀. Low [Ca]₀ did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the -agonist.The results suggest a physiological role of the Ca²⁺-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the -adrenergic cardiac relaxant effect.     

Protein phosphorylation in isolated trabeculae from nonfailing and failing human hearts

Disturbances in the cAMP production during -adrenergic stimulation and alterations of Ca ²⁺ transport controlling proteins and their regulation in the sarcoplasmic reticulum might be involved in the pathogenesis of the failing human heart. Thus, we investigated the cAMP-mediated phosphorylation of phospholamban, troponin I and C-protein in electrically driven, intact isolated trabeculae carneae from nonfailing and failing (NYHA IV) human hearts in parallel to contractile properties on the same tissue samples. The increase in force of contraction induced by isoproterenol (0.2 M) or pimobendan (100 M), a phosphodiesterase inhibitor, was diminished in the failing human hearts compared to nonfailing hearts by 49% and 36%, respectively. Concomitantly the isoproterenol-induced phosphorylation (pmol P/mg homogenate protein) of phospholamban, troponin I and C-protein was reduced from 13.0 ± 2.4 (n = 4), 30.5 ± 1.5 (n = 5) and 11.0 ± 1.3 (n = 5) in the nonfailing heart to 5.2 ±0.6 (n = 13), 14.6 ± 2.2 (n = 16) and 7.1 ± 1.0 (n = 6) in the failing human heart, respectively. Pimobendan changed the phosphorylation state of these proteins similar to isoproterenol. The fact that combined addition of both agents or dibuturyl CAMP (1 mM) alone restored the phosphorylation capacity as observed in the control groups indicates that i) a reduced cAMP generation is related to the reduced phosphorylation of regulatory phosphoproteins located in the sarcoplasmic reticulum and contractile apparatus e.g. phospholamban, troponin I and C-protein, that ii) there is a relationship between protein phosphorylation state and contractile activity and that iii) no changes in the respective content of phosphoproteins are involved in the limitation of cAMP-mediated inotopic activity in the failing human heart. (Mol Cell Biochem 157: 171–179, 1996)     

Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli

A second promoter (P1) was inserted to the PHA (poly--hydroxyalkanoate) operon (pSP2) of Esherichia coli DH5 with an optimal E. coli ribosome binding site and a trc strong promoter (pSJS1) to obtain poly--hydroxybutyrate (PHB) with long chain length. When the inducer, IPTG was added to the culture at 0.4 mM, the average molecular weight was 1.1 × 10⁶ Da. However, an even greater increase of the PHB average molecular weight to 2.5 × 10⁷ Da was observed without IPTG being added.     

Beta-Adrenergic modulation of K+ current in human T lymphocytes

Summary The whole-cell voltage-clamp technique was employed to study the -adrenergic modulation of voltage-gated K⁺ currents in CD8⁺ human peripheral blood lymphocytes. The -receptor agonist, isoproterenol, decreased the peak current amplitude and increased the rate of inactivation of the delayed rectifier K⁺ current. In addition, isoproterenol decreased the voltage dependence of steady-state inactivation and shifted the steady-state inactivation curve to the left. Isoproterenol, on the other hand, had no significant effect on the steady-state parameters of current activation. The isoproterenol-induced decrease in peak current amplitude was inhibited by the -blocker propranolol. Bath application of dibutyryl cAMP (1mm) mimicked the effects of isoproterenol on both K⁺ current amplitude and time course of inactivation. Furthermore, the reduction in the peak current amplitude in response to isoproterenol was attenuated when PKI_5–24 (2–5 m), a synthetic peptide inhibitor of cAMP-dependent protein kinase, was present in the pipette solution. The increase in the rate of inactivation of the K⁺ currents in response to isoproterenol was mimicked by the internal application of GTP--S (300 m) and by exposure of the cell to cholera toxin (1 g/ml), suggesting the involvement of a G protein. These results demonstrate that the voltage-dependent K⁺ conductance in T lymphocytes can be modulated by -adrenergic stimulation. The effects of -agonists, i.e., isoproterenol, appear to be receptor mediated and could involve cAMP-dependent protein kinase as well as G proteins. Since inhibition of the delayed rectifier K⁺ current has been found to decrease the proliferative response in T lymphocytes, the -adrenergic modulation of K⁺ current may well serve as a feedback control mechanism limiting the extent of cellular proliferation.     

Possible modulation of phosphorylation of acetylcholine receptor-enriched membrane preparations

During phosphorylation of acetylcholine receptor (AChR)-enriched membrane preparations fromTorpedo fuscomaculata, phosphate is incorporated into a single protein, with a molecular weight corresponding to that of one of the receptor subunits (37,000 daltons). This protein also seems to contain the receptor binding site. ATP binds to four protein species, one of which corresponds to a different subunit of the receptor (molecular weight 45,000). Phosphorylation of these membrane preparations is affected by several factors, known to be involved in postsynaptic events. Ca²⁺ (10 M) inhibits the reaction, whereas cGMP (20 M), causes stimulation. Furthermore it has been shown that the agonists, acetylcholine, and carbamylcholine (10 M and 1 M) stimulate the phosphorylation reaction, while the antagonists, tubocurarine, hexamethonium, and decamethonium (1 M), cause inhibition.     

Double staining of intracellular cytokine proteins and T-lymphocyte subsets. Evaluation of the method in blood and bronchoalveolar lavage fluid

An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4h with 1ng/ml PMA and 1g/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN- positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4⁺ cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-⁺ cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-⁺. We conclude that this method allows detection of intracellular cytokine proteins in both CD4⁺ and CD8⁺ T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected.     

Cyclic AMP-stimulated protein kinase activity in rabbit peripheral myelin

Cyclic AMP-sensitive protein kinase activity has been found in suspensions of purified rabbit peripheral myelin. The enzyme phosphorylated the P₀, Y, X, P₁, and P₂ myelin proteins. Kinase activity, which was maximal at physiological pH, 2.5 mM Mg²⁺, and 2 M cAMP, was stimulated three-fold over basal levels by cyclic AMP. Addition of calcium or EGTA had no effect on the enzyme activity in the presence or absence of cyclic AMP. Cyclic GMP also did not stimulated endogenous or exogenous protein phosphorylation. Theophylline, an inhibitor of 3,5-cyclic nucleotide phosphodiesterase activity, increased protein kinase activity in the presence of cyclic AMP. These data show that PNS myelin proteins can be phosphorylated in situ by a protein kinase system whose activity is stimulated selectively by cyclic AMP.     

Phosphorylation of heavy sarcoplasmic reticulum vesicles: Identification and characterization of three phosphorylated proteins

Summary Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl₂, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 m -³²P-ATP.³²P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl₂ (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 m sodium dantrolene, an inhibitor of Ca²⁺ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.     

In-vivo phosphorylation of the cardiac L-type calcium channel beta-subunit in response to catecholamines

In canine myocardium, the -subunit of the L-type Ca²⁺ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217–222, 1993). We have assessed the identity of the -subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca²⁺ channel -subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the -subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dt_max. The results imply the involvement of a PKA-mediated phosphorylation of the Ca²⁺ channel -subunit in the adrenergic stimulation of intact canine myocardium.     

Phosphorylation-induced Conformational Changes in Rap1b: ALLOSTERIC EFFECTS ON SWITCH DOMAINS AND EFFECTOR LOOP*

Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser¹⁷⁹ at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser¹⁷⁹ phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.Rap1b, a member of the Ras superfamily of small G proteins, is a GTPase that acts as a molecular on/off switch for the transduction of several external stimuli by alternating from an inactive GDP-bound to an active GTP-bound state (1, 2). Rap1 activation is mediated by several second messengers, growth factors, cytokines, and cell adhesion molecules. The steady-state level of Rap1-GTP is tightly regulated by a family of guanine nucleotide exchange factors that catalyze the otherwise slow dissociation of GDP (i.e. activation) and GTPase-activating proteins, which stimulate the rather slow intrinsic GTPase catalytic activity (i.e. inactivation) (3). GTP binding is coupled to conformational changes in two well defined regions, the switch I (residues 30–40) and switch II (residues 60–76) domains, responsible for high affinity interaction with effector molecules (4, 5) and thus downstream signal transduction.cAMP is one among several pathways leading to Rap1 activation (6). cAMP exerts both mitogenic and anti-mitogenic responses in different cell types, and Rap1 activation is required downstream of cAMP in both scenarios (7, 8). Elevation of intracellular cAMP levels activates cAMP-dependent protein kinase (PKA)⁴ and Epac (exchange protein activated by cAMP), a Rap guanine nucleotide exchange factor (9). Expression of Rap1b in cells where cAMP is mitogenic is associated with an increase in cAMP-mediated G₁/S phase entry (7, 10), and both biochemical events, Rap activation and phosphorylation at Ser¹⁷⁹, are synergistically required for this action (11).PKA substrates able to modulate Rap1 activity (i.e. Src/C3G recruitment and GTPase-activating protein) were recently reported (12, 13). However, the role of PKA-dependent Rap1 phosphorylation at Ser¹⁷⁹ is still unknown. Rap1 phosphorylation does not affect its overall intracellular localization, its basal GTP/GDP exchange reaction, its intrinsic rate of GTP hydrolysis, or its ability to be stimulated by a cytosolic Rap GTPase-activating protein (10); however, several reports suggest that Rap1 phosphorylation is able to modulate its association with some binding partners, namely cytochrome b₅₅₈ (14) and Raf1 (15). The mechanism by which a modification of Ser¹⁷⁹ at the C-terminal end of the molecule affects the regions involved with effector interaction at its N terminus is for the moment unclear.In this study, we report a global assessment of the effects of Ser¹⁷⁹ phosphorylation on conformational change/mobility analyzed by hydrogen/deuterium exchange mass spectrometry (DXMS). The results are consistent with an allosteric effect of the C terminus (containing Ser¹⁷⁹) to the switch loops/effector domain.     

Respiratory stimulus in Rhizobium sp. by legume lectins

High molecular weight lectins (> 100 kDa) from seeds of the legumes Canavalia brasiliensis (CnBr), Cratylia floribunda (CFL), Phaseolus vulgaris (PHA) and Vatairea macrocarpa (VML), temporarily stimulate the respiration of Rhizobium tropici-CIAT899 and R. etli-CFN42. These stimulants were significant (P < 0.05) in bacterial suspensions (> 2.85 mg dry biomass ml^–1), having at least 6200 molecules of lectins per bacteria. The VML (20 g ml^–1), induced specific O₂ demand of 2.3–2.5 M O₂ min^–1 mg dry biomass^–1, in CFN42 and CIAT899, respectively. However, CnBr, CFL and PHA induced smaller demands of O₂ (5×), in both strains. The order of affinities of the lectins was approximately VML > PHA > CFL > CnBr, with regard to respiratory stimuli in CIAT899 strain. The co-administration of 10 g VML ml^–1 and 9.8 M galactose, in CIAT899 suspensions, reduced the respiratory stimuli significantly in relation to the treatment with VML alone. These respiratory stimuli, induced by the lectins, increase the significance of the interaction lectin × Rhizobium in terms of bacterial physiology. Its understanding could be important in relation to bacterial symbiotic behaviour.     

Nucleolar changes in human phytohaemagglutinin-stimulated lymphocytes

Summary The nucleoli of lymphocytes from circulating peripheral blood and from phytohaemagglutinin (PHA)-stimulated cultures (from 2 h–96 h) were studied using a silver method, RNA-specific fluorescent staining, and electron microscopy of ultrathin sections. In peripheral blood about 75% of the lymphocytes have one ring-shaped nucleolus composed of a distinct fibrillar centre surrounded by a dense pars fibrillaris and little granular material; the remaining lymphocytes showing two or more small ring-shaped nucleoli. With PHA stimulation, the number of cells with several nucleoli increases first (from 2 h–12 h). Next, cells containing one or, at most, two large nucleoli with nucleolonema devoid of fibrillar centers are seen (from 4 h on). 34 h after PHA, nucleoli of the compact type containing one or more fibrillar centres appear and comprise about 60% of the cells after 72 h. The appearance of more than one nucleolus per cell shortly after PHA administration suggests an activation of additional nucleolar organizer regions (NOR), which fuse to form one or two large nucleoli with nucleolonema. These are then transformed into compact nucleoli. The fibrillar centers stain preferentially with silver. They contain nonchromosomal proteins and may serve as stores for nucleolar proteins. The fusion of activated NORs during the first cell cycle explains the relatively high frequency of satellite associations in first mitoses compared to later mitoses after stimulation.     

Regulation by lipolytic and antillipolytic compounds of the phosphorylation of specific proteins in isolated intact fat cells.

The effects of various lipolytic and antilipolytic compounds on the phosphorylation of specific proteins, on lipolysis, and on cyclic AMP levels have been studied in isolated intact fat cells of rats. Norepinephrine (NE), adrenocorticotropic hormone (ACTH), 3-isobutyl-1-methylxanthine (IBMX), and monobutyryl cyclic AMP (MBcAMP) each increased the incorporation of [³²P] into three proteins, with apparent molecular weights of approximately 130,000 (protein A), 69,000 (protein B), and 47,000 (protein C), as determined by gel electrophoresis in the presence of sodium dodecyl sulfate (DodSO₄^?). The concentrations of lipolytic agents necessary to obtain a half-maximal increase in phosphorylation of these proteins were similar to the concentrations necessary to obtain a half-maximal stimulation of lipolysis. Propranolol, a β-adrenergic blocking agent, blocked the effects of NE both on protein phosphorylation and on lipolysis, but did not modify the effects of ACTH, IBMX, or MBcAMP on these parameters. When the NE-induced increase in phosphorylation of proteins B and C was maximal, addition of propranolol resulted in a rapid dephosphorylation of these proteins and a rapid cessation of lipolysis; under the same experimental conditions, propranolol had almost no effect on the dephosphorylation of protein A. Concentrations of insulin that prevented or reversed the actions of NE and ACTH on lipolysis also prevented or reversed the NE- and ACTH-induced increase in [³²P] incorporation into proteins B and C. Insulin did not modify the effects of IBMX or MBcAMP either on lipolysis or on [³²P] incorporation into proteins B and C. Insulin increased the incorporation of [³²P] into a protein which, by several criteria, appeared to be protein A. Under a variety of experimental conditions in which lipolytic and antilipolytic hormones were studied, the rate of lipolysis correlated well with the level of phosphorylation of proteins B and C, but not with the level of cyclic AMP.     

Anti-phospholipase C antibodies inhibit the lectin-induced proliferation of human lymphocytes

A novel approach was used to assess the role of phosphoinositide hydrolysis in the mitogenic action of phytohemagglutinin (PHA) or concanavalin A (ConA). The treatment of human peripheral blood leukocytes (PBL) with monospecific antibodies against phospholipase C (PLC) produced a dose-dependent inhibition (up to 100%) of PHA (10 g/ml) or ConA (25 g/ml) proliferative effects. Thus, the activation of membrane-bound PLC is asine-qua-non condition for lectin-induced proliferation of T lymphocytes. The key-role of PLC versus protein kinase C (PKC) is stressed by the fact that the inhibition of PKC with Hidaka's compound H-7 (40 M) produced only a partial blockade (about 25%) of lectin mitogenic effect.To whom correspondence should be addressed.     

Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets

The elevation of [cAMP]_i is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹², in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE₁ and forskolin-induced phosphorylation of Ser³¹² and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE₁-evoked cAMP accumulation by thrombin required both G_i and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹² leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.Upon vascular injury, platelets adhere to the newly exposed subintimal collagen and undergo activation leading to platelet spreading to cover the damaged region and release of thrombogenic factors such as ADP and thromboxane A₂. In addition, platelets are activated by thrombin, which is generated as a result of activation of the coagulation pathway, and stimulates platelets by cleaving the protease-activated receptors (PAR),² PAR-1 and PAR-4. The final common pathway is the exposure of fibrinogen binding sites on integrin α_IIbβ₃ resulting in platelet aggregation and thrombus formation.Thrombin-mediated cleavage of PARs leads to activation of phospholipase C β (PLC), hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and a subsequent increase in [Ca²⁺]_i and activation of protein kinase C (PKC). Protein kinase C contributes to platelet activation both directly, through affinity regulation of the fibrinogen receptor, integrin α_IIbβ₃ (1), and indirectly by enhancing degranulation (2). Thrombin also stimulates activation of PI 3-kinases and subsequent generation of PI (3, 4, 5) trisphosphate and PI (3, 4) bisphosphate (3), which recruit protein kinase B (PKB) to the plasma membrane where it becomes phosphorylated and activated.Platelet activation is opposed by agents that raise intracellular 3′-5′-cyclic adenosine monophosphate ([cAMP]_i). cAMP is a powerful inhibitory second messenger that down-regulates platelet function by interfering with Ca²⁺ homeostasis, degranulation and integrin activation (4). Synthesis of cAMP is stimulated by mediators such as prostaglandin I₂ (PGI₂), which bind to G_s-coupled receptors leading to activation of adenylate cyclase (AC). This inhibitory pathway is opposed by thrombin, which inhibits the elevation of cAMP indirectly via autocrine activation of the G_i-coupled ADP receptor P2Y₁₂. cAMP signaling is terminated by hydrolysis to biologically inert 5′-AMP by 3′-phosphodiesterases. Platelets express two cAMP phosphodiesterase isoforms, cGMP-stimulated PDE2 and cGMP-inhibited PDE3A. PDE3A is the most abundant isoform in platelets and has a ∼250-fold lower K_m for cAMP than PDE2 (4). As a consequence of these properties, PDE3A exerts a greater influence on cAMP homeostasis, particularly at resting levels. The importance of PDE3A in platelet function is further emphasized by the finding that the PDE3A inhibitors cilostamide and milrinone raise basal cAMP levels and strongly inhibit thrombin-induced platelet activation (5). Furthermore, PDE3A^-/- mice demonstrate increased resting levels of platelet cAMP and are protected against a model of pulmonary thrombosis (6). In contrast, the PDE2 inhibitor EHNA has no significant effect on cAMP levels and platelet aggregation (7, 8). The activity of PDE3A is therefore essential to maintain low equilibrium levels of cAMP and determine the threshold for platelet activation (7).Like its paralogue PDE3B, it has recently become clear that PDE3A activity can be positively regulated by phosphorylation in platelets and human oocytes (9, 10). There is some evidence that PKB may be involved in this regulation, although the phosphorylation sites are poorly characterized. In contrast, phosphorylation of PDE3A in HeLa cells was stimulated by phorbol esters and blocked by inhibitors of PKC (11). In this study, we aimed to identify the signaling pathways and phosphorylation sites that are involved in regulation of platelet PDE3A. Here, we show strong evidence that PKC, and not PKB, is involved in agonist-stimulated PDE3A phosphorylation on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹², leading to an increase in PDE3A activity, 14-3-3 binding and modulation of intracellular cAMP levels.