Kinase-dependent Mechanism Research Articles

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.

Stretch and interleukin 1 beta: Pro‐labour factors with similar mitogen‐activated protein kinase effects but differential patterns of transcription factor activation and gene expression

IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism

Bile duct epithelium forms a barrier to the backflow of bile into the liver parenchyma. However, the structure and regulation of the tight junctions in bile duct epithelium is not well understood. In the present study, we evaluated the effect of lipopolysaccharide on tight junction integrity and barrier function in normal rat cholangiocyte monolayers. Lipopolysaccharide disrupts barrier function and increases paracellular permeability in a time- and dose-dependent manner. Lipopolysaccharide induced a redistribution of tight junction proteins, occludin, claudin-1, claudin-4, and zonula occludens (ZO)-1 from the intercellular junctions and reduced the level of ZO-1. Tyrosine kinase inhibitors (genistein and PP2) prevented lipopolysaccharide-induced increase in permeability and subcellular redistribution of ZO-1. Reduced expression of c-Src, TLR4, or LBP by specific small interfering RNA attenuated lipopolysaccharide-induced permeability and redistribution of ZO-1. ML-7, a myosin light chain kinase inhibitor, attenuated LPS-induced permeability. Lipopolysaccharide treatment rapidly increased the phosphorylation of occludin and ZO-1 on tyrosine residues, which was prevented by genistein and PP2. Occludin and ZO-1 were found to be highly phosphorylated on threonine residues in intact cell monolayers. Threonine-phosphorylation of occludin was rapidly reduced by lipopolysaccharide administration. Lipopolysaccharide-induced dephosphorylation of occludin on Thr residues was prevented by genistein and PP2. In conclusion, lipopolysaccharide disrupts the tight junction of a bile duct epithelial monolayer by a c-Src-, TLR4-, LBP-, and myosin light chain kinase-dependent mechanism.

Acetaldehyde dissociates the PTP1B–E-cadherin–β-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism

Interactions between E-cadherin, beta-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell-cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, beta-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and beta-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and beta-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and beta-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of beta-catenin on tyrosine residues, and abolished the interaction of beta-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of beta-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and beta-catenin was reduced by tyrosine phosphorylation of beta-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)-PTP1B. The pairwise binding study showed that GST-E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of beta-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, beta-catenin and PTP1B by a phosphorylation-dependent mechanism.

Alcohol Regulates Eukaryotic Elongation Factor 2 Phosphorylation via an AMP-activated Protein Kinase-dependent Mechanism in C2C12 Skeletal Myocytes

Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.

All-trans retinoic acid induces COX-2 and prostaglandin E2 synthesis in SH-SY5Y human neuroblastoma cells: involvement of retinoic acid receptors and extracellular-regulated kinase 1/2

BackgroundOur recent results show that all-trans retinoic acid (ATRA), an active metabolite of vitamin A, induces COX-dependent hyperalgesia and allodynia in rats. This effect was mediated by retinoic acid receptors (RARs) and was associated with increased COX-2 expression in the spinal cord. Since ATRA also up-regulated COX-2 expression in SH-SY5Y human neuroblastoma cells, the current study was undertaken to analyze in these cells the mechanism through which ATRA increases COX activity.MethodsCultured SH-SY5Y neuroblastoma cells were treated with ATRA. COX expression and kinase activity were analyzed by western blot. Transcriptional mechanisms were analyzed by RT-PCR and promoter assays. Pharmacological inhibitors of kinase activity and pan-antagonists of RAR or RXR were used to assess the relevance of these signaling pathways. Production of prostaglandin E2 (PGE2) was quantified by enzyme immunoabsorbent assay. Statistical significance between individual groups was tested using the non-parametric unpaired Mann-Whitney U test.ResultsATRA induced a significant increase of COX-2 expression in a dose- and time-dependent manner in SH-SY5Y human neuroblastoma cells, while COX-1 expression remained unchanged. Morphological features of differentiation were not observed in ATRA-treated cells. Up-regulation of COX-2 protein expression was followed by increased production of PGE2. ATRA also up-regulated COX-2 mRNA expression and increased the activity of a human COX-2 promoter construct. We next explored the participation of RARs and mitogen-activated peptide kinases (MAPK). Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist LE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059 resulted in the abolition of ATRA-induced COX-2 promoter activity, COX-2 protein expression and PGE2 production whereas the retinoid X receptor pan-antagonist HX531, the p38 MAPK inhibitor SB203580 or the c-Jun kinase inhibitor SP600125 did not have any effect. The increase in RAR-β expression and extracellular-regulated kinase 1/2(ERK1/2) phosphorylation in ATRA-incubated cells suggested that RARs and ERK1/2 were in fact activated by ATRA in SH-SY5Y human neuroblastoma cells.ConclusionThese results highlight the importance of RAR-dependent and kinase-dependent mechanisms for ATRA-induced COX-2 expression and activity.

A Novel BH3 Mimetic Reveals a Mitogen-Activated Protein Kinase–Dependent Mechanism of Melanoma Cell Death Controlled by p53 and Reactive Oxygen Species

The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell-selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-x(L) and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-x(L), and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design.

Ionizing radiation activates IGF-1R triggering a cytoprotective signaling by interfering with Ku-DNA binding and by modulating Ku86 expression via a p38 kinase-dependent mechanism

Ionizing radiation exposure results in the activation of several tyrosine kinase receptors that participate in radiation-induced DNA damage response and radioresistance. We previously showed that insulin-like growth factor 1 receptor (IGF-1R) inhibition enhanced radiosensitivity of non-small-cell lung cancer (NSCLC) cells. In this paper, we demonstrate that in U1810 NSCLC cells gamma-radiation activates IGF-1R within 10 min, with a maximal activation effect 2 h post-irradiation. Impairment of IGF-1R tyrosine kinase activity enhances human lung cancer cells radiosensitivity by a mechanism that involves phosphatidylinositol 3-kinase (PI3-K) and p38 kinase. In an active form, IGF-1R binds and activates p38 kinase, promoting receptor signaling. Conversely, inhibition of IGF-1R phosphorylation results in IGF-1R/p38 complex disruption and p38 kinase inactivation. We have also demonstrated that in insulin-like growth factor-1-stimulated cells, Ku-DNA-binding activation is induced by ionizing radiation within 4 h, reaches a maximum level at 12 h and remains active up to 72 h. Blockade of IGF-1R activity or its downstream signaling through p38 kinase induces a decrease in radiation-mediated Ku-DNA-binding activation and downregulates the level of Ku86, without affecting Ku70 expression in the nucleus of U1810 cells. The IGF-1R signaling via PI3-K does not interfere with the p38 signaling, the Ku-DNA-binding activity or the level of Ku86. Our present study demonstrates for the first time that ionizing radiation activates IGF-1R. Inhibition of IGF-1R signaling via p38 kinase induces radiosensitivity by a novel mechanism involving nuclear Ku86.

IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): Evidence for MAP kinase-dependent and MAP kinase-independent mechanisms

Objective IGF-1 and IGF-1 receptors are major determinants of fetal growth and are expressed primarily on the maternal-facing surface of the syncytiotrophoblast cell membrane in the human placenta. IGF-1 regulates fetal growth, in part, by regulating amino acid transport across the placenta. The objective of these studies was to study the role of IGF-1 and its signaling pathway in regulating neutral amino acid transport in a human trophoblast cell culture model. Design The regulation of neutral amino acid transport by IGF-1 was studied in cultured BeWo b30 choriocarcinoma cells using the non-metabolizing amino acid analog, [ 3H]-α-aminoisobutyric acid (AIB). Transport in the absence of Na was used to distinguish system L from total AIB transport. Similarly, Na-dependent transport in the presence of excess methyl-AIB (MeAIB) permitted discrimination of systems A (MeAIB-sensitive) and ASC (MeAIB-insensitive). Specific inhibitors of intracellular signaling pathways were then used to determine the signaling pathway utilized by IGFs to regulate each amino acid transport system. Specificity of inhibition was assessed using specific markers of p70 S6 kinase activity and MAP kinase activation. Results Maximal stimulating concentrations of IGF-I (100 ng/ml) stimulated AIB transport by 30–40% exclusively through system A. Wortmannin (100 nM), an inhibitor of PI-3-kinase activity, inhibited all IGF-I-stimulated transport. Rapamycin (100 ng/ml), an inhibitor of p70 S6 kinase, and bisindolylmaleimide, an inhibitor of protein kinase C (PKC), had no effect. PD-098059 (50 μM), an inhibitor of MAP kinase activation, inhibited 20–30% of basal AIB transport but did not inhibit IGF-I-stimulated transport under the conditions studied. IGF-1 did not increase steady state mRNA levels of the system A transporters, SNAT1 and SNAT2, suggesting IGF-1 stimulates transport via post-transcriptional mechanisms. Conclusions These data demonstrate that IGF-I stimulates neutral amino acid transport system A by a PI3-kinase dependent, post-transcriptional pathway in the BeWo b30 cell line. Additionally, system A activity appear to be sensitive to MAP kinase-dependent pathways not regulated by IGFs.

Protocatechuic acid induces cell death in HepG2 hepatocellular carcinoma cells through a c-Jun N-terminal kinase-dependent mechanism

Protocatechuic acid induces cell death in HepG2 hepatocellular carcinoma cells through a c-Jun N-terminal kinase-dependent mechanism

C-Jun N-terminal kinase-dependent mechanisms in respiratory disease

Respiratory diseases pose a multifaceted dilemma. Although the symptoms and pathology are obvious and provide multiple opportunities for therapeutic investigation, at the same time, the molecular complexities and prioritisation are overwhelming. Even within a disease such as asthma, the number of inducers, cell types, secondary mediators, chemical changes, immune responses and tissue modifications is remarkable. One means of therapeutically targeting this complexity is to identify individual factors responsible for regulating multiple disease processes. The mitogen-activated protein kinase family integrates multiple diverse stimuli, and, in turn, initiates a cell response by phosphorylating and thereby modulating the activity of many target proteins. The c-Jun N-terminal kinase is a critical regulator of pro-inflammatory genes, tissue remodelling and apoptosis, and, therefore, represents an attractive target for novel therapies. Pre-clinical and clinical investigation into the efficacy of c-Jun N-terminal kinase inhibitors has been ongoing since the late 1990s. Over the course of this work, hypotheses have shifted as to the role of c-Jun N-terminal kinase in the many processes that promote allergic, inflammatory, obstructive and fibrotic diseases of the lung. Inhibition of c-Jun N-terminal kinase may indeed provide a means of suppressing more pathological mechanisms in respiratory disease than first suspected.

Distinct Clathrin‐Coated Pits Sort Different G Protein‐Coupled Receptor Cargo

Upon activation, many G protein-coupled receptors (GPCRs) internalize by clathrin-mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin-coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y(1) and P2Y(12), which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase-dependent processes because internalization of P2Y(12) is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y(1) internalization is GRK- and arrestin-independent but requires protein kinase C. Importantly, the beta(2) adrenoceptor which also internalizes in a GRK-dependent manner also traffics exclusively to P2Y(12)-containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase-dependent mechanisms.

Protocatechuic acid induces cell death in HepG2 hepatocellular carcinoma cells through a c-Jun N-terminal kinase-dependent mechanism

Protocatechuic acid (PCA), chlorogenic acid (CA) and luteolin (LT) are plant phenols found in Chinese medicinal herbs such as Lonicera japonica. Cytotoxicity assays showed that PCA, CA and LT (at 100 micromol/L) effectively killed the HepG2 hepatocellular carcinoma cells. Among these three naturally occurring compounds, only PCA was capable of stimulating the c-Jun N-terminal kinase (JNK) and p38 subgroups of the mitogen-activated protein kinase (MAPK) family. Coincidently, PCA-induced cell death was rescued by specific inhibitors for JNK and p38, while the cytotoxicities of CA and LT were partially eliminated by the antioxidant effect of N-acetyl-L-cysteine (NAC). Further investigation demonstrated that the aqueous extract of Lonicera japonica also triggered HepG2 cell death in a JNK-dependent manner, but the amount of PCA alone in this herbal extract was insufficient to contribute the subsequent cytotoxic effect. Collectively, our results suggest that PCA is a naturally occurring compound capable of inducing JNK-dependent hepatocellular carcinoma cell death.

Distinct Roles for Protein Kinase C Isoforms in Regulating Platelet Purinergic Receptor Function

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.

Rapid inhibition of neural excitability in the nucleus tractus solitarii by leptin: implications for ingestive behaviour

The fat-derived peptide leptin regulates cellular activity in areas of the CNS related to feeding, and application of leptin to the fourth ventricle or the nucleus tractus solitarii (NTS) inhibits food intake and weight gain. The hypothesis that leptin modulates cellular activity in the NTS was tested using whole-cell patch-clamp recordings in brainstem slices. Leptin caused a rapid membrane hyperpolarization in 58% of rat NTS neurones, including neurones receiving tractus solitarius input (i.e. viscerosensory) and those involved in regulating output to the stomach, identified after gastric inoculation with a transneuronal retrograde viral label. The hyperpolarization was accompanied by a decrease in input resistance and cellular responsiveness, reversed near the K(+) equilibrium potential, and was prevented by intracellular Cs(+). Perfusion of tolbutamide (200 microm) or wortmannin (100-200 nm) prevented the hyperpolarization, indicating activation of an ATP-sensitive K(+) channel via a PI3 kinase-dependent mechanism. Constant latency tractus solitarius-evoked EPSCs were decreased in amplitude by leptin, and the paired-pulse ratio was increased, suggesting effects on evoked EPSCs involved activation of receptors on vagal afferent terminals. Leptin reduced the frequency of spontaneous and miniature EPSCs, whereas IPSCs were largely unaffected. Leptin's effects were observed in neurones from lean, but not obese, Zucker rats. Neurones that expressed enhanced green fluorescent protein (EGFP) in a subpopulation of putative GABAergic neurones in transgenic mice did not respond to leptin, whereas unlabelled murine neurones responded similarly to rat neurones. Leptin therefore directly and rapidly suppresses activity of excitatory NTS neurones likely to be involved in viscerosensory integration and/or premotor control of the stomach.

The Ser/Thr kinase activity of the Yersinia protein kinase A (YpkA) is necessary for full virulence in the mouse, mollifying phagocytes, and disrupting the eukaryotic cytoskeleton

The Yersinia protein kinase A (YpkA) is injected into host cells by the yersinial type three secretion system (TTSS). YpkA is widely believed to function within the host cell based on the fact that its kinase domain is clearly homologous to eukaryotic Ser/Thr kinases and that its enzymatic activity, when assayed in vitro, is dependent on eukaryotic-derived host factors. Whether this activity is required for virulence has not been addressed. Here, we report that a Yersinia pseudotuberculosis strain expressing a kinase-inactive YpkA D270A variant is greatly attenuated in the mouse model of infection compared to the isogenic wild-type strain. The ypkA D270A mutant strain was likewise attenuated in a cell culture infection assay indicating that the kinase activity of YpkA enhances the viability of host cell-associated bacteria. To begin to understand what cellular activities are targeted, we expressed YpkA and its variants in two different yeast model systems. In agreement with previous studies, we found that when rapidly induced and expressed at high levels in Saccharomyces cerevisiae, YpkA-mediated toxicity occurred extremely swiftly. Under these conditions toxicity was dependent on the structurally distinct GTPase-binding domain of YpkA and was entirely independent of its kinase activity. Therefore, to probe for kinase-dependent effects we expressed YpkA and its kinase-inactive variant at comparatively moderate levels in the fission yeast Schizosaccharomyces pombe. S. pombe is particularly well suited for actin cytoskeletal studies due to its easily quantifiable, well defined pattern of actin localization. S. pombe transformed with a wild-type YpkA-encoding plasmid displayed a pronounced actin mislocalization phenotype, the severity of which was directly proportional to the level of YpkA expressed in the cell. In cells expressing the kinase-inactive YpkA variant, the degree of actin mislocalization was reduced, but not entirely abrogated, suggesting that YpkA affects the eukaryotic cytoskeleton through kinase-dependent and kinase-independent mechanisms. Collectively, our yeast-derived results show how critical expression levels and exposure periods are for assaying virulence factor activities in heterologous model systems. More generally, our finding that the ‘eukaryotic-like’ kinase domain of YpkA is important for virulence illustrates how a bacterium can utilize a host-like factor or activity in order to enhance its survival following host cell contact.

117 The stimulation of purinergic P2Y receptors promotes hepatocyte preconditioning via a SRC and p38 map kinase-dependent mechanism

117 The stimulation of purinergic P2Y receptors promotes hepatocyte preconditioning via a SRC and p38 map kinase-dependent mechanism

Advanced glycation end products activate a chymase-dependent angiotensin II-generating pathway in diabetic complications.

Angiotensin II is a key mediator of diabetes-related vascular disease. It is now recognized that in addition to angiotensin-converting enzyme, chymase is an important alternative angiotensin II-generating enzyme in hypertension and diabetes. However, the mechanism of induction of chymase in diabetes remains unknown. Here, we report that chymase is upregulated in coronary and renal arteries in patients with diabetes by immunohistochemistry. Upregulation of vascular chymase is associated with deposition of advanced glycation end products (AGEs), an increase in expression of the receptor for AGEs (RAGE), and activation of ERK1/2 MAP kinase. In vitro, AGEs can induce chymase expression and chymase-dependent angiotensin II generation in human vascular smooth muscle cells via the RAGE-ERK1/2 MAP kinase-dependent mechanism. This is confirmed by blockade of AGE-induced vascular chymase expression with a neutralizing RAGE antibody and an inhibitor to ERK1/2 and by overexpression of the dominant negative ERK1/2. Compared with angiotensin-converting enzyme, chymase contributes to the majority of angiotensin II production (>70%, P<0.01) in response to AGEs. Furthermore, AGE-induced angiotensin II production is blocked by the anti-RAGE antibody and by inhibition of ERK1/2 MAP kinase activities. AGEs, a hallmark of diabetes, induce chymase via the RAGE-ERK1/2 MAP kinase pathway. Chymase initiates an important alternative angiotensin II-generating pathway in diabetes and may play a critical role in diabetic vascular disease.

Rho/ROCK is Up‐regulated in Pulmonary Arteries of Pregnant Ewes in Chronic High Altitude Hypoxia

We have shown that in pulmonary vessels, cGMP acts through cGMP-dependent protein kinase (PKG) dependent and independent mechanisms to mediate vasodilation (JAP, 2000), and that cGMP-induced relaxation of pulmonary vessels is greater in normoxia compared to acute hypoxia (AJP, 2003). In this study, we examined the effect of chronic high altitude hypoxia (HAH) on cGMP-PKG-mediated relaxation. Isolated ovine intrapulmonary arteries (PAs) from pregnant ewes exposed to HAH (ewes kept at 12,470 ft altitude from ~35d to 145d gestation; term 150d) or matched normoxic controls were preconstricted with endothelin-1 and relaxed to 8-Bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), a cell membrane-permeable analog of cGMP, or to DETA NONOate, a stable nitric oxide donor. We found that PAs relaxed to NO through both cGMP-dependent and independent mechanisms in normoxia. In chronic hypoxia, NO-mediated relaxation increased significantly, suggesting that guanylyl cyclase activity may be upregulated in these vessels. We observed minimal PKG-mediated relaxation both in normoxia or hypoxia. Furthermore, the inhibition of ROCK with a Rho-kinase-specific inhibitor (Y27632), induced increased relaxation of PAs to cGMP in chronic HAH PAs, but not in normoxic PAs, suggesting altered Rho/ROCK signaling under chronic HAH. We hypothesize that chronic high altitude hypoxia may induce sustained pulmonary vasoconstriction via increased protein expression and/or post-translational modification of ROCK or through altered protein-protein interactions involving Rho/ROCK, and that there is a compensatory upregulation of NO-cGMP mediated relaxation. HL059435, MIRS to S. Negash.

Hydrogen peroxide inhibits colonic epithelial ion transport by MAP kinase and PI3‐kinase (PI3K) independently of activated epidermal growth factor receptor (EGFr)

Reactive oxygen species contribute to epithelial damage and dysfunction in inflammatory bowel disease (IBD). We have shown that hydrogen peroxide (H2O2) inhibits intestinal Ca2+-dependent ion transport by PI3K-and MAP kinase-dependent mechanisms, while also activating the EGFr. Here, we studied whether EGFr regulates H2O2-mediated inhibition of ion transport in colonic epithelial cells. Protein phosphorylation was measured in T84 colonic epithelial monolayers by Western blot, and ion transport was measured in Ussing chambers. H2O2 (500 μM) increased EGFr tyrosine phosphorylation and its association with the catalytic p110β subunit of PI3-K in T84 cells. However, neither an EGFr kinase inhibitor, tyrphostin AG1478 (1 μM), nor a neutralizing anti-EGFr antibody that blocks the EGFr ligand binding domain (5 μg/ml), had any significant effect on H2O2-induced activation of ERK or the PI3-K target, Akt1. Both AG1478 and anti-EGFr inhibited H2O2-induced EGFr activation indicating that H2O2 can activate EGFr via EGFr ligand release. Tyrphostin AG1478 also had no effect on H2O2 inhibition of CCh-stimulated Cl- secretion across T84 monolayers. In summary, H2O2 activates EGFr in colonic epithelial cells, probably via the release of an EGFr ligand, but this effect does not account for inhibition of Ca2+-dependent Cl− secretion by H2O2. Because we have previously shown that EGFr activated by other means can lead to inhibition of Cl− secretion, our data imply selective outcomes of EGFr signaling depending on its mode of activation. Supported by the Crohn's and Colitis Foundation of America and NIH.