PKA) And C Research Articles

Protein kinases A and C are involved in the mechanisms underlying consolidation of cocaine place conditioning

Using a balanced conditioned place preference (CPP) paradigm, we studied the role of protein kinases A (PKA) and C (PKC) on the acquisition, consolidation and expression of cocaine place conditioning. H7, a non-selective inhibitor of protein kinases, was administered intracerebroventricularly at 1 and 10 μg/10 μl. The higher dose significantly reduced the time spent by rats in the cocaine compartment when given immediately after each conditioning session (consolidation), whereas it had no effect when administered before cocaine during the training phase (acquisition) or before testing for place preference in the absence of cocaine (expression). The same effect was found on administering immediately after each training session 3 μg/10 μl chelerythrine, a selective PKC inhibitor, or 10 μg/10 μl H89, a selective PKA inhibitor, suggesting that both kinases contribute to the consolidation of stimulus-reward association which determines rats' behavior in the cocaine CPP. Changes in the activity of PKA and PKC may thus be part of the cascade of events that contribute to enhancing synaptic responses in the consolidation phase of cocaine CPP and determine rats' behavior associated with the memory of the rewarding effect of cocaine during cocaine CPP expression. These findings may have implications for the study of cocaine `craving' and relapse.

Protein synthesis-dependent potentiation by thyroxine of antiviral activity of interferon-gamma.

We have studied the prenuclear signal transduction pathway by which thyroid hormone potentiates the antiviral activity of human interferon-gamma (IFN-gamma) in HeLa cells, which are deficient in thyroid hormone receptor (TR). The action of thyroid hormone was compared with that of milrinone, which has structural homologies with thyroid hormone. L-Thyroxine (T4), 3,5,3'-L-triiodothyronine (T3), and milrinone enhanced the antiviral activity of IFN-gamma up to 100-fold, a potentiation blocked by cycloheximide. The 5'-deiodinase inhibitor 6-n-propyl-2-thiouracil did not block the T4 effect. 3,3',5,5'-Tetraiodothyroacetic acid prevented the effect of T4 but not of milrinone. The effects of T4 and milrinone were blocked by inhibitors of protein kinases C (PKC) and A (PKA) and restored by PKC and PKA agonists; only the effect of T4 was blocked by genistein, a tyrosine kinase inhibitor. In separate models, milrinone was shown not to interact with nuclear TR-beta. T4 potentiation of the antiviral activity of IFN-gamma requires PKC, PKA, and tyrosine kinase activities but not traditional TR.

Protein kinase and Ca2+ modulation of myo-inositol transport in cultured retinal pigment epithelial cells.

The acute regulation of inwardly directed Na(+)-myo-inositol (MI) cotransporter activity and basal and volume-sensitive MI efflux by protein kinases C (PKC) and A (PKA), cytosolic Ca2+, and phosphoinositide (PI) turnover were characterized in cultured human retinal pigment epithelial cells using 2-[3H]MI and liquid scintillation spectrometry. Kinetic analysis revealed two distinct Na(+)-MI cotransporter components differing in apparent Michaelis constant and maximal velocity. Composite Na(+)-MI cotransport activity was stimulated by PKA activation, the muscarinic agonist carbachol, and the Ca2+ ionophore A-23187 and was inhibited by PKC activation. PKC activation also increased MI efflux, but only the volume-sensitive component, whereas PKA activation increased both basal and volume-sensitive MI efflux. These studies implicate PKC as a negative modulator of MI content through Na(+)-MI cotransport inhibition and potentiation of volume-sensitive MI efflux. PKA is a positive modulator of both Na(+)-MI cotransport and basal and volume-sensitive MI efflux. Cytosolic Ca2+ release through receptor-mediated PI hydrolysis may facilitate Na(+)-MI cotransport activity.

Elevation of protein kinase A and protein kinase C activities in malignant as compared with normal human breast tissue.

Because of their central role in the transduction of extracellular signals, protein kinases A (PKA) and C (PKC) are critical enzymes in the control of cellular proliferation and differentiation. We have measured the catalytic activity of PKA and PKC, as well as the regulatory subunit expression for PKA, in paired samples of normal and malignant breast tissue from 13 patients with breast cancer. Paired non-parametric (Wilcoxon) analysis revealed significantly higher values for both basal (P = 0.0002) and total (P = 0.0002) PKA catalytic activity in malignant compared with normal breast in all 13 paired tissue samples. Expression of both R1- and RII-PKA regulatory subunits were also higher in malignant tissue from 12 (P = 0.0005) and 9 (P = 0.01) of the 13 pairs, respectively. However, the degree of RI-subunit overexpression in malignant tissue was greater than that of the RII-subunit, as demonstrated by an increase in the RI/RII subunit ratio in 10 of the 13 paired samples (P = 0.017). Total PKC catalytic activity was elevated in 11 of the 13 malignant tissue specimens when compared with corresponding normal breast tissue (P = 0.01). This was accounted for by an increase in Ca(2+)-dependent PKC activity (P = 0.01), there being no significant increase in Ca(2+)-independent PKC activity. These data suggest that the activities of both PKA and PKC signalling pathways are intrinsically higher in malignant compared with normal breast tissue and these may therefore represent targets for interventive treatment of breast cancer.

Adducin Regulation: DEFINITION OF THE CALMODULIN-BINDING DOMAIN AND SITES OF PHOSPHORYLATION BY PROTEIN KINASES A AND C

Adducin promotes association of spectrin with actin and caps the fast growing end of actin filaments. Adducin contains N-terminal core, neck, and C-terminal tail domains, is a substrate for protein kinases A (PKA) and C (PKC), and binds to Ca2+/calmodulin. Ser-726 and Ser-713 in the C-terminal MARCKS-related domains of alpha- and beta-adducin, respectively, were identified as the major phosphorylation sites common for PKA and PKC. PKA, in addition, phosphorylated alpha-adducin at Ser-408, -436, and -481 in the neck domain. Phosphorylation by PKA, but not PKC, reduced the affinity of adducin for spectrin-F-actin complexes as well as the activity of adducin in promoting binding of spectrin to F-actin. The myristoylated alanine-rich protein kinase C substrate-related domain of beta-adducin was identified as the dominant Ca2+-dependent calmodulin-binding site. Calmodulin-binding was inhibited by phosphorylation of beta-adducin and of a MARCKS-related domain peptide by PKA and PKC. Calmodulin in turn inhibited the rate, but not the extent, of phosphorylation of beta-adducin, but not alpha-adducin, by PKA and that of each subunit by PKC. These findings suggest a complex reciprocal relationship between regulation of adducin function by calmodulin binding and phosphorylation by PKA and PKC.

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.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

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.

Neonatal Meningitis

The function of many ion channels is under dynamic control by coincident activation of G-protein-coupled receptors (GPCRs), particularly those coupled to the G<i>_α</i>_s and G<i>_α</i>_q family members. Such regulation is typically dependent on the subunit composition of the ionotropic receptor or channel as well as the GPCR subtype and the cell-specific panoply of signaling pathways available. Because GPCRs and ion channels are so highly represented among targets of U.S. Food and Drug Administration-approved drugs, functional cross-talk between these drug target classes is likely to underlie many therapeutic and adverse effects of marketed drugs. GPCRs engage a myriad of signaling pathways that involve protein kinases A and C (PKC) and, through PKC and interaction with <i>β</i>-arrestin, Src kinase, and hence the mitogen-activated–protein-kinase cascades. We focus here on the control of ionotropic glutamate receptor function by GPCR signaling because this form of regulation can influence the strength of synaptic plasticity. The amino acid residues phosphorylated by specific kinases have been securely identified in many ionotropic glutamate (iGlu) receptor subunits, but which of these sites are GPCR targets is less well known even when the kinase has been identified. <i>N</i>-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and heteromeric kainate receptors are all downstream targets of GPCR signaling pathways. The details of GPCR–iGlu receptor cross-talk should inform a better understanding of how synaptic transmission is regulated and lead to new therapeutic strategies for neuropsychiatric disorders.

Characterization of two second messenger pathways and their interactions in eliciting the human sperm acrosome reaction.

The human sperm acrosome reaction (AR) occurs via the activation of at least two signal transduction pathways. The purpose of this investigation was to characterize two of the pathways, the protein kinase A (PKA) and C (PKC) pathways, and determine whether pathway "crosstalk" occurs between them in eliciting the AR in capacitated spermatozoa. Stimulators of each pathway were tested in a dose-dependent manner. ARmax, ED50, and delta ARmax (%ARmax-%ARcontrol) values were calculated. The PKA pathway stimulators forskolin and dibutyryl cyclic AMP (dbcAMP) induced an ARmax at 1.0 microM and 1.0 mM, respectively. The ED50 and delta ARmax values were: 0.01 microM and 17% for forskolin and 0.069 mM and 13% for dbcAMP. Two stimulator types of the PKC pathway were tested: synthetic diacylglycerols (DG) and a phorbol diester. 1,2-dioleoyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol, analogues of the PKC-activating second messenger DG, each induced an ARmax at 50 microM. The ED50 and delta AR max values were: 33 microM and 24% for 1,2-dioleoyl and 34.8 microM and 34% for 1,2-dioctanoyl. 4 beta-Phorbol-12,13-didecanoate, a PKC stimulator, induced an ARmax at 0.1 microM. The ED50 and delta ARmax were 0.021 microM and 26%. An inhibitor of each kinase was added at the end of the capacitation period and prior to stimulation by inducers at their ARmax dose. KT5720, a PKA inhibitor, caused a dose-dependent reduction of the forskolin and dbcAMP-induced AR. Calphostin C, a PKC inhibitor, prevented stimulation of the AR by 1,2-dioleoyl and 4 beta-phorbol-12,13-didecanoate. To investigate pathway "crosstalk," the following experiments were conducted: (1) stimulators of each pathway were combined and tested at the ARmax and ED50 concentrations for each; (2) spermatozoa were pretreated with a kinase inhibitor and then stimulated using an alternative pathway stimulator; and (3) a PKA or PKC inhibitor and a combination of PKA and PKC stimulators, at ED50 concentrations, were tested. The results for (1) indicate an additive AR response of ED50 concentrations but not for ARmax doses. The results for (2) demonstrate that a kinase inhibitor for one pathway prevents induction of the AR by a stimulator of the alternative pathway. Finally, the results for (3) show that a kinase inhibitor for one pathway prevents induction of the AR by the combined use of separate pathway stimulators. When taken collectively, the present results suggest a convergent mechanism of crosstalk between the PKA and PKC pathways leading to the human sperm AR.

Regulation of riboflavin intestinal uptake by protein kinase A: studies with Caco-2 cells.

Although the mechanism of riboflavin (RF) intestinal uptake has been the subject of many studies, virtually nothing is known about the cellular regulation of the uptake process. In the present study, we investigated the role of protein kinase A (PKA)- and C (PKC)-mediated pathways in the regulation of RF intestinal uptake using the confluent Caco-2 monolayers. Treatment of Caco-2 cells with 3-isobutyl-1-methylxanthine (IBMX), forskolin, cholera toxin, or dibutyryl adenosine 3',5'-cyclic monophosphate caused a significant inhibition in RF uptake. The inhibitory effect of IBMX was reversible and resulted from a significant decrease in the maximal velocity of the RF uptake process with no change in its apparent Michaelis constant. The IBMX-induced inhibition in RF uptake was not mediated via inhibition in the synthesis of the RF carrier protein or through inhibition in the recruitment of preexisting carrier protein into the plasma membrane. Calyculin A also inhibited RF uptake when added alone and further potentiated the inhibitory effect of IBMX when added together. Phorbol 12-myristate 13-acetate or chelerythrine, on the other hand, showed no significant effect on RF uptake. These results demonstrate for the first time that compounds that increase intracellular cAMP levels downregulate RF intestinal uptake and that this effect is mediated via a decrease in the activity of the RF uptake carrier. It is suggested that a PKA-mediated pathway(s) plays an important role in regulating RF intestinal uptake.

Muscarinic regulation of Ca2+ currents in rat sensory neurons: channel and receptor types, dose-response relationships and cross-talk pathways.

We studied, in rat sensory neurons, the modulation of high voltage-activated Ca2+ currents (ICa) mediated by the pertussis toxin-sensitive activation of muscarinic receptors, which were found to be of subtypes M2 or M4. Muscarine reversibly blocked somatic Ca2+ spikes but strong predepolarizations only partially relieved the inhibited Ca2+ current. On the other hand, the putative coupling messenger could not rapidly diffuse towards channels whose activity was recorded from a macro-patch. The perforated patch technique virtually prevented the response rundown present during whole-cell experiments. Both omega-conotoxin GVIA (omega-CgTx)-sensitive channels and omega-CgTx- and dihydropyridine-resistant channels are coupled to the muscarinic receptor, but not the L-channel. When measured in the same neuron, dose-response relationships for the first and subsequent agonist applications differed; maximal inhibition, the reciprocal of half-maximal concentration and the Hill coefficient were always highest in the first trial. Muscarine and oxotremorine exhibited monotone dose-response curves, but oxotremorine-M showed non-linear relationships which became monotonic when cells were intracellularly perfused with inhibitors of protein kinase A (PKA) and C (PKC), suggesting that either PKA or receptor-induced PKC could phosphorylate and thus inactive G-proteins or other unknown proteins involved in inhibitory muscarinic actions on ICa. In summary, these data provide a preliminary pharmacological characterization of the muscarinic inhibition of the Ca2+ channels in sensory neurons, with implications about agonist specificity and the interplay between signalling pathways.

Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.