cAMP-dependent Protein Kinase Activity Research Articles

Epinephrine Protects Cancer Cells from Apoptosis via Activation of cAMP-dependent Protein Kinase and BAD Phosphorylation

The stress hormone epinephrine is known to elicit multiple systemic effects that include changes in cardiovascular parameters and immune responses. However, information about its direct action on cancer cells is limited. Here we provide evidence that epinephrine reduces sensitivity of cancer cells to apoptosis through interaction with beta(2)-adrenergic receptors. The antiapoptotic mechanism of epinephrine primarily involves phosphorylation and inactivation of the proapoptotic protein BAD by cAMP-dependent protein kinase. Moreover, BAD phosphorylation was observed at epinephrine concentrations found after acute and chronic psychosocial stress. Antiapoptotic signaling by epinephrine could be one of the mechanisms by which stress promotes tumorigenesis and decreases the efficacy of anti-cancer therapies.

CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation

A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.

New tricks teach old flies

Mutations that reduce cAMP-dependent protein kinase activity suppress memory loss in middle-aged Drosophila.

Extracellular Activity of Cyclic AMP–Dependent Protein Kinase as a Biomarker for Human Cancer Detection: Distribution Characteristics in a Normal Population and Cancer Patients

The overexpression of cyclic AMP (cAMP)-dependent protein kinase (PKA) has been reported in patients with cancer, and PKA inhibitors have been tested in clinical trials as a novel cancer therapy. The present study was designed to characterize the population distribution of extracellular activity of cAMP-dependent protein kinase (ECPKA) and its potential value as a biomarker for cancer detection and monitoring of cancer therapy. The population distribution of ECPKA activity was determined in serum samples from a Chinese population consisting of a total of 603 subjects (374 normal healthy volunteers and 229 cancer patients). The serum ECPKA was determined by a validated sensitive radioassay, and its diagnostic values (including positive and negative predictive values) were analyzed. The majority of normal subjects (>70%) have undetectable or very low levels of serum ECPKA. In contrast, the majority of cancer patients (>85%) have high levels of ECPKA. The mean ECPKA activity in the sera of cancer patients was 10.98 units/mL, 5-fold higher than that of the healthy controls (2.15 units/mL; P < 0.001). In both normal subjects and cancer patients, gender and age had no significant influence on the serum ECPKA. Among factors considered, logistic analysis revealed that the disease (cancer) is the only factor contributing to the elevation of ECPKA activity in cancer patients. In conclusion, ECPKA may function as a cancer marker for various human cancers and can be used in cancer detection and for monitoring response to therapy with other screening or diagnostic techniques.

A Bioluminescent-Based, HTS-Compatible Assay to Monitor G-Protein-Coupled Receptor Modulation of Cellular Cyclic AMP

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 +/- 5 fmol [mean +/- standard error of the mean] of cAMP per well) and reproducibility (Z' of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferase/luciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Galpha(s) or Galpha(i) forms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50% effective concentration (EC(50)) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC(50) values for agonists and 50% inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Galpha(s) and Galpha(i)). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications.

α1-Antitrypsin, Old Dog, New Tricks: α1-ANTITRYPSIN EXERTS IN VITRO ANTI-INFLAMMATORY ACTIVITY IN HUMAN MONOCYTES BY ELEVATING cAMP

Regulation of serine protease activity is considered to be the sole mechanism for the function of alpha1-antitrypsin (AAT). However, recent reports of the anti-inflammatory effects of AAT are hard to reconcile with this classical mechanism. We discovered that two key activities of AAT in vitro, namely inhibition of endotoxin-stimulated tumor necrosis factor-alpha and enhancement of interleukin-10 in human monocytes, are mediated by an elevation of cAMP and activation of cAMP-dependent protein kinase A. As expected with this type of mechanism, the AAT-mediated rise in cAMP and the impact on endotoxin-stimulated tumor necrosis factor-alpha and interleukin-10 was enhanced when the catabolism of cAMP was blocked by the phosphodiesterase inhibitor rolipram. These effects were still observed with modified forms of AAT lacking protease inhibitor activity.

Role for Non-Proteolytic Control of M-phase Promoting Factor Activity at M-phase Exit

M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca++/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.

Effect of matrine on HeLa cell adhesion and migration

This study sought to explore the role of matrine in the metastasis of cancer cells and to gain insight into the possible mechanism of matrine's ability to inhibit cancer metastasis. Accordingly, changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation and in cAMP-dependent protein kinase (PKA) activity during cell detachment and reattachment were first investigated. After administration of matrine (50 microg/ml), the decrease in VASP phosphorylation paralleled the decrease in PKA activity. Matrine was found to significantly inhibit HeLa cell adhesion to collagen I. To determine the effect of matrine on the migration of HeLa cells, we analysed the migratory behaviour of HeLa cells in a two and three-dimensional cell migration assay. In a two-dimensional cell migration assay, the average cell migration velocity was significantly reduced by matrine compared with the control. Moreover, in a three-dimensional cell migration assay performed with the Transwell system, HeLa cells treated with matrine (50 microg/ml) were found to migrate less than the control cells. These data suggest that the inhibitory effect of matrine may be produced by decreased phosphorylation of VASP due to inhibition of the activity of PKA during HeLa cell adhesion and migration.

Downregulation of Bone Morphogenetic Protein 4 Expression in Coronary Arterial Endothelial Cells

Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor beta family member cytokine that exerts proinflammatory effects on the endothelium and is likely to play a role in atherogenesis. Recent studies suggested that atheroprotective levels of shear stress control endothelial BMP-4 expression; however, the underlying mechanisms remained unknown. We found that shear stress downregulated BMP-4 expression in human and rat coronary arterial endothelial cells (CAECs) as well as in cultured mesenteric arterioles, although it had no effect on the expression of BMP-2, a related cytokine. In human coronary arterial endothelial cells, 8-bromo-cAMP, the adenylate cyclase activator forskolin, or a cAMP-dependent protein kinase (PKA) activator effectively decreased BMP-4 expression, mimicking the effects of shear stress. Indeed, shear stress induced the nuclear translocation of PKA-c, and inhibition of PKA attenuated the effects of shear stress and forskolin on BMP-4 expression. RNA decay assay and BMP-4 promoter-driven luciferase reporter gene assay showed that cAMP regulates BMP-4 expression at the transcriptional level. Laminar shear stress and the cAMP/PKA pathway are important negative regulators of BMP-4 expression in the vascular endothelium. Because BMP-4 elicits endothelial activation and dysfunction, hypertension, and vascular calcification, inhibition of BMP-4 expression by shear stress and the cAMP/PKA pathway is likely to exert antiatherogenic and vasculoprotective effects.

Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function

PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.

Maximal β3-Adrenergic Regulation of Lipolysis Involves Src and Epidermal Growth Factor Receptor-dependent ERK1/2 Activation

Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.

The interaction between prion protein and laminin modulates memory consolidation

Cellular prion protein (PrPc) has a pivotal role in prion diseases. PrPc is a specific receptor for laminin (LN) gamma1 peptide and several lines of evidence indicate that it is also involved in neural plasticity. Here we investigated whether the interaction between PrPc and LN plays a role in rat memory formation. We found that post-training intrahippocampal infusion of PrPc-derived peptides that contain the LN binding site (PrPc163-182 and PrPc173-192) or of anti-PrPc or anti-LN antibodies that inhibit PrPc-LN interaction impaired inhibitory avoidance memory retention. The amnesic effect of anti-PrPc antibodies and PrPc173-192 peptide was reversed by co-infusion of a LN gamma1 chain-derived peptide containing the PrPc-binding site, suggesting that PrPc-LN interaction is indeed crucial for memory consolidation. In addition, PrPc173-192 peptide and anti-PrPc or anti-LN antibodies also inhibited the activation of hippocampal cAMP-dependent protein kinase A (PKA) and extracellular regulated kinase (ERK1/2), two kinases that mediate the up-regulation of signaling pathways needed for consolidation of inhibitory avoidance memory. Our findings show that, through its interaction with LN, hippocampal PrPc plays a critical role in memory processing and suggest that this role is mediated by activation of both PKA and ERK1/2 signaling pathways.

Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.

Adipokinetic hormone‐induced mobilization of fat body triglyceride stores in Manduca sexta: Role of TG‐lipase and lipid droplets

Triglycerides (TG) stores build up in the insect fat body as lipid droplets at times of excess of food. The mobilization of fat body triglyceride (TG) is stimulated by adipokinetic hormones (AKH). The action of AKH involves a rapid activation of cAMP-dependent protein kinase (PKA). Recent in vitro studies have shown that PKA phosphorylates and activates the TG-lipase substrate, the lipid droplets. Conversely, purified TG-lipase from Manduca sexta fat body is phosphorylated by PKA in vitro but is not activated. This study was directed to learn whether or not AKH promotes a change in the state of phosphorylation of the lipase in vivo, and what are the relative contributions of cytosol and lipid droplets to the overall increase of lipolysis triggered by AKH. TG-lipase activity of fat body cytosols isolated from control and AKH-treated insects was determined against the native substrate, in vivo [3H]-TG radiolabeled lipid droplets, obtained from control and AKH-treated insects. The lipase activity of the system composed of AKH-cytosol and AKH-lipid droplets (11.1 +/- 2.1 nmol TG/min-mg) was 3.1-fold higher than that determined with control cytosol and lipid droplets (3.6 +/- 0.5 nmol TG/min-mg). Evaluation of the role of AKH-induced changes in the lipid droplets on lipolysis showed that changes in the lipid droplets are responsible for 70% of the lipolytic response to AKH. The remaining 30% appears to be due to AKH-dependent changes in the cytosol. However, the phosphorylation level of the TG-lipase was unchanged by AKH, indicating that phosphorylation of the TG-lipase plays no role in the activation of lipolysis induced by AKH.

Expression of Serotonin7Receptor and Coupling of Ectopic Receptors to Protein Kinase A and Ionic Currents in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia Causing Cushing’s Syndrome

In ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushing's syndrome, cortisol secretion is controlled by illegitimate membrane receptors. The aim of the present study was to characterize the pharmacological properties and the transduction mechanisms of illegitimate receptors, i.e. receptors for serotonin (5-HT), gastric inhibitory polypeptide (GIP), and LH/human chorionic gonadotropin (hCG), expressed by AIMAH tissues to evaluate the role of ectopic receptors in the physiopathology of Cushing's syndrome. We used in vitro studies on cultured adrenal hyperplasia cells. The setting was a university research laboratory. AIMAH tissues (H1-H3) were removed from three patients previously screened for illegitimate receptors. The main outcome measures were steroidogenic and electrical activities of cultured adrenal hyperplasia cells. In vitro studies showed that the corticotropic effect of 5-HT was mediated by ectopic 5-HT7 receptors in H1 and H2. GIP and hCG stimulated cortisol production via activation of cAMP-dependent protein kinase A in H2. On the contrary, the protein kinase A inhibitor H-89 did not affect hCG-induced cortisol production in H3. Activation of 5-HT7 or GIP receptors enhanced T-type calcium current in H1 or H2 and H3, respectively. In addition, GIP reduced the amplitude of transient and sustained potassium currents in H2. Conversely, hCG did not modify T-type calcium current in H3. These data show that, besides their coupling to the cAMP pathway, illegitimate adrenal receptors can activate additional transduction mechanisms, including modulation of membrane channels.

Exchange Protein Activated by Cyclic AMP (Epac)-Mediated Induction of Suppressor of Cytokine Signaling 3 (SOCS-3) in Vascular Endothelial Cells

Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.

Association of 14-3-3 Proteins to β1-Adrenergic Receptors Modulates Kv11.1 K+ Channel Activity in Recombinant Systems

We identify a new mechanism for the beta(1)-adrenergic receptor (beta(1)AR)-mediated regulation of human ether-a-go-go-related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3epsilon proteins is competed by wild type beta(1)AR by means of a novel interaction between this receptor and 14-3-3epsilon. The association between beta(1)AR and 14-3-3epsilon is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The beta(1)AR/14-3-3epsilon association is direct, since it can be recapitulated using purified 14-3-3epsilon and beta(1)AR fusion proteins and is abolished in cells expressing beta(1)AR phosphorylation-deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that beta(1)AR phosphorylation-deficient mutants do not recruit 14-3-3epsilon away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type beta(1)AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both beta(1)AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness.

Dopamine D2 Receptor Modulation of K+ Channel Activity Regulates Excitability of Nucleus Accumbens Neurons at Different Membrane Potentials

The nucleus accumbens (NAc) is a forebrain area in the mesocorticolimbic dopamine (DA) system that regulates many aspects of drug addiction. Neuronal activity in the NAc is modulated by different subtypes of DA receptors. Although DA signaling has received considerable attention, the mechanisms underlying D(2)-class receptor (D(2)R) modulation of firing in medium spiny neurons (MSNs) localized within the NAc remain ambiguous. In the present study, we performed whole cell current-clamp recordings in rat brain slices to determine whether and how D(2)R modulation of K(+) channel activity regulates the intrinsic excitability of NAc neurons in the core region. D(2)R stimulation by quinpirole or DA significantly and dose-dependently decreased evoked Na(+) spikes. This D(2)R effect on inhibiting evoked firing was abolished by antagonism of D(2)Rs, reversed by blockade of voltage-sensitive, slowly inactivating A-type K(+) currents (I(As)), or eliminated by holding membrane potentials at levels in which I(As) was inactivated. It was also mimicked by inhibition of cAMP-dependent protein kinase (PKA) activity, but not phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Moreover, D(2)R stimulation also reduced the inward rectification and depolarized the resting membrane potentials (RMPs) by decreasing "leak" K(+) currents. However, the D(2)R effects on inward rectification and RMP were blocked by inhibition of PI-PLC, but not PKA activity. These findings indicate that, with facilitated intracellular Ca(2+) release and activation of the D(2)R/G(q)/PLC/PIP(2) pathway, the D(2)R-modulated changes in the NAc excitability are dynamically regulated and integrated by multiple K(+) currents, including but are not limited to I(As), inwardly rectifying K(+) currents (I(Kir)), and "leak" currents (I(K-2P)).

Phosphorylation of Protein Phosphatase Inhibitor-1 by Protein Kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.

In vivo footprinting of the carbamoylphosphate synthetase I cAMP-response unit indicates important roles for FoxA and PKA in formation of the enhanceosome

The expression of carbamoylphosphate synthetase-I (CPS), the first and rate-determining enzyme of the urea cycle, is regulated at the transcriptional level by glucocorticoids and glucagon, the latter acting via cyclic AMP (cAMP). The hormonal response is mediated by a distal enhancer located 6.3 kb upstream of the transcription-start site. Within this enhancer, a cAMP-response unit (CRU) is responsible for mediating cAMP-dependent transcriptional activity. The CPS CRU contains binding sites for cAMP-response element (CRE)-binding protein (CRE-BP), forkhead box A (FoxA), CCAAT/enhancer-binding protein (C/EBP), and an unidentified protein P1. To gain insight in the protein–DNA interactions that activate the CPS CRU in living cells, we have employed in vivo footprinting assays. Comparison of the fibroblast cell line Rat-1 and the hepatoma cell lines FTO-2B and WT-8 showed that FoxA binds the CPS CRU constitutively in CPS-expressing cells only. Comparison of FTO-2B and WT-8 hepatoma cells, which only differ in cAMP responsiveness, demonstrated that the binding of the other transcription factors is dependent on cAMP-dependent protein kinase (PKA) activity. Finally, we observed a footprint between the CRE and the P1-binding site in the in vivo footprint assay that was not detectable by in vitro footprint assays, implying a major change in CRU-associated chromatin conformation upon CRU activation. These findings indicate that activation of the CRU is initiated in a tissue-specific manner by the binding of FoxA. When cellular cAMP and glucocorticoid levels increase, CRE-BP becomes activated, allowing the binding of the remaining transcription factors and the transactivation of the CPS promoter.