Dephosphorylation Of Protein Kinase Research Articles

Oxygen-dependent regulation of permeability in low resistance intestinal epithelial cells infected with Giardia lamblia

Intestinal epithelial cells (IECs) reside in a highly anaerobic environment that is subject to daily fluctuations in partial oxygen pressure (pO2), depending on intestinal tissue perfusion. This condition, known as physiological hypoxia, has a major impact on the maintenance of gut homeostasis, such as effects on the integrity and function of the intestinal epithelial barrier. Giardia lamblia is a microaerophilic protozoan parasite that infects and colonizes the small intestine of its host, causing watery diarrhea. The disease, known as giardiasis, is associated with enhanced intestinal permeability and disruption or reorganization of tight junction (TJ) proteins between IECs. Given the central role of oxygen in gut homeostasis, in this study, we aimed to evaluate whether pO2 affects intestinal permeability (flux of ions and macromolecules) and TJ protein expression in human IECs during G. lamblia infection. Using human cell lines HuTu-80 and Caco-2 as models of “loose” (low resistance) and “tight” (high resistance) intestines, respectively, we elucidated that low pO2 drives intestinal barrier dysfunction in IECs infected with trophozoites through dephosphorylation of protein kinase C (PKC α/β II). Additionally, we demonstrated that IECs infected with trophozoites in the presence of a pharmacological PKC activator (phorbol 12-myristate 13-acetate) partially restored the barrier function, which was correlated with increased protein expression levels of zonula occludens (ZO)-2 and occludin. Collectively, these results support the emerging theory that molecular oxygen impacts gut homeostasis during Giardia infection via direct host signaling pathways. These findings further our knowledge regarding Giardia-host interactions and the pathophysiological mechanisms of human giardiasis.

Intracellular antibody targeting HBx suppresses invasion and metastasis in hepatitis B virus-related hepatocarcinogenesis via protein phosphatase 2A-B56γ-mediated dephosphorylation of protein kinase B.

Hepatitis B virus X (HBx) is closely associated with HBV-related hepatocarcinogenesis via the inactivation of tumour suppressors. Protein phosphatase 2A (PP2A) regulatory subunit B56 gamma (B56γ), as a tumour suppressor, plays a critical role in regulating cellular phosphorylation signals via dephosphorylation of signalling proteins. However, the underlying mechanism that B56γ involved in regulating HBx-associated hepatocarcinogenesis phenotypes and mediating anti-HBx antibody-mediated tumour suppression remains unknown. We used bioinformatics analysis, paired HCC patient specimens, HBx transgenic (HBx-Tg) mice, xenograft nude mice, HBV stable replication in the HepG2.2.15 cells, and anti-HBx antibody intervention to systematically evaluate the biological function of protein kinase B (AKT) dephosphorylation through B56γ in HBx-associated hepatocarcinogenesis. Bioinformatics analysis revealed that AKT, matrix metalloproteinase 2 (MMP2), and MMP9 were markedly upregulated, while cell migration and viral carcinogenesis pathways were activated in HBV-infected liver tissues and HBV-associated HCC tissues. Our results demonstrated that HBx-expression promotes AKT phosphorylation (p-AKTThr308/Ser473 ), mediating the migration and invasion phenotypes in vivo and in vitro. Importantly, in clinical samples, HBx and B56γ were downregulated in HBV-associated HCC tumour tissues compared with peritumor tissues. Moreover, intervention with site-directed mutagenesis (AKTT308A , AKTS473A ) of p-AKTThr308/Ser473 mimics dephosphorylation, genetics-based B56γ overexpression, and intracellular anti-HBx antibody inhibited cell growth, migration, and invasion in HBx-expressing HCC cells. Our results demonstrated that B56γ inhibited HBV/HBx-dependent hepatocarcinogenesis by regulating the dephosphorylation of p-AKTThr308/Ser473 in HCC cells. The intracellular anti-HBx antibody and the activator of B56γ may provide a multipattern chemopreventive strategy against HBV-related HCC.

Memory regulation in feeding habit transformation to dead prey fish of Chinese perch (Siniperca chuatsi).

Memory drove a critical process of feeding habit transformation in Chinese perch when they re-trained to eat dead prey fish. To investigate the regulatory mechanism of cAMP-response element-binding protein (CREB) signaling pathway on the memory of Chinese perch during feeding habit transformation, the phosphorylation levels of upstream signal proteins of CREB between the control group (trained once) and the experimental group (trained twice) were measured. The results illustrated that the re-training was correlated to phosphorylation of extracellular regulated protein kinase (ERK1/2) and calcium/calmodulin-dependent protein kinase II (CaMKII), and dephosphorylation of protein kinase A (PKA) of Chinese perch. Inhibition of ERK1/2-CREB pathway decreased the mRNA levels of memory-related genes ((fos-related antigen 2 (fra2), CCAAT enhancer-binding protein delta (c/ebpb), immediate-early gene zif268 (zif268), proto-oncogenes c-fos (c-fox) and synaptotagmin-IV (sytIV)) and mRNA levels of appetite-related genes (agouti-related peptide (agrp) and ghrelin), and activation of PP1-CREB pathway increased the phosphorylated levels of CREB, the mRNA levels of memory-related genes (fra2, c/ebpb, zif268, and c-fox), and the mRNA levels of appetite-related genes (pro-opiomelanocortin (pomc) and leptin) in primary brain cells of Chinese perch. The memory in Chinese perch feeding habit transformation was associated with the ERK1/2-CREB and PP1-CREB pathways, which could regulate the transcription of memory-related genes and appetite-related genes.

Protein Phosphatase 1 Regulates Human Cytomegalovirus Protein Translation by Restraining AMPK Signaling

Human cytomegalovirus (HCMV) carries the human protein phosphatase 1 (PP1) and other human proteins important for protein translation in its tegument layer for a rapid supply upon infection. However, the biological relevance behind PP1 incorporation and its role during infection is unclear. Additionally, PP1 is a difficult molecular target due to its promiscuity and similarities between the catalytic domain of multiple phosphatases. In this study, we circumvented these shortcomings by using 1E7-03, a small molecule protein–protein interaction inhibitor, as a molecular tool of noncatalytic PP1 inhibition. 1E7-03 treatment of human fibroblasts severely impaired HCMV replication and viral protein translation. More specifically, PP1 inhibition led to the deregulation of metabolic signaling pathways starting at very early time points post-infection. This effect was at least partly mediated by the prevention of AMP-activated protein kinase dephosphorylation, leading to elongation factor 2 hyperphosphorylation and reduced translation rates. These findings reveal an important mechanism of PP1 for lytic HCMV infection.

CONDITION OF RIG-IAND NF-KB-SIGNAL PATHWAYS IN MONONUCLEAR CELLS OF WHOLE BLOOD OF PRACTICALLY HEALTHY PEOPLE AND RECONVENCENTS OF PNEUMONIA AFFECTED BY MITOGENIC STIMULATION

The key role in the antiviral and antimicrobial defense of the body is played by the RIG-I and NF-kB signaling pathways. The RIG-I signaling pathway activates interferon-regulated factors IRF3 and IRF7, and the central component of the NF-kB signaling pathway, the nuclear transcription factor NF-kB, determines the production of endogenous antimicrobial peptides and interferons by cells. One of the key regulators of the RIG-I signaling pathway is the mitochondrial protein MAVS, which integrates signals from receptors that recognize pathogenicity patterns. The influence of various factors, such as bacterial toxins, free radicals, reactive oxygen species, leads to MAVS dysfunction, impaired antiviral resistance and the progression of viral infection. However, despite the important role of the proteins of the RIG-I-pathway and the components of the NF-KB-signaling pathway in ensuring the body's resistance to infections and sanogenesis, their significance in the postclinical phase has not been fully studied. The aim of the study was to evaluate the content of the components of RIG-I and NF-kB signaling pathways in mononuclear cells of whole blood of healthy individuals and pneumonia convalescents after exposure to a complex mitogen. The enzyme-linked immunosorbent assay determined the content of components of the NF-kB signaling pathway (p50, p65, c-Rel, RelB, NF-kB2), protein kinases of the NF-kB nuclear transcription factor inhibitor (IkB), and RIG-I- proteins in mononuclear cells of whole blood the signal path (TAK1, TBK1, TRIM25, TMEM173, RNF125, IRF3, IRF7, MAVS), RIG-I-dependent helicase (LGP2), the level of phosphorylation of protein kinase p38 and IkB, as well as the production of whole blood cells IL-4, IL-, were evaluated 12, RANTES, cathelicidin and interferons (IFN-p and IFNa). It was established that in the subclinical phase of community-acquired bacterial pneumonia in mononuclear cells of whole blood after stimulation with a complex mitogen containing lipopolysaccharide, the content of RelB, MaVS, DHX58, and IRF7 decreased compared to practically healthy individuals, p38 protein kinase dephosphorylation was noted. In contrast, the concentrations of IKKa, IKKp, the level of phosphorylation of kBa, the protein content of TRIM25, TMEM173, OTuD5, RNF125 and tBk1 were increased. These changes were accompanied by a statistically significant decrease in the production of IL-4, IL-12, RANTES, cathelicidin and IFN-p against the background of an increase in the level of IFNa. The effect on the mononuclear cells of whole blood of a complex mitogen led to a change in the ratio of the components of the signaling pathways that determine the antibacterial and antiviral defense of the body. In patients with pneumonia, against the background of mitogenic stimulation, the production of cathelicidin,

Isobavachalcone inhibits the proliferation and invasion of tongue squamous cell carcinoma cells.

Isobavachalcone (2′,4′,4-trihydroxy-3′-[3′-methylbut-3′-ethyl] chalcone or IBC) exhibits anticancer activities in a number of types of cancer cell. However, its role in tongue squamous cell carcinoma (TSCC) cells remains unclear. The aim of the present study was to investigate the biological effect of IBC in TSCC Tca8113 cells. The function of IBC on Tca8113 cell apoptosis and apoptosis-associated signaling pathways was determined using an MTT assay, morphological staining, annexin V-propidium iodide (PI) staining and Western blot analysis. The effects of IBC on Tca8113 cell migration, invasion and relative protein expression were confirmed using wound healing analysis, Transwell invasion analysis and Western blot analysis, respectively. The results of the MTT assay and annexin V-PI staining indicated that IBC is able to significantly inhibit proliferation and induce apoptosis of Tca8113 cells in vitro. IBC treatment resulted in typical apoptotic morphology of nuclear fragmentation and apoptotic bodies in Tca8113 cells. Western blot analysis further demonstrated that IBC caused downregulation of the expression of B-cell lymphoma 2 (Bcl-2) protein, upregulation of the expression of Bcl-2-associated X protein (Bax), activation of caspases, and dephosphorylation of protein kinase B (Akt) and extracellular-signal-regulated kinase (ERK) proteins in a concentration- and time-dependent manner. The results of the present study suggest that IBC induces apoptosis in Tca8113 cells and that the induction may be associated with the activation of Bcl-2, Bax and caspase-3, and the inactivation of Akt and ERK. Furthermore, IBC inhibited migration and invasion of Tca8113 cells in vitro by downregulating matrix metalloproteinase (MMP)-2 and MMP-9 protein expression. The results of the present study indicate that IBC may be a potential anticancer drug for the treatment of TSCC.

Activation of transient receptor potential vanilloid 1 decreases endothelial nitric oxide synthase phosphorylation at Thr497 by protein phosphatase 2B‐dependent dephosphorylation of protein kinase C (696.1)

We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium‐permeable non‐selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα at Serine 657, and PKCβ2 at Serine 660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B‐PKC complex. Inhibition of TRPV1 activation, removal of extracellular calcium or pharmacological inhibition of PI3K/Akt/CaMKII/AMPK signaling pathway abolished the evodiamine‐induced alterations in phosphorylation of eNOS at Thr497, PKCα, PKCβ2 and PP2B activity, as well as the formation of a PP2B‐PKC complex. Inhibition of PP2B activation partially reduced the evodiamine‐induced NO bioavailability and tube formation in ECs and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα, and PKCβ2 in apolipoprotein E (ApoE)‐deficient mouse aortas but not TRPV1‐deficient or ApoE/TRPV1 double‐knockout mice. TRPV1 activation in ECs may elicit a calcium‐dependent effect on PP2B‐PKC signaling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.Grant Funding Source: National Science Council, Taiwan

Activation of transient receptor potential vanilloid 1 decreases endothelial nitric oxide synthase phosphorylation at Thr497 by protein phosphatase 2B‐dependent dephosphorylation of protein kinase C

We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium (Ca(2+) )-permeable non-selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. Western blotting and immunoprecipitation were used for the evaluation of protein phosphorylation; protein phosphatase 2B (PP2B) activity was assessed by convention kit; Griess assay was for NO production; tube formation and Matrigel plug assay were used for angiogenesis. In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα) at Serine 657 (Ser657) and PKCβ2 at Ser660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B-PKC complex. Inhibition of TRPV1 activation by the pharmacological antagonists, removal of extracellular Ca(2+) or pharmacological inhibition of PI3K/Akt/calmodulin-dependent protein kinase II/AMP-activated protein kinase signalling pathway abolished the evodiamine-induced alterations in phosphorylation of eNOS at Thr497, PKCα at Ser657, PKCβ2 at Ser660 and PP2B activity, as well as the formation of a PP2B-PKC complex. Inhibition of PP2B activation partially reduced the evodiamine-induced NO bioavailability and tube formation in endothelial cells (ECs) and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα at Ser657 and PKCβ2 at Ser660 in apolipoprotein E (ApoE)-deficient mouse aortas but not TRPV1-deficient or ApoE/TRPV1 double-knockout mice. TRPV1 activation in ECs may elicit a Ca(2+) -dependent effect on PP2B-PKC signalling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.

Aldosterone Modulates Endothelial Permeability and Endothelial Nitric Oxide Synthase Activity by Rearrangement of the Actin Cytoskeleton

Aldosterone (Aldo) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Aldo alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells were exposed to Aldo (10(-9) mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions and activation of intracellular signaling pathways were determined. Aldo increased endothelial permeability for molecules ≤ 70 kDa within 60 minutes. A transient loss of cortical actin with formation of actin stress fibers and disruption of continuous adherens and tight junction strands accompanied these changes. Mineralocorticoid receptor blockade, inhibition of RhoA, or disruption of extracellular-regulated protein kinase1/2 signaling pathways attenuated the Aldo-related effects. Moreover, Aldo-induced cytoskeletal rearrangement led to rapid dephosphorylation of protein kinase B and subsequent deactivation of endothelial nitric oxide synthase. Ex vivo tracer flux experiments with Evans blue-conjugated albumin demonstrated a concordant response to Aldo in freshly isolated umbilical arteries. Furthermore, low-dose cortisol (3 × 10(-10) to 3 × 10(-9) mol/L) mimics the effect of Aldo on endothelial integrity, and Aldo, by upregulating11β-hydroxysteroid dehydrogenase type 2, might even aggravate this deleterious effect of low-dose cortisol. We suggest that these mechanisms may contribute to the vasculopathy induced by inappropriate mineralocorticoid receptor activation.

Abstract 515: Aldosterone Modulates Endothelial Paracellular Permeability by Opening Intercellular Junctions

Aldosterone (Ald) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Ald alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells (HUVEC) were exposed to Ald (10 -9 mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions, and activation of intracellular signaling pathways were determined. Ald increased endothelial paracellular permeability for molecules up to 70 kDa within 60 min. A transient loss of cortical actin with formation of actin stress fibers, and disruption of continuous adherens and tight junction strands, accompanied the changes. Mineralocorticoid receptor (MR) blockade, inhibition of RhoA or disruption of extracellular regulated protein kinase (ERK) 1/2 signaling pathways attenuated the Ald-related effects. Moreover, the observed changes of the actin cytoskeleton and intercellular junction architecture were accompanied by a rapid dephosphorylation of protein kinase B (AKT) as well as deactivation of endothelial NO synthase (eNOS). Ex vivo tracer flux experiments with Evans Blue (EB)-conjugated albumin demonstrated concordant response to Ald in freshly isolated umbilical arteries. In summary, Ald leads to increased permeability of endothelial monolayer or isolated arteries. These alterations are associated with a temporal opening of Intercellular junctions and diminished eNOS activity, and depend on activation of the RhoA/ERK pathway. We conclude that these mechanisms could contribute to Ald-induced vasculopathy.

Regulation of Protein Kinase C Inactivation by Fas-associated Protein with Death Domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.

Sphingosine Kinase 1 Is Overexpressed and Promotes Proliferation in Human Thyroid Cancer

Sphingosine kinase 1 (SphK1), an oncogenic kinase, has been previously found to be elevated in various types of human cancer and play a role in tumor development and progression. Nevertheless, the biological and clinical significance of SphK1 in thyroid cancer is largely unknown. Here, we demonstrate that the expression of SphK1 is generally up-regulated in thyroid cancer and that its expression level is correlated with the degree of thyroid malignancy. Silencing SphK1 by specific RNA interference is able to suppress the proliferation of thyroid cancer cells, and SphK1 expression level is strongly associated with the expression of proliferation cell nuclear antigen in thyroid cancer tissues. Of particular note is that depletion of SphK1 results in dephosphorylation of protein kinase B and glycogen synthase kinase-3β and subsequent inactivation of β-catenin-T-cell factor/lymphoid enhancing factor transcriptional activity. Hence, taken together, our study has identified SphK1 as a proproliferative oncogenic kinase, an Akt/glycogen synthase kinase-3β/β-catenin activator, and probably a biomarker for thyroid cancer as well.

Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg 2+ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn 2+); and (3) by inducing reactive oxygen species (ROS). Hg 2+ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn 2+ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn 2+ or Hg 2+. Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg 2+-induced oxidation, because phosphatase activity is inhibited at concentrations of Hg 2+ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

Cadmium ions induce monocytic production of tumor necrosis factor-alpha by inhibiting mitogen activated protein kinase dephosphorylation

Cadmium ions (Cd 2+) are carcinogenic and have cytotoxic effects in a variety of organisms. In addition to its direct cytotoxicity, Cd 2+ acts as an immunomodulator at sub-toxic concentrations. Among other influences Cd 2+ can induce inflammation, but the molecular basis for this effect is not well investigated. In this manuscript, we analyze the impact of Cd 2+ on monocytes/macrophages, which are potent producers of pro-inflammatory cytokines, finding that Cd 2+ treatment induced tumor necrosis factor (TNF)-α secretion. Based on the observation that another group IIb metal, zinc (Zn 2+), has a physiological role in these events, we investigated if Cd 2+ acts on the same molecular targets. Like Zn 2+, Cd 2+ inhibits phosphatases, and hereby dephosphorylation of mitogen activated protein kinases (MAPK). Consequently, treatment of cells with Cd 2+ resulted in stimulation of ERK 1/2 and p38 MAPK phosphorylation. Furthermore, Cd 2+-induced release of TNF-α from primary human monocytes was blocked by inhibitors for ERK 1/2 (U0126) and p38 MAPK (SB202190), demonstrating that MAPKs are involved in the induction of TNF-α by Cd 2+.

Exercise-induced immunosuppression: roles of reactive oxygen species and 5′-AMP-activated protein kinase dephosphorylation within immune cells

We previously proposed 5'-AMP-activated protein kinase (AMPK) dephosphorylation within immune cells as an intracellular mechanism linking exercise and immunosuppression. In this study, AMPK phosphorylation underwent transient (<1 h) decreases (53.8+/-7.2% basal) immediately after exercise (45 min of cycling at 70% VO2max) in a cohort of 16 adult male participants. Similar effects were seen with running. However, because exercise-induced inactivation of AMPK was previously shown to occur in an AMP-independent manner, the means by which AMPK is inactivated in this context is not yet clear. To investigate the hypothesis that exercise-induced inactivation of AMPK is mediated via signaling mechanisms distinct from changes in cellular AMP-to-ATP ratios, reactive oxygen species (ROS) and intracellular Ca2+ signaling were investigated in mononuclear cells before and after exercise and in cultured monocytic MM6 cells. In in vitro studies, treatment with an antioxidant (ascorbic acid, 4 h, 50 microM) decreased MM6 cell intracellular ROS levels (88.0+/-5.2% basal) and induced dephosphorylation of AMPK (44.7+/-17.6% basal). By analogy, the fact that exercise decreased mononuclear cell ROS content (32.8+/-16.6% basal), possibly due to downregulation (43.4+/-8.0% basal) of mRNA for NOX2, the catalytic subunit of the cytoplasmic ROS-generating enzyme NADPH oxidase, may provide an explanation for the AMPK-dephosphorylating effect of exercise. In contrast, exercise-induced Ca2+ signaling events did not seem to be coupled to changes in AMPK activity. Thus we propose that the exercise-induced decreases in both intracellular ROS and AMPK phosphorylation seen in this study constitute evidence supporting a role for ROS in controlling AMPK, and hence immune function, in the context of exercise-induced immunosuppression.

Enhanced developmental potential of heat-shocked porcine parthenogenetic embryos is related to accelerated mitogen-activated protein kinase dephosphorylation

Recently, we demonstrated that a 9-h heat shock of 42 degrees C can have marked stimulatory effects on porcine parthenogenetic embryo development if applied immediately after oocyte activation. Developmental discrepancies between heat-shocked (HS) and non-HS embryos were manifest as early as 3 h after activation, suggesting involvement of maturation promoting factor (MPF) and/or mitogen-activated protein kinase (MAPK). Analysis of cdc2 kinase activity showed that MPF inactivation occurred at similar rates in HS and control embryos upon oocyte activation. However, MAPK dephosphorylation was accelerated in HS embryos compared with controls. Okadaic acid, a protein phosphatase inhibitor, maintained MAPK activity at high levels in both non-HS and HS embryos and sensitised HS embryos to the effects of elevated temperatures. No increase in heat shock proteins was observed in pronuclear-stage HS embryos. These data suggest that the acceleration of development observed in HS porcine parthenogenetic embryos is associated with a precocious inactivation of the MAPK signalling cascade. The faster cleavage divisions observed in HS embryos may be linked physiologically to their enhanced developmental potential in vitro.

Signaling Functions of Free Radicals Superoxide &amp; Nitric Oxide under Physiological &amp; Pathological Conditions

Superoxide and nitric oxide are ubiquitous physiological free radicals that are responsible for many pathological disorders. Both radicals by themselves are relatively harmless but are the precursors of many toxic species such as peroxy and hydroxyl radicals, hydrogen peroxide, and peroxynitrite. However, it has been shown now that both superoxide and nitric oxide are also able to perform important signaling functions in physiological and pathophysiological processes. Wrongly named "superoxide," the radical anion of dioxygen is not a super-oxidant but the strong super-nucleophile, an efficient catalyst of heterogenic nucleophilic reaction. Due to this, superoxide plays an important role in many enzymatic processes such as the phosphorylation and activation of numerous protein kinases. On the other hand, superoxide inhibits the activation of phosphatases, the enzymes catalyzed by dephosphorylation of protein kinases. We suggest that superoxide catalyzes these enzymatic processes as a result of its nucleophilic properties. Another important physiological function of superoxide and nitric oxide is their competition for the interaction with mitochondrial cytochrome c oxidase. Disturbance of superoxide/nitric oxide balance leads to the dysfunction of mitochondria and the enhancement of apoptosis and oxidative stress, which are primary causes of various pathological disorders and aging. In conclusion, interplay between superoxide and nitric oxide, one of major factors of aging development, is considered.

Leishmania donovani-Induced Ceramide as the Key Mediator of Akt Dephosphorylation in Murine Macrophages: Role of Protein Kinase Cζ and Phosphatase

Leishmania donovani is an intracellular protozoan parasite that impairs the host macrophage immune response to render it suitable for its survival and establishment. L. donovani-induced immunosuppression and alteration of host cell signaling is mediated by ceramide, a pleiotropic second messenger playing an important role in regulation of several kinases, including mitogen-activated protein kinase and phosphatases. We observed that the endogenous ceramide generated during leishmanial infection led to the dephosphorylation of protein kinase B (PKB) (Akt) in infected cells. The study of ceramide-mediated Akt phosphorylation revealed that Akt was dephosphorylated at both Thr308 and Ser473 sites in infected cells. Further investigation demonstrated that ceramide was also responsible for the induction of PKCzeta, an atypical Ca-independent stress kinase, as well as the ceramide-activated protein phosphatases (e.g., protein phosphatase 2A [PP2A]). We found that Akt dephosphorylation was mediated by ceramide-induced PKCzeta-Akt association and PP2A activation. In addition, treatment of L. donovani-infected macrophages with PKCzeta-specific inhibitor peptide could restore the translocation of phosphorylated Akt to the cell membrane. This study also revealed that ceramide is involved in the inhibition of proinflammatory cytokine tumor necrosis factor alpha release by infected macrophages. These observations strongly suggest the importance of ceramide in the alteration of normal cellular functions, impairment of the kinase/phosphatase balance, and thereby establishment of leishmaniasis in the hostile macrophage environment.

Enhanced Dephosphorylation of cAMP-dependent Protein Kinase by Oxidation and Thiol Modification

The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.

Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms

The mechanisms underlying the inhibition of bile acid-induced apoptosis by cyclic AMP (cAMP) were studied in 24-h-cultured rat hepatocytes. Taurolithocholate 3-sulfate (TLCS, 100 μmol/l) led to a sustained activation of mitogen activated protein (MAP) kinases (JNK, p38 MAPK, and ERKs), dephosphorylation of protein kinase B (PKB), activation of caspases 3 and 8, and hepatocyte apoptosis. cAMP prevented TLCS-induced apoptosis, shifted the persistent TLCS-induced MAP kinase response to a transient pattern, and prevented PKB dephosphorylation. TLCS-induced CD95 and TRAIL receptor-2 trafficking to the plasma membrane were significantly inhibited. Blockade of protein kinase A (PKA) abolished the inhibitory effect of cAMP on TLCS-induced CD95 membrane targeting, but not TRAIL receptor-2 membrane targeting, PKB and MAP kinase responses. H89, an inhibitor of PKA, had no effect on cAMP-induced inhibition of TLCS-triggered poly(ADP) ribose polymerase (PARP) cleavage and caspase activation, but abolished the cAMP-induced inhibition of TLCS-triggered TUNEL- and Annexin V staining. It is concluded that cAMP inhibits bile acid-induced apoptosis via PKA-dependent and -independent mechanisms.