Role Of Protein Kinase Research Articles

Roles of protein kinase R in cancer: Potential as a therapeutic target.

Double‐stranded (ds) RNA‐dependent protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. It was initially identified as an innate immune antiviral protein induced by interferon (IFN) and activated by dsRNA. PKR is recognized as a key executor of antiviral host defense. Moreover, it contributes to inflammation and immune regulation through several signaling pathways. In addition to IFN and dsRNA, PKR is activated by multiple stimuli and regulates various signaling pathways including the mitogen‐activated protein kinase (MAPK) and nuclear factor kappa‐light‐chain‐enhancer of activated B cells pathways. PKR was initially thought to be a tumor suppressor as a result of its ability to suppress cell growth and interact with major tumor suppressor genes. However, in several types of malignant disease, such as colon and breast cancers, its role remains controversial. In hepatocellular carcinoma, hepatitis C virus (HCV) is the main cause of liver cancer, and PKR inhibits HCV replication, indicating its role as a tumor suppressor. However, PKR is overexpressed in cirrhotic patients, and acts as a tumor promoter through enhancement of cancer cell growth by mediating MAPK or signal transducer and activator of transcription pathways. Moreover, PKR is reportedly required for the activation of inflammasomes and influences metabolic disorders. In the present review, we introduce the multifaceted roles of PKR such as antiviral function, tumor cell growth, regulation of inflammatory immune responses, and maintaining metabolic homeostasis; and discuss future perspectives on PKR biology including its potential as a therapeutic target for liver cancer.

Phosphoproteomics: Protein Phosphorylation in Regulation of Seed Germination and Plant Growth

Seed germination and plant growth are key stages in the plant life cycle and they are affected by abiotic stresses. Protein phosphorylation is commonly involved in key regulatory process, which mediates plant growth/development and stress responses. In plant, phosphoproteins play roles in stress responses and provide basic backbone in complex signaling networks. The mechanism of plant responses to abiotic stresses has been uncovered through study of protein phosphorylation. Recently, new methods for enrichment of phosphopeptides and phosphoproteins have been developed. Phosphoproteomic techniques are widely applied to studies of seed germination and plant growth. The signaling networks of phytohormones have been explored using the phosphoproteomic approach. In this review, the enrichment methods of phosphopeptides and phosphoproteins are summarized for phosphoproteomics. The roles of protein kinases and phosphatases in plant are described. The significant achievements of phosphoproteomics in the areas of seed germination and plant growth under abiotic stresses are reviewed. Applications of phosphoproteomics are valuable resources to uncover functional proteins regulating plant growth and stress responses.

Translating Neuronal Activity at the Synapse: The Role of the Protein Kinase C Cascade in Short-Term Plasticity

Neurons dynamically regulate neurotransmitter release through many processes known collectively as synaptic plasticity. Post-tetanic potentiation (PTP) is a widespread form of synaptic plasticity that lasts for tens of seconds, and which may have important computational roles and contribute to short-term memory. According to a leading mechanism, the high-frequency stimulation that is necessary to induce PTP increases presynaptic calcium, which activates calcium sensors to regulate the amplitude and time-course of increased release. At the “Synaptic Vesicle Fusion and Retrieval” symposium, I will discuss our investigations into the role of the calcium-sensitive protein kinase C (PKC) as an essential regulator of PTP at 3 mammalian excitatory synapses: the parallel fiber - Purkinje cell synapse in the cerebellar cortex, the calyx of Held in the auditory brainstem, and the CA3 - CA1 synapse in the hippocampus. I will introduce a conditional knock-in approach to selectively mutate all PKC phosphorylation sites in the prominent PKC phosphorylation substrate Munc18-1, an SM family protein that is essential for release. Finally, I will describe initial efforts using this new mouse model to delineate the molecular events underlying enhanced neurotransmission during PTP.

Role of protein kinase C, PI3 kinase, tyrosine kinases, NO-synthase, KATP channels and MPT pore in the signaling pathway of the cardioprotective effect of chronic continuous hypoxia.

It was established that adaptation to chronic continuous normobaric hypoxia (CCNH) increases cardiac tolerance to ischemia and reperfusion. It was performed coronary artery occlusion (20 min) and reperfusion (3 h) in Wistar rats. CCNH promoted a decrease in the infarct size/area at risk ratio in 2-fold. CCNH promoted an increase in the nitrite/nitrate levels in blood serum and myocardium. Pretreatment with protein kinase C (PKC) inhibitor chelerythrine, NO-synthase (NOS) inhibitor L-NAME, iNOS inhibitor S-methylisothiourea, KATP channel blocker glibenclamide, mitoKATP channel blocker 5-hydroxydecanoic acid abolished the infarct-reducing effect of CCNH. The non-selective tyrosine kinase inhibitor genistein attenuated but not eliminated infarct-sparing effect of CCNH. The nNOS inhibitor 7-nitroindazole, sarcKATP channel blocker HMR 1098, MPT pore inhibitor atractyloside, PI3 kinase inhibitor wortmannin did not reverse infarct-limiting effect of CCNH. It was concluded that infarct-reducing effect of CCNH is mediated via PKC, iNOS activation and mitoKATP channel opening. While nNOS, PI3 kinase, sarcKATP channel, MPT pore are not involved in the development of CCNH-induced cardiac tolerance to impact of ischemia-reperfusion.

PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

Background/Aims: The voltage-gated potassium channel K_V11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation. Methods: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting. Results: Using brain tissue from rat and mouse, we mapped several phosphorylated K_V11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of K_V11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four K_V11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in K_V11.1. Conclusions: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of K_V11.1. We propose that PKD1 mediates the PKC effects on K_V11.1 and we found that PKD targets S284 in the N-terminus of the channel.

Clinical manifestations and basic mechanisms of myocardial ischemia/reperfusion injury.

Acute myocardial ischemia/reperfusion (I/R) injury is a significant, unsolved clinical puzzle. In the disease context of acute myocardial infarction, reperfusion remains the only effective strategy to salvage ischemic myocardium, but it also causes additional damage. Myocardial I/R injury is composed of four types of damage, and these events attenuate the benefits of reperfusion therapy. Thus, inventing new strategies to conquer I/R injury is an unmet clinical need. A variety of pathological processes and mediators, including changes in the pH, generation of reactive oxygen radicals, and intracellular calcium overload, are proposed to be crucial in I/R-related cell injury. Among the intracellular events that occur during I/R, we stress the importance of protein phosphorylation signaling and elaborate its regulation. A variety of protein kinase pathways could be activated in I/R, including reperfusion injury salvage kinase and survivor-activating factor enhancement pathways, which are critical to cardiomyocyte survival. In addition to serine/threonine phosphorylation signaling, protein tyrosine phosphorylation is also critical in multiple cell functions and survival. However, the roles of protein kinases and phosphatases in I/R have not been extensively studied yet. By better understanding the mechanisms of I/R injury, we may have a better chance to develop new strategies for I/R injury and apply them in the clinical patient care.

Glutamatergic Regulation of Hypothalamic Presympathetic Neurons in Hypertension.

Elevated sympathetic vasomotor tone emanating from the brain is a major mechanism involved in the development of hypertension. Increased glutamatergic excitatory input to presympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus leads to increased sympathetic outflow in various animal models of hypertension. Recent studies have revealed molecular and cellular mechanisms underlying enhanced glutamatergic synaptic input to PVN presympathetic neurons in hypertension. In this review article, we summarize recent findings on changes in inotropic and metabotropic glutamate receptors, at both presynaptic and postsynaptic sites, responsible for increased glutamatergic input to PVN presympathetic neurons in hypertension. Particular emphasis is placed on the role of protein kinases and phosphatases in the potentiated activity of synaptic NMDA receptors in the PVN in hypertension. New findings about glutamatergic synaptic plasticity in the PVN not only improve the understanding of molecular mechanisms involved in heightened activity of the sympathetic nervous system but also suggest new therapeutic targets for treating drug-resistant, neurogenic hypertension.

Role of PKCα/HO-1 signaling pathway in endotoxin-induced damage to alveolar macrophages of rats: the relationship with mitofusin-1

Objective To evaluate the role of protein kinase C α (PKCα)/heme oxygenase-1 (HO-1) signaling pathway in endotoxin-induced damage to alveolar macrophages and the relationship with mitofusin-1 (Mfn1) in rats. Methods Rat alveolar macrophages NR8383 cells cultured in vitro were seeded in 96-well plates at a density of 1×104 cells/ml.NR8383 cells were divided into 5 groups (n=15 each) using a random number table: control group (group C), endotoxin challenge model group (group E), PKCα inhibitor Go6976 group (group G), PKCα agonist PMA group (group P) and dimethyl sulfoxide group (group D). NR8383 cells were stimulated with 10 μg/ml lipopolysaccharide (LPS) to establish the model of endotoxin challenge in alveolar macrophages.In G, P and D groups, cells were pretreated with 5 μmol/L Go6976, 100 nmol/L PMA and 0.1% dimethyl sulfoxide, respectively, for 30 min starting from 30 min before stimulation with LPS, and 10 μg/ml LPS was then given.The cells were collected after 24 h of incubation for measurement of malondialdehyde (MDA) and reactive oxygen species (ROS) contents, superoxide dismutase (SOD) activity and expression of PKCα, HO-1 and Mfn1 protein and mRNA (by fluorescent quantitative polymerase chain reaction or Western blot). Results Compared with group C, MDA and ROS contents were significantly increased, the SOD activity was decreased, the expression of PKCα and HO-1 protein and mRNA was up-regulated, and the expression of Mfn1 protein and mRNA was down-regulated in E, G, P and D groups (P 0.05). Conclusion Promotion of Mfn1 expression following PKCα/HO-1 signaling pathway activation is the endogenous protective mechanism of endotoxin-induced damage to alveolar macrophages of rats. Key words: Protein kinase C-alpha; Heme oxygenase-1; Endotoxins; Mitochondrial proteins; Membrane fusion proteins

Role of Akt/mTOR signaling pathway in ulinastatin-induced reduction of oxidative stress injury to cardiomyocytes of rats

Objective To evaluate the role of protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway in ulinastatin-induced reduction of oxidative stress injury to cardiomyocytes of rats. Methods H9c2 cells were inoculated in 96-well plates at a density of 104 cells/well.The cells were divided into 5 groups using a random number table: control group(group C, n=12), H2O2 group(n=12), H2O2 plus ulinastatin group(group HU, n=12), H2O2 plus ulinastatin plus LY294002 group(group HUL, n=9)and H2O2 plus LY294002 group(group HL, n=9). In group H2O2, H2O2(final concentration 200 μmol/L)was added, and the cells were incubated for 12 h in the incubator.In HU, HL and HUL groups, ulinastatin(final concentration 50 U/ml), LY294002(final concentration 10 μmol/L), and ulinastatin(final concentration 50 U/ml)plus LY294002(final concentration 10 μmol/L)were added, respectively, the cells were incubated for 2 h, H2O2(final concentration 200 μmol/L)was then added, and the cells were incubated for 12 h in the incubator.The cell survival rate was measured by methyl thiazolyl tetrazolium assay.The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were determined by using water soluble tetrazolium-1 and thiobarbituric acid colorimetric assays.Cell apoptosis was measured by flow cytometry, and apoptosis rate(AR)was calculated.The expression of Akt, phosphorylated Akt(p-Akt), mTOR, phosphorylated mTOR(p-mTOR), activated caspase-3 and activated poly-ADP-ribose polymerase(PARP)was detected by Western blot.The p-Akt/AKT and p-mTOR/mTOR ratios were calculated. Results Compared with group C, the cell survival rate was significantly decreased, AR was increased, the SOD activity was decreased, the MDA content was increased, the expression of activated caspase-3 and PARP was up-regulated(P 0.05). Compared with group H2O2, the cell survival rate was significantly increased, AR was decreased, the SOD activity was increased, the MDA content was decreased, the p-Akt/AKT and p-mTOR/mTOR ratios were increased, and the expression of activated caspase-3 and PARP was down-regulated in group HU(P<0.05). Compared with group HU, the cell survival rate was significantly decreased, AR was increased, and the expression of activated caspase-3 and PARP was up-regulated in group HUL(P<0.05). Conclusion The mechanism by which ulinastatin reduces oxidative stress injury to cardiomyocytes is related to activation of Akt/mTOR signaling pathway in rats. Key words: Trypsin inhibitors; Protein-serine-threonine kinases; Oxidative stress; Myocytes; Cardiac

Prostaglandin E2 stimulates the expression of cumulus expansion-related genes in pigs: the role of protein kinase B

The production of prostaglandin E2 (PGE2) seems to play an important role in the ovulation process. PGE2 was found to induce cumulus expansion and meiosis resumption in mice, but little is known about its role in pigs. The goals of this study were (a) to assess the effect of PGE2 on the expression levels of cumulus expansion-related genes, (b) to define the signaling pathways that drive the PGE2-stimulated expression of cumulus expansion-related genes, (c) to measure the effect of PGE2 on the activation of key signaling molecules (MAPK3/1, PKB) and on hyaluronan production in cumulus cells, and (d) to assess the effect of PGE2 on meiosis resumption. We documented that PGE2 is able to induce the expression of cumulus expansion-related genes (HAS2, TNFAIP6) as well as genes involved in steroidogenesis (CYP11A1) or prostaglandin production (PTGS2). PGE2 is able to activate PKB and MAPK3/1 and induce mild cumulus expansion and meiosis resumption, but less efficiently than FSH.

PKCβ: Expanding role in hepatic adaptation of cholesterol homeostasis to dietary fat/cholesterol.

Cholesterol homeostasis relies on an intricate network of cellular processes whose deregulation in response to Western type high-fat/cholesterol diets can lead to several life-threatening pathologies. Significant advances have been made in resolving the molecular identity and regulatory function of transcription factors sensitive to fat, cholesterol, or bile acids, but whether body senses the presence of both fat and cholesterol simultaneously is not known. Assessing the impact of a high-fat/cholesterol load, rather than an individual component alone, on cholesterol homeostasis is more physiologically relevant because Western diets deliver both fat and cholesterol at the same time. Moreover, dietary fat and dietary cholesterol are reported to act synergistically to impair liver cholesterol homeostasis. A key insight into the role of protein kinase C-β (PKCβ) in hepatic adaptation to high-fat/cholesterol diets was gained recently through the use of knockout mice. The emerging evidence indicates that PKCβ is an important regulator of cholesterol homeostasis that ensures normal adaptation to high-fat/cholesterol intake. Consistent with this function, high-fat/cholesterol diets induce PKCβ expression and signaling in the intestine and liver, while systemic PKCβ deficiency promotes accumulation of cholesterol in the liver and bile. PKCβ disruption results in profound dysregulation of hepatic cholesterol and bile homeostasis and imparts sensitivity to cholesterol gallstone formation. The available results support involvement of a two-pronged mechanism by which intestine and liver PKCβ signaling converge on liver ERK1/2 to dictate diet-induced cholesterol and bile acid homeostasis. Collectively, PKCβ is an integrator of dietary fat/cholesterol signal and mediates changes to cholesterol homeostasis.

Protein Kinase C Activation Promotes α1B-Adrenoceptor Internalization and Late Endosome Trafficking through Rab9 Interaction. Role in Heterologous Desensitization

Upon agonist stimulation, α1B-adrenergic receptors couple to Gq proteins, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized, and internalized. Internalization seems to involve scaffolding proteins, such as β-arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, in α1B-AR vesicular traffic were investigated by studying α1B-adrenergic receptor-Rab protein interactions, using Förster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In human embryonic kidney 293 cells overexpressing Discosoma spp. red fluorescent protein (DsRed)-tagged α1B-ARs and enhanced green fluorescent protein--tagged Rab proteins, pharmacological protein kinase C activation mimicked α1B-AR traffic elicited by nonrelated agents, such as sphingosine 1-phosphate (i.e., transient α1B-AR-Rab5 FRET signal followed by a sustained α1B-AR-Rab9 interaction), suggesting brief receptor localization in early endosomes and transfer to late endosomes. This latter interaction was abrogated by blocking protein kinase C activity, resulting in receptor retention at the plasma membrane. Similar effects were observed when a dominant-negative Rab9 mutant (Rab9-GDP) was employed. When α1B-adrenergic receptors that had been mutated at protein kinase C phosphorylation sites (S396A, S402A) were used, phorbol ester-induced desensitization of the calcium response was markedly decreased; however, interaction with Rab9 was only partially decreased and internalization was observed in response to phorbol esters and sphingosine 1-phosphate. Finally, Rab9-GDP expression did not affect adrenergic-mediated calcium response but abolished receptor traffic and altered desensitization. Data suggest that protein kinase C modulates α1B-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off.

Regulation of Cardiac Voltage-Gated Sodium Channel by Kinases: Roles of Protein Kinases A and C.

In the heart, voltage-gated sodium (Nav) channel (Nav1.5) is defined by its pore-forming α-subunit and its auxiliary β-subunits, both of which are important for its critical contribution to the initiation and maintenance of the cardiac action potential (AP) that underlie normal heart rhythm. The physiological relevance of Nav1.5 is further marked by the fact that inherited or congenital mutations in Nav1.5 channel gene SCN5A lead to altered functional expression (including expression, trafficking, and current density), and are generally manifested in the form of distinct cardiac arrhythmic events, epilepsy, neuropathic pain, migraine, and neuromuscular disorders. However, despite significant advances in defining the pathophysiology of Nav1.5, the molecular mechanisms that underlie its regulation and contribution to cardiac disorders are poorly understood. It is rapidly becoming evident that the functional expression (localization, trafficking and gating) of Nav1.5 may be under modulation by post-translational modifications that are associated with phosphorylation. We review here the molecular basis of cardiac Na channel regulation by kinases (PKA and PKC) and the resulting functional consequences. Specifically, we discuss: (1) recent literature on the structural, molecular, and functional properties of cardiac Nav1.5 channels; (2) how these properties may be altered by phosphorylation in disease states underlain by congenital mutations in Nav1.5 channel and/or subunits such as long QT and Brugada syndromes. Our expectation is that understanding the roles of these distinct and complex phosphorylation processes on the functional expression of Nav1.5 is likely to provide crucial mechanistic insights into Na channel associated arrhythmogenic events and will facilitate the development of novel therapeutic strategies.

Role of Protein Kinase C and Nox2-Derived Reactive Oxygen Species Formation in the Activation and Maturation of Dendritic Cells by Phorbol Ester and Lipopolysaccharide.

Aims. Activation/maturation of dendritic cells (DCs) plays a central role in adaptive immune responses by antigen processing and (cross-) activation of T cells. There is ongoing discussion on the role of reactive oxygen species (ROS) in these processes and with the present study we investigated this enigmatic pathway. Methods and Results. DCs were cultured from precursors in the bone marrow of mice (BM-DCs) and analyzed for ROS formation, maturation, and T cell stimulatory capacity upon stimulation with phorbol ester (PDBu) and lipopolysaccharide (LPS). LPS stimulation of BM-DCs caused maturation with moderate intracellular ROS formation, whereas PDBu treatment resulted in maturation with significant ROS formation. The NADPH oxidase inhibitors apocynin/VAS2870 and genetic gp91phox deletion both decreased the ROS signal in PDBu-stimulated BM-DCs without affecting maturation and T cell stimulatory capacity of BM-DCs. In contrast, the protein kinase C inhibitors chelerythrine/Gö6983 decreased PDBu-stimulated ROS formation in BM-DCs as well as maturation. Conclusion. Obviously Nox2-dependent ROS formation in BM-DCs is not always required for their maturation or T cell stimulatory potential. PDBu/LPS-triggered BM-DC maturation rather relies on phosphorylation cascades. Our results question the role of oxidative stress as an essential “danger signal” for BM-DC activation, although we cannot exclude contribution by other ROS sources.

Role of protein kinase C in metabolic regulation of the cardiac Na+ channel

The reduced form of nicotinamide adenine dinucleotide (NADH) increases in cardiomyopathy, activates protein kinase C (PKC), up-regulates mitochondrial reactive oxygen species (mitoROS), and down-regulates the cardiac Na+ channel (NaV1.5). The purpose of this study was to determine how NADH signals down-regulation of NaV1.5. Isolated mouse cardiomyocytes were used for patch-clamp recording and for monitoring mitoROS with MitoSOX Red. HEK293 cells were used for transient transfections. HEK293 cells stably expressing human NaV1.5 were used for single channel recording, whole-cell patch-clamp recording, activity measurements of phospholipase C and phospholipase D (PLD), channel protein purification, and co-immunoprecipitation with PKC isoforms. HL-1 cells were used for mitochondria isolation. NADH enhanced PLD activity (1.6- ± 0.1-fold, P <.01) and activated PKCδ. Activated PKCδ translocated to mitochondria and up-regulated mitoROS (2.8- ± 0.3-fold, P <.01) by enhancing the activities of mitochondrial complexes I, II, and IV (1.1- to 1.5-fold, P <.01). PKCδ also interacted with NaV1.5 to down-regulate Na+ current (INa). Reduction in INa by activated PKCδ was prevented by antioxidants and by mutating the known PKC phosphorylation site S1503. At the single channel level, the mechanism of current reduction by PKC and recovery by protein kinase A was a change in single channel conductance. NADH activated PKCδ by enhancing PLD activity. PKCδ modulated both mitoROS and NaV1.5. PKCδ elevated mitoROS by enhancing mitochondrial oxidative phosphorylation complex activities. PKCδ-mediated channel phosphorylation and mitoROS were both required to down-regulate NaV1.5 and alter single channel conductance.

Mimicking the Phenomenon of Ischemic Preconditioning by Actions on Cannabinoid Receptors: The Roles of Protein Kinase C and NO Synthase

Stimulation of CB1 receptors was found to mimic the phenomenon of preconditioning. As the cardioprotective effect of the cannabinoid HU-210 is seen in both in vivo experiments and in vitro studies, there are grounds for suggesting that the protective effects of HU-210 are mediated by activation of cardiac CB1 receptors. The cardioprotective effect of the cannabinoid HU-210 was found to be linked with stimulation of protein kinase C, while NO synthase has no role in the protective action of stimulation of CB1 receptors.

Effects of heterologous expression of human cyclic nucleotide phosphodiesterase 3A (hPDE3A) on redox regulation in yeast.

Oxidative stress plays a pivotal role in pathogenesis of cardiovascular diseases and diabetes; however, the roles of protein kinase A (PKA) and human phosphodiesterase 3A (hPDE3A) remain unknown. Here, we show that yeast expressing wild-type (WT) hPDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous hydrogen peroxide (H2O2), respectively. H2O2-stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A (Oxidative stress Sensitive 1, OxiS1), compared with yeast expressing WT hPDE3A (Oxidative stress Resistant 1, OxiR1). In OxiR1, YAP1 and YAP1-dependent antioxidant genes were up-regulated, accompanied by a reduction in thioredoxin peroxidase. In OxiS1, expression of YAP1 and YAP1-dependent genes was impaired, and the thioredoxin system malfunctioned. H2O2 increased cyclic adenosine monophosphate (cAMP)-hydrolyzing activity of WT hPDE3A, but not K13R hPDE3A, through PKA-dependent phosphorylation of hPDE3A, which was correlated with its ubiquitinylation. The changes in antioxidant gene expression did not directly correlate with differences in cAMP-PKA signaling. Despite differences in their capacities to hydrolyze cAMP, total cAMP levels among OxiR1, OxiS1, and mock were similar; PKA activity, however, was lower in OxiS1 than in OxiR1 or mock. During exposure to H2O2, however, Sch9p activity, a target of Rapamycin complex 1-regulated Rps6 kinase and negative-regulator of PKA, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol, with PKA activation. In OxiS1, Sch9p activity was unchanged during exposure to H2O2, consistent with reduced activation of PKA. These results suggest that, during oxidative stress, TOR-Sch9 signaling might regulate PKA activity, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress.

Participation of protein kinases andphosphatases in the progesterone-induced acrosome reaction and calcium influx in human spermatozoa.

In human spermatozoa, protein kinases have a role in the acrosome reaction (AR) induced by a variety of stimuli. However, there is disagreement or a lack of information regarding the role of protein kinases and phosphatases in the progesterone (P)-induced increase in intracellular calcium concentration ([Ca2+ ]i ). In addition, there are no studies regarding the role of Ser/Thr and Tyr phosphatases and there are contradictory results regarding the role of Tyr kinases in the P-induced acrosome reaction. Here, we performed a simultaneous evaluation of the involvement of protein kinases and phosphatases in the P-induced acrosome reaction and in the P-induced calcium influx. Motile spermatozoa were capacitated for 18h and different aliquots were allocated to treated or control groups and then evaluated for their ability to undergo the acrosome reaction and to increase [Ca2+ ]i in response to P. The acrosome reaction was evaluated using Pisum sativum agglutinin (PSA)-FITC, and [Ca2+ ]i was evaluated using fura 2AM. At all of the concentrations tested, PKA inhibitors significantly reduced the percentage of the P-induced acrosome reaction (p<0.001). However, only the highest concentrations of PKA inhibitors reduced the P-induced calcium influx; lower concentrations of PKA inhibitors did not affect it. Similar results were apparent for PKC inhibitors and for tyrosine kinase inhibitors. None of the Ser/Thr phosphatase inhibitors affected the P-induced acrosome reaction or the P-induced calcium influx, except for the PP2B inhibitors that significantly reduced the P-induced acrosome reaction without affecting calcium influx. Finally, the protein tyrosine phosphatase inhibitors significantly blocked the P-induced acrosome reaction and reduced the amplitude of the P-induced calcium transient (p<0.001) as well as the amplitude of the plateau phase (p<0.01). The data suggest that protein kinases and possibly PP2B have a role on the acrosome reaction at some point downstream of calcium entry and that Tyr phosphatases have a role on the acrosome reaction upstream of calcium entry.

Role of Protein Kinase G and Reactive Oxygen Species in the Regulation of Podocyte Function in Health and Disease.

Podocytes and their foot processes form an important cellular layer of the glomerular barrier involved in the regulation of glomerular permeability. Disturbing the function of podocytes plays a central role in the development of proteinuria in diabetic nephropathy. Retraction of the podocyte foot processes that form slit diaphragms is a common feature of proteinuria; although, the correlation between these events in not well understood. Notably, it is unclear whether podocyte foot processes are able to regulate slit diaphragm permeability and glomerular ultrafiltration. The occurrence of reactive oxygen species generation, insulin resistance, and hyperglycemia characterizes early stages of type 2 diabetes. Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. It is activated in both cGMP-dependent and cGMP-independent manners. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes. J. Cell. Physiol. 232: 691-697, 2017. © 2016 Wiley Periodicals, Inc.

Regulation of phosphatidylinositol 4-kinase activity in rat pancreatic acini.

Previous data showed that carbachol (CCh) elicits a concentration-dependent increase in phosphatidylinositol (PtdIns) 4-kinase activity in homogenates derived from agonist-stimulated pancreatic acini. In the present study CCh elicited a time-dependent increase in PtdIns 4-kinase activity, which was blocked by n-methylscopolamine and mimicked by muscarine. A membrane-associated PtdIns 4-kinase activity was identified that displayed maximal activity in the pH range of 5.5-8.5. The zymogen granule fraction possessed relatively high specific enzyme activity, which was sensitive to CCh stimulation. The enzyme had apparent Km values for PtdIns and ATP of 4 and 60 microM, respectively. CCh caused no discernible change in the Km for either PtdIns or ATP but increased Vmax. Dioctanoylglycerol and oleoyl acetylglycerol augmented PtdIns 4-kinase activity, which was blocked by staurosporine, thus suggesting a possible role for protein kinase C in the regulation of PtdIns 4-kinase. These collective findings demonstrate a muscarinic receptor-mediated regulation of PtdIns 4-kinase activity in exocrine pancreas, involving a protein kinase C-dependent process.