Activation Of PKA) And C Research Articles

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA

Isoform 3 of the Na+-Ca2+ exchanger (NCX3) participates in the Ca2+ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca2+ uptake and extrusion of the two murine variants was measured using fura-2-based Ca2+ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca2+ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca2+]i or [Na+]i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca2+ uptake after a rise in [Ca2+]i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca2+ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca2+-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.

Glycine receptor internalization by protein kinases activation

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.

Relaxin alters cardiac myofilament function through a PKC-dependent pathway

The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLX's positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular Ca(2+) without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-delta, decreases in PKC-alpha and -beta(II), but no effect on PKC-epsilon. Inhibition of PKC with chelerythrine chloride or PKC-delta with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects.

Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and C Modulates Distinct Na,K-ATPase Isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.

Arginine vasopressin stimulates 11beta-hydroxysteroid dehydrogenase type 2 expression in the mineralocorticosteroid target cells

11Beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) deficiency causes sodium retention and severe hypertension by allowing glucocorticoids access to the non-selective mineralocorticosteroid receptor. Understanding regulation of the HSD11B2 gene is thus of fundamental importance in hypertension research. A number of studies have suggested that second messenger pathways may be important in this regard. In the present study we show that HSD11B2 expression in human renal epithelial P58 cells is regulated at the mRNA and protein level, and that protein kinases A (PKA) and C (PKC) are involved in this process. PKA stimulation resulted in almost two-fold increase while PKC activation in almost two-fold decrease in the HSD11B2 mRNA and protein level. Western blot analysis revealed a dimeric form of 11beta-HSD2 of about 80 kDa. Arginine vasopressin (AVP), acting through the AVP2 receptor, as well as 11beta-HSD2 substrates, corticosterone and dexamethasone, up-regulate HSD11B2 expression, suggesting their role as possible factors affecting blood pressure. We show that the activators of the PKA pathway induce, while activators of PKC pathway repress the expression of HSD11B2 in human renal epithelial cells. AVP, acting via the PKA pathway, might be a physiological stimulator of the HSD11B2 expression. The 11beta-HSD2 substrates, both natural (corticosterone) and synthetic (dexamethasone), might protect the mineralocorticosteroid-target cells against cortisol excess.

Sperm capacitation is regulated by the crosstalk between protein kinase A and C

The binding of capacitated sperm to the egg's zona pellucida stimulates it to undergo the acrosome reaction, a process which enables the sperm to penetrate the egg. Mammalian sperm capacitation and the acrosome reaction require remodeling of actin filaments. An increase in phospholipase D (PLD)-dependent actin polymerization occurs during capacitation whereas the increase in sperm intracellular calcium after its binding to the egg causes very fast actin depolymerization prior to the acrosome reaction. Protein kinase A (PKA) and C (PKC) can both activate sperm PLD and actin polymerization under in vitro incubation, however under physiological conditions, actin polymerization depends primarily on PKA activity. We suggest that PKA indirectly activates phosphatidylinositol 4-kinase to produce phosphatidylinositol 4,5-bisphosphate which is a cofactor for PLD activation. In addition, activation of PKA during capacitation inactivates phospholipase C resulting in preventing PKC activation. It appears that PKA activation promotes sperm capacitation whereas too early activation of PKC during capacitation would jeopardize this process. Thus, a refined balance between the two pathways is required for optimal and sustained activation during sperm capacitation.

Protein Kinase C Inhibits ROMK1 Channel Activity via a Phosphatidylinositol 4,5-Bisphosphate-dependent Mechanism

The activity of apical K(+) channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the cloned CCD K(+) channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K(+) secretion in CCD by hormones that activate PKC.

Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness.

1. The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastomaxrat glioma hybrid cells. 2. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5'-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A(2) adenosine receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA). 3. In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of G(s)-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. 4. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. 5. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells.

Ca2+]i determines the effects of protein kinases A and C on activity of rat renal Na+,K+-ATPase.

1. It is well established that the activity of Na+,K+-ATPase (NKA) is regulated by protein kinases A (PKA) and C (PKC), but results on their effects have been conflicting. The aim of this study was to examine if this is ascribed to the intracellular concentration of Ca2+ ([Ca2+]i). 2. Rat renal NKA was stably expressed in COS cells (green monkey kidney cells). Increases in [Ca2+]i were achieved with the Ca2+ ionophore A23187 and verified by direct measurements of [Ca2+]i using fura-2 AM as an indicator. The activity of NKA was measured as ouabain-sensitive 86Rb+ uptake and the state of phosphorylation of NKA was monitored with two site-directed phosphorylation state-specific antibodies. 3. Activation of PKA with forskolin decreased NKA activity by 45.5 +/- 8.9 % at low [Ca2+]i (120 nM) and increased it by 40.5 +/- 6.4 % at high [Ca2+]i (420 nM). The change in NKA activity by forskolin correlated with the level of increase in [Ca2+]i. 4. The effect of 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a specific PKC activator, on the activity of NKA was also Ca2+ dependent, being inhibitory when [Ca2+]i was low (29.3 +/- 3.6 % decrease at 120 nM Ca2+) and stimulatory when [Ca2+]i was high (36.6 +/- 10.1 % increase at 420 nM Ca2+). 5. The alpha subunit of NKA was phosphorylated under both low and high [Ca2+]i conditions upon PKA or PKC activation. PKA phosphorylates Ser943. PKC phosphorylates Ser23. 6. To see if the observed effects on NKA activity are secondary to changes in Na+ entry, we measured NKA hydrolytic activity using permeabilized membranes isolated from cells under controlled Na+ conditions. A decreased activity at low [Ca2+]i and no change in activity at high [Ca2+]i were observed following forskolin or OAG treatment. 7. Purified NKA from rat renal cortex was phosphorylated and inhibited by PKC. This phosphorylation-associated inhibition of NKA was neither affected by Ca2+ nor by calmodulin, tested alone or together. 8. We conclude that effect of PKA/PKC on NKA activity is dependent on [Ca2+]i. This Ca2+ dependence may provide an explanation for the diversity of responses of NKA to activation of either PKA or PKC.

In vitro Secretion of Gonadotropin-Releasing Hormone (GnRH) and [Hydroxyproline9]GnRH from the Rat Hypothalamus Exhibits a Differential Sensitivity to Castration and Second Messengers

The decapeptide [hydroxyproline⁹]GnRH (HypGnRH) has been characterized as an endogenous posttranslational product of the gonadotropin-releasing hormone (GnRH) precursor in a wide range of mammalian brains. Despite consistent biological effects, its secretion by the hypothalamus remains hypothetical. We report here in vitro secretion of HypGnRH and GnRH by the hypothalamus from intact and castrated male rats and provide evidence that they are differentially regulated. Both peptides were identified by two anti-GnRH antibodies of different specificities after separation under two high-performance liquid chromatography conditions. Calcium dependency of HypGnRH release was demonstrated under stimulation with KCl in the absence or presence of Ca²⁺, as well as with Bay K 8644, veratridine, methoxyverapamil, or tetrodotoxin. Activation of signaling pathways involving adenylate cyclase and protein kinases A and C (PKC) induced HypGnRH release. Expression of data as percentage of release over tissue stores revealed a two- to threefold higher release of HypGnRH than of GnRH under the different modes of stimulation used, except under PKC activation which triggered a comparable recruitment of both peptides. Castration selectively affected PKC-coupled GnRH secretion which showed a twofold lesser release than in intact rats, while the HypGnRH release was unaffected. We conclude that HypGnRH and GnRH are not secreted from the hypothalamus according to the same mechanisms.

Long-term changes in excitability induced by protein kinase C activation in Aplysia sensory neurons.

Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.

Regulation of Na+/Glucose Cotransporter Expression by Protein Kinases in Xenopus laevis Oocytes

Cotransporters are proteins responsible for the accumulation of nutrients, neurotransmitters, and drugs in cells. As forskolin has been shown to stimulate intestinal Na+/glucose cotransport, we have used electrophysiological techniques to examine the role of protein kinases in regulating Na+/glucose cotransporters, SGLT1, expressed in Xenopus laevis oocytes. We monitored SGLT1 kinetics, the number of SGLT1 cotransporters in the plasma membrane, and plasma membrane area before and after activation of protein kinases. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and sn-1, 2-dioctanoylglycerol (DOG) were used as membrane permeable activators of protein kinases A (PKA) and C (PKC), respectively. In oocytes expressing rabbit SGLT1 8-Br-cAMP increased by 28 +/- 4% (n = 10), and DOG decreased by 51 +/- 5% (n = 13) the maximum rate of Na+/glucose cotransport. These reversible changes in the maximum transport rate occurred within minutes, and were accompanied by proportional changes in the number of cotransporters in the membrane and area of the plasma membrane. This suggests that protein kinases regulate rabbit SGLT1 activity by controlling the distribution of transporters between intracellular compartments and the plasma membrane, and that this occurs by exo- and endocytosis. Similar increases in maximum transport were obtained with activation of PKA in oocytes expressing rabbit, human, and rat SGLT1 isoforms, but with activation of PKC the response was isoform-dependent. PKC activation decreased the maximum rate of transport by rabbit and rat SGLT1, but increased transport by human SGLT1. We conclude that: (i) the regulation of SGLT1 expression in oocytes by protein kinases occurs mainly by regulated endo- and exocytosis; (ii) it is independent of consensus phosphorylation sites in the transporter; and (iii) the effect of a given kinase depends upon the actual sequence of the cotransporter expressed. These considerations may also apply to the regulation of other cotransporters by protein kinases in oocytes, cells, and tissues.

Regulation of the Human Brush Border Na<sup>+</sup>/Phosphate Cotransporter (NaPi-3) Expressed in Xenopus Oocytes by Intracellular Calcium and Protein Kinase C

Tubular phosphate reabsorption is strongly regulated by parathyroid hormone (PTH) via intracellular activation of protein kinase A (PKA) and C (PKC). In the present study, we expressed the human renal brush border Na⁺-dependent phosphate (Pi) transporter (NaPi-3) in <i>Xenopus </i>oocytes and analyzed its regulation by coexpressed PTH receptors, activation of PKA and PKC and increase of [Ca²⁺]i. Stimulation of coexpressed PTH receptors (with 10 nMPTH 1-34) or activation of PKA (with dibutyryl-cAMP) had no effect on I_p. Increasing [Ca²⁺]i with the calcium ionophore A23187 reduced I_p. Ca²⁺-mediated I_p inhibition was prevented in the presence of staurosporine, but not by the calmodulin-antagonist calmidazo-lium. Activation of PKC by the racemate of phorbol-12, 13-didecanoate (PDD) mimicked the effect of A23187 and decreased Pi-induced currents (I_p). The inactive enantiomere α-phorbol-12, 12-didecanoate (α-PDD) had no effects on I_P. An increase in [Ca²⁺]i accelerated PDD-mediated I_P-inhibition, but [Ca²⁺]i and PDD effects were not additive. It is concluded that [Ca²⁺]i and PDD inhibit I_P via activation of PKC, while activation of PKA has no functional consequence for NaPi-3 expressed <i>Xenopus </i>oocytes.