Search

This study evaluated whether improved insulin-stimulated glucose uptake in recovery from acute exercise coincides with reduced malonyl-CoA (MCoA) content in human muscle. Furthermore, we investigated whether a high-fat diet [65 energy-% (Fat)] would alter the content of MCoA and insulin action compared with a high-carbohydrate diet [65 energy-% (CHO)]. After 4 days of isocaloric diet on two occasions (Fat/CHO), 12 male subjects performed 1 h of one-legged knee extensor exercise (~80% peak workload). Four hours after exercise, insulin-stimulated glucose uptake was determined in both legs during a euglycemic-hyperinsulinemic clamp. Muscle biopsies were obtained in both legs before and after the clamp. Four hours after exercise, insulin-stimulated glucose uptake was improved (~70%, P < 0.001) independent of diet composition and despite normal insulin-stimulated regulation of insulin receptor substrate-l-associated phosphatidylinositol 3-kinase, Akt, GSK-3, and glycogen synthase. Interestingly, exercise resulted in a sustained reduction (~20%, P < 0.05) in MCoA content 4 h after exercise that correlated (r = 0.65, P < 0.001) with improved insulin-stimulated glucose uptake. Four days of Fat diet resulted in an increased content of intramyocellular triacylglycerol (P < 0.01) but did not influence muscle MCoA content or whole body insulin-stimulated glucose uptake. However, at the muscular level proximal insulin signaling and insulin-stimulated glucose uptake appeared to be compromised, although to a minor extent, by the Fat diet. Collectively, this study indicates that reduced muscle MCoA content in recovery from exercise may be part of the adaptive response leading to improved insulin action on glucose uptake after exercise in human muscle. AMP-activated protein kinase; acetyl coenzyme A carboxylase; intramuscular triacylglycerol; Akt; diet

Protein kinase B (PKB) is known to mediate a number of biological responses to insulin and growth factors, its role in glucose uptake being one of the most extensively studied. In this work, we have employed a recently described allosteric inhibitor of PKB, Akti, to clarify the role of PKB in lipid metabolism in adipocytes--a subject that has received less attention. Pretreatment of primary rat and 3T3L1 adipocytes with Akti resulted in dose-dependent inhibition of PKB phosphorylation and activation in response to insulin, without affecting upstream insulin signaling [insulin receptor (IR), insulin receptor substrate ORS)] or the insulin-induced phosphoinositide 3-kinase (PI3K)-dependent activation of the ERK/p90 ribosomal kinase (RSK) pathway. PKB activity was required for the insulin-induced activation of phosphodiesterase 3B (PDE3B) and for the antilipolytic action of insulin. Moreover, inhibition of PKB activity resulted in a reduction in de novo lipid synthesis and in the ability of insulin to stimulate this process. The regulation of the rate-limiting lipogenic enzyme acetyl-CoA carboxylase (ACC) by insulin through dephosphorylation of S79, which is a target for AMP-activated protein kinase (AMPK), was dependent on the presence of active PKB. Finally, AMPK was shown to be phosphorylated by PKB on S485 in response to insulin, and this was associated with a reduction in AMPK activity. In summary, we propose that PKB is required for the positive effects of insulin on lipid storage and that regulation of PDE3B and AMPK by PKB is important for these effects. Akt; phosphodiesterase 3B; acetyl-coenzyme A carboxylase; adenosine 5'-monophosphate-activated protein kinase; lipogenesis

The sarcolemmal [Na.sup.+]-[K.sup.+] pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to [epsilon]-protein kinase C ([epsilon]PKC), previously implicated in ANG II-induced [Na.sup.+]-[K.sup.+] pump inhibition. A role for [epsilon]PKC was also supported by an ANG H-induced increase in coimmunoprecipitation of [epsilon]PKC with the receptor for the activated kinase and with the cytosolic [p47.sup.phox] subunit of NADPH oxidase. ANG II decreased electrogenic [Na.sup.+]-[K.sup.+] pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by [epsilon]PKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the [Na.sup.+]-[K.sup.+] pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The [[alpha].sub.1]-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated [p22.sup.phox] and cytosolic [p47.sup.phox] NADPH oxidase subunits at baseline. ANG II had no effect on [[alpha].sub.1]/caveolin 3 or [[alpha].sub.1]/[p22.sup.phox] interaction, but it increased [[alpha].sub.1]/[p47.sup.phox] coimmunoprecipitation. We conclude that ANG II inhibits the [Na.sup.+]-[K.sup.+] pump via PKC-dependent NADPH oxidase activation. reduced nicotinamide adenine dinucleotide phosphatase oxidase; oxidant signaling; glutathionylation; protein kinase C; [Na.sup.+]-[K.sup.+]-ATPase

Acidic luminal pH and low [HC[O.sup.-.sub.3]] maintain sperm quiescent during maturation in the epididymis. The vacuolar [H.sup.+]-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HC[O.sup.-.sub.3], induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and nonhuman primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by [N.sup.6]-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [[sup.32]P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. vas deferens; male reproductive tract; metabolism; soluble adenylyl cyclase; acid secretion

The present study was aimed at evaluating the molecular mechanisms associated with the differences in muscle glycogen content and breast meat quality between 2 experimental lines of chicken divergently selected on abdominal fatness. The glycogen at death (estimated through the glycolytic potential) of the pectoralis major muscle and the quality of the resulting meat were estimated in the 2 lines. The fat chickens exhibited greater glycolytic potential, and in turn lower ultimate pH than the lean chickens. Consequently, the breast meat of fat birds was paler and less colored (i.e., less red and yellow), and exhibited greater drip loss compared with that of lean birds. In relation to these variations, transcription and activation levels of adenosine monophosphate-activated protein kinase (AMPK) were investigated. The main difference observed between lines was a 3-fold greater level of AMPK activation, evaluated through phosphorylation of AMPKa-([Thr.sup.172]), in the muscle of lean birds. At the transcriptional level, data indicated concomitant down-and upregulation for the y1 and y2 AMPK subunit isoforms, respectively, in the muscle of lean chickens. Transcriptional levels of enzymes directly involved in glycogen turnover were also investigated. Data showed greater gene expression for glycogen synthase, glycogen phosphorylase, and the y subunit of phosphorylase kinase in lean birds. Together, these data indicate that selection on body fatness in chicken alters the muscle glycogen turnover and content and consequently the quality traits of the resulting meat. Alterations of AMPK activity could play a key role in these changes. Key words: adenosine monophosphate-activated protein kinase, chicken, fatness, glycogen, meat quality, pH

B23/NPM is a major nucleolar phosphoprotein that has a critical role in cell proliferation and cell death. Here, we show that it forms a complex with Akt on growth factor (GF) stimulation in both the cytoplasm and the nucleus, for which Akt activation is indispensable. The C terminus of B23 (239-294 residues) potently binds pleckstrin homology (PH) domain of Akt. Akt binding to B23 protects it from proteolytic degradation by caspase-3, leading to the up-regulation of cell survival. Interestingly, unsumoylated B23 K263R, but not wild-type B23, strongly interacts with Akt in the nucleoplasm in the absence of GFs. Furthermore, we show that Akt2, but not other isoforms, specifically regulates B23 sumoylation and protein stability. Also, nuclear Akt regulates the cell cycle progression activity of B23. Therefore, our findings support that nuclear Akt binds and stabilizes B23 in the nucleoplasm, and regulates its activities in cell survival and cell cycle. Akt/PKB | caspase-3 | stability | sumoylation

The role of the endoplasmic reticulum stress-regulated kinase, PERK, in mammary gland function was assessed through generation of a targeted deletion in mammary epithelium. Characterization revealed that PERK is required for functional maturation of milk-secreting mammary epithelial cells. PERK-dependent signaling contributes to lipogenic differentiation in mammary epithelium, and perk deletion inhibits the sustained expression of lipogenic enzymes FAS, ACL, and SCD1. As a result, mammary tissue has reduced lipid content and the milk produced has altered lipid composition, resulting in attenuated pup growth. Consistent with PERK-dependent regulation of the lipogenic pathway, Loss of PERK inhibits expression of FAS, ACL, and SCD1 in immortalized murine embryonic fibroblasts when cultured under conditions favoring adipocyte differentiation. These findings implicate PERK as a physiologically relevant regulator of the lipogenic pathway. lipid metabolism | SREBP1 | FAS | ACL | fatty acids

Skeletal muscle fibers differ considerably in their metabolic and physiological properties. Skeletal muscle displays a high degree of metabolic flexibility, which allows the myofibers to adapt to various physiological demands by shifting energy substrate utilization. Transcriptional events play a pivotal role in the metabolic adaptations of skeletal muscle. The expression of genes essential for skeletal muscle glucose and lipid metabolism is tightly coordinated in support of a shift in substrate utilization. AMP-activated protein kinase (AMPK) and calcineurin (a calcium-regulated serine/threonine protein phosphatase) regulate skeletal muscle metabolic gene expression programs in response to changes in the energy status and levels of neuronal input, respectively. AMPK and calcineurin activate transcriptional regulators such as peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] and myocyte enhancer factor as well as increase skeletal muscle oxidative capacity and mitochondrial gene expression. Activation of either the AMPK or calcineurin pathway can also enhance the glycogen storage capacity and insulin sensitivity in skeletal muscle. Characterization of pathways governing skeletal muscle metabolism offers insight into physiological and pharmacological strategies to prevent or ameliorate peripheral insulin resistance associated with metabolic disorders such as type 2 diabetes. adenosine 5'-monophosphate-activated protein kinase

Membrane-associated guanylate kinase (MAGUK) proteins interact with several synaptogenesistriggering adhesion molecules. However, direct evidence for the involvement of MAGUK proteins in synapse formation is lacking. In this study, we investigate the function of calcium/calmodulin-dependent serine protein kinase (CASK), a MAGUK protein, in dendritic spine formation by RNA interference. Knockdown of CASK in cultured hippocampal neurons reduces spine density and shrinks dendritic spines. Our analysis of the time course of RNA interference and CASK overexpression experiments further suggests that CASK stabilizes or maintains spine morphology. Experiments using only the CASK PDZ domain or a mutant lacking the protein 4.1-binding site indicate an involvement of CASK in linking transmembrane adhesion molecules and the actin cytoskeleton. We also find that CASK is SUMOylated. Conjugation of small ubiquitin-like modifier 1 (SUMO1) to CASK reduces the interaction between CASK and protein 4.1. Overexpression of a CASK-SUMO1 fusion construct, which mimicks CASK SUMOylation, impairs spine formation. Our study suggests that CASK contributes to spinogenesis and that this is controlled by SUMOylation.

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-[delta] and PKC-[epsilon] reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-[delta] and -[epsilon], reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-[delta] and -[epsilon] isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-[epsilon] redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-[epsilon] overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-[delta] and -[epsilon] isoforms translocate to specific acinar cell compartments and modulate zymogen activation. translocation; PKC activator phorbol ester; PKC inhibitor

Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial [Ca.sup.2+] reabsorption in kidney. Many hormones alter renal [Ca.sup.2+] reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal [Ca.sup.2+] reabsorption by PTH. dynamin; caveolin; parathyroid hormone; phospholipase C; Madin-Darby canine kidney cells

Obese conditions increase the expression of adipocytokine monocyte chemoattractant protein-1 (MCP-1) in adipose tissue as well as MCP-1 plasma levels. To investigate the mechanism behind increased MCP-1, we used a model in which 3T3-L1 adipocytes were artificially hypertrophied by preloading with palmitate in vitro. As observed in obesity, under our model conditions, palmitate-preloaded ceils showed significantly increased oxidative stress and increased MCP-1 expression relative to control cells. This increased MCP-1 expression was enhanced by adding exogenous tumor necrosis factor-[alpha] (TNF-[alpha]; 17.8-fold vs. control cells, P < 0.01 ) rather than interleukin-1[beta] (IL-1[beta]; 2.6-fold vs. control cells, P < 0.01). However, endogenous TNF-[alpha] and IL-1[beta] release was not affected in hypertrophied cells, suggesting that these endogenous cytokines do not mediate hypertrophy-induced increase in MCP-1. MCP-1 secretion from hypertrophied cells was significantly decreased by treatment with antioxidant N-acetyl-cysteine, JNK inhibitors SP600125 and JIP-1 peptide, and I[kappa]B phosphorylation inhibitors BAY 11-7085 and BMS-345541 (P < 0.01). MCP-1 secretion was not affected by peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) antagonists assayed. Adiponectin, another adipocytokine studied in parallel, also showed increased release in hypertrophy relative to control cells. But in contrast to MCP-1, adiponectin release was significantly suppressed by both exogenous TNF-[alpha] and IL-1[beta] as well as by PPAR[gamma] antagonists bisphenol A diglycidyl ether and T0070907 (P < 0.01). JNK inhibitors and I[kappa]B phosphorylation inhibitors showed no significant effect on adiponectin. We conclude that adipocyte hypertrophy through palmitate loading causes oxidative stress, which in turn increases MCP-1 expression and secretion through JNK and I[kappa]B signaling. In contrast, the parallel increase in adiponectin expression appears to be related to the PPAR[gamma] ligand properties of palmitate. c-Jun N[H.sub.2]-terminal kinase; monocyte chemoattractant protein-1; tumor necrosis factor-[alpha]; interleukin-1[beta]

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum long us (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of [H.sub.2][O.sub.2], EDL muscles from rats were preincubated with the [H.sub.2][O.sub.2] scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that [H.sub.2][O.sub.2] is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct [H.sub.2][O.sub.2] also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 [micro]mol/l [H.sub.2][O.sub.2]. [H.sub.2][O.sub.2]-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor [N.sup.G]-monomethyl-L-arginine. [H.sub.2][O.sub.2] stimulated the phosphorylation of Akt [Set.sup.473] (7-fold) and [Thr.sup.308] (2-fold) in isolated EDL muscles. [H.sub.2][O.sub.2] at 600 [micro]mol/l had no effect on ATP concentrations and did not increase the activities of either the [alpha]1 or [alpha]2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism. hydrogen peroxide; Akt phosphorylation; adenosine monophosphate-activated protein kinase

we develop a probabilistic method for analyzing global features of a cellular network under intrinsic statistical fluctuations, which is important when there are finite numbers of molecules. By making a self-consistent mean field approximation of splitting the variables in order to reduce the large number of degrees of freedom, which is reasonable for a not very strongly interacting network, we discovered that the underlying energy landscape of the mitogen-activated protein kinases (MAPKs) signal transduction network (with experimentally measured of inferred parameters such as chemical reaction rate coefficients in the network) is funneled toward a global minimum characterized by the nonequilibrium steady-state fixed point of the system at the end of the signal transduction process. For this system, we also show that the energy landscape is robust against intrinsic fluctuations and random perturbation to the inherent chemical reaction rates. The ratio of the slope versus the roughness of the energy landscape becomes a quantitative measure of robustness and stability of the network. Furthermore, we quantify the dissipation cost of this nonequilibrium system through entropy production, caused by the nonequilibrium flux in the system. We found that a lower dissipation cost corresponds to a more robust network. This least dissipation property might provide a design principle for robust and functional networks. Finally, we find the possibility of bistable and oscillatory-like solutions, which are important for cell fate decisions, upon perturbations. The method described here can be used in a variety of biological networks. funnel | stability | potential landscape | function

Protein kinase CK2 (also known as casein kinase 2) is present in the cytoplasm, nuclei, and several other organelles. In addition, this enzyme has been found bound to the external side of the cell membrane where it acts as an ectokinase phosphorylating several extracellular proteins. Previous experiments with transfection of HEK-293T cells demonstrated that expression of both subunits, CK2[alpha] (catalytic) and CK2[beta] (regulatory), was necessary for the appearance of the ectopic enzyme as an ectokinase. In this work, using deletion and point mutations of CK2[beta], it was possible to demonstrate that the region between amino acids 20 and 33 was necessary for the export of the enzyme as an ectokinase. Phenylalanines 21 and 22 and acidic residues in positions 26-28 are involved in the structural aspects that are required for export. However, the region encompassing amino acids 20-33 of CK2[beta] is not sufficient to make the carboxyl half of this subunit functional in bringing CK2 to the ectokinase locus. In cells transfected with only CK2[beta], it was demonstrated that 3-4% of the subunit is exported to the cell medium, but the subunit is not bound to the external membrane. casein kinase 2 | export of proteins | shedding

Plant recognition of pathogens leads to rapid activation of MPK3 and MPK6, two Arabidopsis mitogen-activated protein kinases (MAPKs), and their orthologs in other species. Here, we report that synthesis of camalexin, the major phytoalexin in Arabidopsis, is regulated by the MPK3/MPK6 cascade. Activation of MPK3/MPK6 by expression of active upstream MAPK kinase (MAPKK) or MAPKK kinase (MAPKKK) was sufficient to induce camalexin synthesis in the absence of pathogen attack. Induction of camalexin by Botrytis cinerea was preceded by MPK3/MPK6 activation, and compromised in mpk3 and mpk6 mutants. Genetic analysis placed the MPK3/MPK6 cascade upstream of PHYTOALEXIN DEFICIENT 2 (PAD2) and PAD3, but independent or downstream of PAD1 and PAD4. Camalexin induction after MPK3/MPK6 activation was preceded by rapid and coordinated up-regulation of multiple genes encoding enzymes in the tryptophan (Trp) biosynthetic pathway, in the conversion of Trp to indole-3-acetaldoxime (IAOx, a branch point between primary and secondary metabolism), and in the camalexin biosynthetic pathway downstream of IAOx. These results indicate that the MPK3/MPK6 cascade regulates camalexin synthesis through transcriptional regulation of the biosynthetic genes after pathogen infection. defense signaling | fungal resistance | camalexin biosynthesis | Botrytis cinerea

MYt1 was originally identified as an inhibitory kinase for Cdc2 (Cdk1), the master engine of mitosis, and has been thought to function, together with Wee1, as a negative regulator of mitotic entry. In this study, we report an unexpected finding that Myt1 is essential for Golgi and endoplasmic reticulum (ER) assembly during telophase in mammalian cells. Our analyses reveal that both cyclin B1 and cyclin B2 serve as targets of Myt1 for proper Golgi and ER assembly to occur. Thus, our results show that Myt1 -mediated suppression of Cdc2 activity is not indispensable for the regulation of a broad range of mitotic events but is specifically required for the control of intracellular membrane dynamics during mitosis.

Focal adhesion kinase (FAK) plays a critical role during vascular development because knockout of FAK in endothelial cells (ECs) is embryonic lethal. Surprisingly, tamoxifen-inducible conditional knockout of FAK in adult blood vessels (inducible EC-specific FAK knockout [i-EC-FAK-KO]) produces no vascular phenotype, and these animals are capable of developing a robust growth factor-induced angiogenic response. Although angiogenesis in wild-type mice is suppressed by pharmacological inhibition of FAK, i-EC-FAK-KO mice are refractory to this treatment, which suggests that adult i-EC-FAK-KO mice develop a compensatory mechanism to bypass the requirement for FAK. Indeed, expression of the FAK-related proline-rich tyrosine kinase 2 (Pyk2) is elevated and phosphorylated in i-EC-FAK-KO blood vessels. In cultured ECs, FAK knockdown leads to increased Pyk2 expression and, surprisingly, FAK kinase inhibition leads to increased Pyk2 phosphorylation. Pyk2 can functionally compensate for the loss of FAK because knockdown or pharmacological inhibition of Pyk2 disrupts angiogenesis in i-EC-FAK-KO mice. These studies reveal the adaptive capacity of ECs to switch to Pyk2-dependent signaling after deletion or kinase inhibition of FAK.

Multiple signaling pathways are engaged by the type I and II IFN receptors, but their specific roles and possible coordination in the generation of IFN-mediated biological responses remain unknown. We provide evidence that activation of Akt kinases is required for IFN-inducible engagement of the mTOR/p70 S6 kinase pathway. Our data establish that Akt activity is essential for up-regulation of key IFN-[alpha]- and IFN-[gamma]-inducible proteins, which have important functional consequences in the induction of IFN responses. Such effects of the Akt pathway are unrelated to regulatory activities on IFN-dependent STAT phosphorylation/activation or transcriptional regulation. By contrast, they reflect regulatory activities on mRNA translation via direct control of the mTOR pathway. In studies using Aktl and Akt2 double knockout cells, we found that the absence of Akt kinases results in dramatic reduction in IFN-induced antiviral responses, establishing a critical role of the Akt pathway in IFN signaling. Thus, activation of the Akt pathway by the IFN receptors complements the function of IFN-activated JAK--STAT pathways, by allowing mRNA translation of IFN-stimulated genes and, ultimately, the induction of the biological effects of IFNs.

A hallmark of the pathology of Alzheimer's disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PARI/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimer's disease. Alzheimer's disease | cell signaling | chaperone | MARK2/PAR1

Articles in recent years have described two separate and distinct NF-[kappa]B activation pathways that result in the differential activation of p50- or p52-containing NF-[kappa]B complexes. Studies examining tumor-necrosis factor receptor-associated factors (TRAFs) have identified positive roles for TRAF2, TRAF5, and TRAF6, but not TRAF3, in canonical (p50-dependent) NF-[kappa]B activation. Conversely, it recently was reported that TRAF3 functions as an essential negative regulator of the noncanonical (p52-dependent) NF-[kappa]B pathway. In this article, we provide evidence that TRAF3 potently suppresses canonical NF-[kappa]B activation and gene expression in vitro and in vivo. We also demonstrate that deregulation of the canonical NF-[kappa]B pathway in TRAF3-deficient cells results from accumulation of NF-[kappa]B-inducing kinase (NIK), the essential kinase mediating noncanonical NF-[kappa]B activation. Thus, our data demonstrate that inhibition of TRAF3 results in coordinated activation of both NF-[kappa]B activation pathways. lymphotoxin-[beta] receptor | p50 | tumor-necrosis factor receptor-associated factor 3 (TRAF3)

Inositol pyrophosphates, also designated inositol diphosphates, possess high-energy [beta]-phosphates that can pyrophosphorylate proteins and regulate various cellular processes. They are formed by a family of inositol hexakisphosphate kinases (IP6Ks). We have created mice with a targeted deletion of IP6K1 in which production of inositol pyrophosphates is markedly diminished. Defects in the mutants indicate important roles for IP6K1 and inositol pyrophosphates in several physiological functions. Male mutant mice are sterile with defects in spermiogenesis. Mutant mice are smaller than wild-type despite normal food intake. The mutants display markedly lower circulating insulin. inosito | phosphate kinase | inositol polyphosphate | knockout mouse

Lipopolysaccharide (LPS) endotoxin is a causative agent of sepsis. The aim of this study was to examine LPS effects on intestinal fructose absorption and to decipher mechanisms. Sepsis was induced by intravenous injection of LPS in rabbits. The ultrastructural study and DNA fragmentation patterns were identical in the intestine of LPS and sham animals. LPS treatment reduced fructose absorption altering both mucosal-to-serosal transepithelial fluxes and uptake into brush border membrane vesicles (BBMVs). Cytochalasin B was ineffective on fructose uptake, indicating that GLUT5, but not GLUT2, transport activity was targeted. GLUT5 protein levels in BBMvs were lower in LPS than in sham-injected rabbits. Thus lower fructose transport resulted from lower levels of GLUT5 protein. LPS treatment decreased GLUT5 levels by proteasome-dependent degradation. Specific inhibitors of PKC, PKA, and MAP kinases (p38MAPK, JNK, MEK 1/2) protected fructose uptake from adverse LPS effect. Moreover, a TNF-[alpha] antagonist blocked LPS action on fructose uptake. We conclude that intestinal fructose transport inhibition by LPS is associated with diminished GLUT5 numbers in the brush border membrane of enterocytes triggered by activation of several interrelated signaling cascades and proteasome degradation. GLUT5; small intestine; LPS; intracellular signals; rabbit

Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK), which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied overnight-fasted adult male rats by use of an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU x [min.sup.-1] x [kg.sup.-1]) was begun either 10 min before ischemia ([Insuli.sub.BI]), 5 min before reperfusion ([Insulin.sub.BR]), or 30 min after the onset of reperfusion ([Insulin.sub.AR]), and continued until the end of the study. Compared with saline control, insulin decreased the infarct size in both [Insuli.sub.BI] (P < 0.001) and [Insulin.sub.BR] (P < 0.02) rats hut not in [Insulin.sub.AR] rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK compared with the nonischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg iv 10 min before ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in [Insulin.sub.BI] and [Insulin.sub.BR] rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB-239063, a potent and specific inhibitor of p38 MAPK, 10 rain before reperfusion enabled insulin-mediated myocardial protection in [Insulin.sub.AR] rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion, and activation of p38 MAPK abolishes insulin's cardioprotective effect. insulin; myocardial infarction; anisomycin

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular [Ca.sup.2+] handling in hydrogen sulfide ([H.sub.2]S)-preconditioned cardiomyocytes and its consequent effects on iscbemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an [H.sub.2]S donor, [10.sup.-4] M) preconditioning (SP, 30 min). Intracellular [Ca.sup.2+] was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKC[alpha], PKC[epsilon], and PKC[delta] to membrane fraction but only translocation of PKC[epsilon] and PKC[delta] was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [[Ca.sup.2+]] transients, which were reversed by a selective PKC inhibitor cbelerythrine. These data suggest that SP facilitated [Ca.sup.2+] removal via both accelerating uptake of [Ca.sup.2+] into sarcoplasmic reticulum and enhancing [Ca.sup.2+] extrusion through [Na.sup.+]/[Ca.sup.2+] exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against [Ca.sup.2+] overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKC[alpha], PKC[epsilon], and PKC[delta] in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular [Ca.sup.2+] handling. protein kinase C isoforms; ischemia and reperfusion; cardioprotection; ATP-sensitive potassium channel

The vacuolar [H.sup.+]-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland ceils energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of [V.sub.0] and [V.sub.1] subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of [V.sub.1] components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced [V.sub.1] translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA. vacuolar [H.sup.+]-adenosine 5'-triphosphatase; adenosine 3',5'-cyclic monophosphate; exchange protein directly activated by adenosine 3',5'-cyclic monophosphate; insect

Elevated expression and activation of the focal adhesion kinase (FAK) occurs in a large proportion of human breast cancers. Although several studies have implicated FAK as an important signaling molecule in cell culture systems, evidence supporting a role for FAK in mammary tumor progression is lacking. To directly assess the role of FAK in this process, we have used the Cre/IoxP recombination system to disrupt FAK function in the mammary epithelium of a transgenic model of breast cancer. Using this approach, we demonstrate that FAK expression is required for the transition of premalignant hyperplasias to carcinomas and their subsequent metastases. This dramatic block in tumor progression was further correlated with impaired mammary epithelial proliferation. These observations provide direct evidence that FAK plays a critical role in mammary tumor progression. breast cancer | Cre recombinase | transgenic

In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar [H.sup.+]-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2[DELTA]). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2[DELTA] cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.

Circadian clocks are composed of central oscillators, input pathways that transduce external information to the oscillators, and output pathways that allow the oscillators to temporally regulate cellular processes. Little is known about the output pathways. In this study, we show that the Neurospora crassa osmosensing MAPK pathway, essential for osmotic stress responses, is a circadian output pathway that regulates daily rhythms in the expression of downstream genes. Rhythmic activation of the highly conserved stress-activated p38-type MAPK [Osmotically Sensitive-2 (OS-2)] by the N. crassa circadian clock allows anticipation and preparation for hyperosmotic stress and desiccation that begin at sunrise. These results suggest a conserved role for MAPK pathways in circadian rhythmicity. circadian output pathway | circadian rhythm | Neurospora crassa | osmotic stress phosphorelay

Activation of the p38 mitogen-activated protein kinase (MAPK) pathway induces inflammation, apoptosis, and fibrosis. However, little is known of the contribution of the upstream kinases, MMK3 and MKK6, to activation of the p38 kinase in the kidney and consequent renal injury. This study investigated the contribution of MKK3 to p38 MAPK activation and renal injury in the obstructed kidney. Groups of eight wild-type (WT) or Mkk3-/- mice underwent unilateral ureteric obstruction (UUO) and were killed 3 or 7 days later. Western blotting showed a marked increase in phospho-p38 (p-p38) MAPK in UUO WT kidney. The same trend of increased p-p38 MAPK was seen in the UUO Mkk3-/- kidney, although the actual level of p-p38 MAPK was significantly reduced compared with WT, and this could not be entirely compensated for by the increase in MKK6 expression in the Mkk3-/- kidney. Apoptosis of tubular and interstitial cells in WT UUO mice was reduced by 50% in Mkk3-/- UUO mice. Furthermore, cultured Mkk3-/- tubular epithelial cells showed resistance to [H.sub.2][O.sub.2]-induced apoptosis, suggesting a direct role for MKK3-p38 signaling in tubular apoptosis. Upregulation of MCP-1 mRNA levels and macrophage infiltration seen on day 3 in WT UUO mice was significantly reduced in Mkk3-/- mice, but this difference was not evident by day 7. The development of renal fibrosis in Mkk3-/-UUO mice was not different from that seen in WT UUO mice. In conclusion, these studies identify discrete roles for MKK3-p38 signaling in renal cell apoptosis and the early inflammatory response in the obstructed kidney. MKK6; macrophage; MCP-1, fibrosis

The androgen-secreting Leydig cells produce cGMP, but the pathways responsible for generation and actions of this intracellular messenger have been incompletely characterized in these cells. Here, we show the presence of mRNA transcripts for the membrane-bound and soluble guanylyl cyclases (sGC), the cGMP-specific phosphodiesterase 5, and the cGMP-dependent protein kinase I (PKG I) and PKG II in purified rat Leydig cells from adult animals. Stimulation of both guanylyl cyclases and inhibition of phosphodiesterase 5 in vitro were accompanied by elevations in cGMP and androgen production, whereas inhibition of sGC and PKG led to a decrease in steroidogenesis. The stimulatory action of cGMP on steroidogenesis was preserved in cells with inhibited cAMP-dependent protein kinases. Experiments with exogenously added substrates revealed the dependence of cGMP-induced progesterone and androgen synthesis on cholesterol but not on 22-OH cholesterol, pregnenolone, progesterone, and [[DELTA].sup.4]-androstenedione. Treatment with nitric oxide donor increased phosphorylation of the steroidogenic acute regulatory protein (STAR). In contrast, inhibition of sGC and PKG, but not protein kinase A, significantly reduced StAR phosphorylation. These results suggest that cGMP contributes to the control of basal steroidogenesis in Leydig cells through the PKG-dependent modification of the StAR protein. cGMP; steroidogenic acute regulatory protein

5'-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pymvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKcd activity in [[alpha].sup.2]KO mice (90%, P < 0.01) and increased AMPK[alpha]2 activity in wild-type (WT) mice (110%, P < 0.05), leading to increased AMPK[alpha] [Thr.sup.172] (WT: 40%, [[alpha].sup.2]KO: 100%, P < 0.01) and ACC[beta] [Ser.sup.227] phosphorylation (WT: 70%, [[alpha].sub.2]KO: 210%, P < 0.01). Compared with rest, exercise significantly induced PDH-[E.sub.1] [alpha] site 1 (WT: 20%, [[alpha].sub.2]KO: 62%, P < 0.01) and site 2 (only [[alpha].sub.2]KO: 83%, P < 0.01) dephosphorylation and [PDH.sub.a] [~200% in both genotypes (P < 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the [[alpha].sub.2]KO mice both at rest and during exercise. The increased [PDH.sub.a] activity during exercise was associated with elevated glycolytic flux, and muscles from the [[alpha].sub.2]KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of [[alpha].sub.2]KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-[E.sub.1] [alpha] phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPK[alpha]2 expression. During exercise, [[alpha].sub.2]KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and [PDH.sub.a] activity. This may be linked to decreased mitochondrial oxidative capacity. adenosine 5'-monophosphate-activated protein kinase; 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside; treadmill running; glucose metabolism

Airway goblet cell mucin secretion is controlled by agonist activation of [P2.sub.2] purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (I[P.sub.3]), diacylglycerol, [Ca.sup.2+] and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC[alpha], nPKC[delta], nPKC[epsilon], and nPKC[eta]; of these, only nPKC[delta] translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC[alpha], nPK[delta], and nPKC[eta] had the same levels of ATP[gamma]S- and phorbol-l,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC[epsilon] (14.6 and 23.5%, for ATP[gamma]S and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC[epsilon] exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATP[gamma]S-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in [P2Y.sub.2]-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC[delta] knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC[epsilon] KO mice relative to its WT littermates. We conclude that nPKC[epsilon] is the effector isoform downstream of [P2Y.sub.2]-R activation in the goblet cell secretory response. The translocation of nPKC[delta] observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung

Activation of macrophages and subsequent 'killing' effector functions against infectious pathogens are essential for the establishment of protective immunity. NF-IL6 is a transcription factor downstream of IFN-[gamma] and TNF in the macrophage activation pathway required for bacterial killing. Comparison of microarray expression profiles of Listeria monocytogenes (LM)-infected macrophages from WT and NF-IL6-deficient mice enabled us to identify candidate genes downstream of NF-IL6 involved in the unknown pathways of LM killing independent of reactive oxygen intermediates and reactive nitrogen intermediates. One differentially expressed gene, PKC[delta], had higher mRNA levels in the LM-infected NF-IL6-deficient macrophages as compared with WT. To define the role of PKC[delta] during listeriosis, we infected PKC[delta]-deficient mice with LM. PKC[delta]-deficient mice were highly susceptible to LM infection with increased bacterial burden and enhanced histopathology despite enhanced NF-IL6 mRNA expression. Subsequent studies in PKC[delta]-deficient macrophages demonstrated that, despite elevated levels of proinflammatory cytokines and NO production, increased escape of LM from the phagosome into the cytoplasm and uncontrolled bacterial growth occurred. Taken together these data identified PKC[delta] as a critical factor for confinement of LM within macrophage phagosomes. microarray | phagosomal escape | bacterial killing

To determine whether ceramide mediates regulation of the renal epithelial sodium channel (ENaC) by tumor necrosis factor-[alpha] (TNF-[alpha]), confocal microscopy and patch-clamp experiments were performed in A6 distal nephron cells. We found that TNF-[alpha] (100 ng/ml) had no effect on ENaC activity and ceramide level when the cells were grown in the presence of aldosterone, but significantly inhibited ENaC and induced ceramide production after the cells were pretreated with LY 294002, an inhibitor of phosphatidylinositol 3-kinase, for 24 h. The inhibition of ENaC induced by TNF-[alpha] was mimicked by exogenous sphingomyelinase (0.1 U/ml) and [C.sub.2]-ceramide (50 [micro]M), but neither [C.sub.2]-dihydroceramide, a membrane-impermeable analog of [C.sub.2]-ceramide, nor choline, and abolished by pretreatment with GF109203X, a protein kinase C (PKC) inhibitor. [C.sub.2]-ceramide failed to affect ENaC in the cells pretreated with GF109203X, but not in the cells pretreated with PD-98059, a mitogen-activated protein kinase kinase inhibitor. [C.sub.2]-ceramide induced the externalization of phosphatidylserine (PS) in control A6 cells, but not in the cells pretreated with GFI09203X. Together with our previous finding that cytosolic PS maintains ENaC activity in A6 cells, these data suggest that ceramide mediates TNF-[alpha] inhibition of the renal ENaC via a pathway associated with PKC-dependent externalization of PS. A6 cells; single-channel recordings; confocal microscopy; sphingomyelinase; [C.sub.2]-ceramide; phosphatidylserine

[PGE.sub.2] plays a critical role in colorectal carcinogenesis. We have previously shown that COX-2 expression and [PGE.sub.2] synthesis are mediated by IGF-II/IGF-I receptor signaling in the Caco-2 cell line and that the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt protects the cell from apoptosis. In the present study, we demonstrate that [PGE.sub.2] has the ability to increase Ras and PI3K association and decrease the level of apoptosis in the same experimental system. The effect of [PGE.sub.2] on PI3K/Ras association is dependent on the activation of EP4 receptor, the increase of cAMP levels, and the activation of PKA. In fact, treatment of cells with the PKA inhibitor H89 decreases the association of Ras and PI3K and Ras-associated PI3K activity. PKA inhibitor H89 is able to decrease threonine phosphorylation of Akt and to increase serine phosphorylation of Akt by p38 MAPK and counteracts the cytoprotective effect induced by [PGE.sub.2]. In addition, [PGE.sub.2] is able to activate p38 MAPK and the inhibition of p38 MAPK, with SB203580 specific inhibitor or with dominant negative MKK6 kinase, is able to revert the apoptotic effect of H89 and serine phosphorylation of Akt. The effect of [PGE.sub.2] on Caco-2 cell survival through PKA activation is mediated and regulated by the balance of threonine/serine phosphorylation of Akt by p38 kinase and PI3K. In conclusion, our data elucidate a novel mechanism for regulation of colon cancer cell survival and provide evidences for new combinatory treatments of colon cancer. colon cancer; prostaglandin [E.sub.2]; Akt; protein kinase A; phosphatidylinositol 3-kinase

Secretin stimulates ductal secretion by activation of cAMP [right arrow] PKA [right arrow] CFTR [right arrow] [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger in cholangiocytes. We evaluated the expression of [[alpha].sub.2A-], [[alpha].sub.2B-], and [[alpha].sub.2C-]-adrenergic receptors in cholangiocytes and the effects of the selective [[alpha].sub.2]-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the [Na.sup.+]/[H.sup.+] exchanger isoform NHE3; and 3) cAMP levels, PKA activity, [Cl.sup.-] efflux, and [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger activity in purified cholangiocytes. [[alpha].sub.2]-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, [Cl.sup.-] efflux, and [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. [[alpha].sub.2]-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases. bicarbonate secretion; chloride efflux; gastrointestinal hormones; intrahepatic biliary epithelium: protein kinase A

5'AMP-activated protein kinase (AMPK) signaling initiates adaptive changes in skeletal muscle fibers that restore homeostatic energy balance. The purpose of this investigation was to examine, in rats, the fiber-type protein expression patterns of the [alpha]-catalytic subunit isoforms in various skeletal muscles, and changes in their respective contents within the tibialis anterior (TA) after chronic low-frequency electrical stimulation (CLFS; 10 Hz, 10 h daily), applied for 4 [+ or -] 1.2 or 25 [+ or -] 4.8 days. Immunocytochemical staining of soleus (SOL) and medial gastrocnemius (MG) showed that 86 [+ or -] 4.1 to 97 [+ or -] 1.4% of type IIA fibers stained for both the [alpha]1- and [alpha]2-isoforms progressively decreased to 63 [+ or -] 12.2% of type IID/X and 9 [+ or -] 2.4% of IIB fibers. 39 [+ or -] 11.4% of IID/X and 83 [+ or -] 7.9% of IIB fibers expressed only the [alpha]2 isoform in the MG, much of which was localized within nuclei, [alpha]1 and [alpha]2 contents, assessed by immunoblot, were lowest in the white gastrocnemius [WG; 80% myosin heavy chain (MHC) IIb; 20% MHCIId/x]. Compared with the WG, [alpha]1 content was 1.6 [+ or -] 0.08 (P < 0.001) and 1.8 [+ or -] 0.04 (P < 0.0001)-fold greater in the red gastrocnemius (RG: 13%, MHCIIa) and SOL (21%, MHCIIa), respectively, and increased in proportion to MHCIIa content. Similarly, [alpha]2 content was 1.4 [+ or -] 0.10 (P < 0.02) and 1.5 [+ or -] 0.07 (P < 0.001)-fold greater in RG and SOL compared with WG. CLFS induced 1.43 [+ or -] 0.13 (P < 0.007) and 1.33 [+ or -] 0.08 (P < 0.009)-fold increases in the [alpha]1 and [alpha]2 contents of the TA and coincided with the transition of faster type IIB and IID/X fibers toward IIA fibers. These findings indicate that fiber types differ with regard to their capacity for AMPK signaling and that this potential is increased by CLFS. myosin heavy chain; fast-twitch muscle; slow-twitch muscle

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing 'rescue' studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of 'combining' functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing movphogenesis, thereby revealing the first postembryonic function for Pkn.

Inhibition of Convergent extension movements in xenopus gastrulation by cyclic adenosine monophosphate -dependent protein kinase is presented.

Arterial smooth muscle constriction in response to pressure, i.e., myogenic tone, may involve calcium-dependent and calcium-sensitization mechanisms. Calcium sensitization in vascular smooth muscle is regulated by kinases such as PKC and Rho kinase, and activity of these kinases is known to be altered in cardiovascular disorders. In the present study, we evaluated the relative contribution of PKC and Rho kinase to myogenic tone in cerebral arteries in hypertension. Myogenic tone and arterial wall calcium in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were measured simultaneously, and the effect of PKC and Rho kinase inhibitors on myogenic tone was evaluated. SHR arteries showed significantly greater myogenic tone than WKY arteries. Pressure/wall tension-arterial wall calcium curves showed a hyperbolic relation in WKY rats, but the curves for SHR arteries were parabolic. Myogenic tone was decreased by the Rho kinase inhibitors Y-27632 and HA-1077, with a significantly greater effect in SHR than in WKY arteries. Reduction in myogenic tone produced by the PKC inhibitor bisindolylmaleimide I in WKY and SHR arteries was significantly less than that produced by Rho kinase inhibition. The pressure-dependent increase in myogenic tone was significantly decreased by Y-27632, and the decrease was markedly greater than that produced by bisindolylmaleimide I in SHR arteries. In WKY arteries, the pressure-dependent increase in myogenic tone was decreased to a similar extent by Y-27632 and bisindolylmaleimide I. These results suggest greater myogenic tone with increased calcium sensitization in SHR arteries, largely because of Rho kinase activation, with a minor contribution of PKC activation. spontaneously hypertensive rats; cerebral arteries; myogenic tone

Byline: V. P. Nikitin (1), S. A. Kozyrev (1) Keywords: snail; neuron; sensitization; nociception; synaptic plasticity; protein kinase C Abstract: Studies of defensive behavior command neurons LPl1 and RPl1 in semi-intact common snail preparations addressed the effects of the protein kinase C antagonist polymyxin B on the effect of nociceptive sensitization. Neurons in control snails responded to application of nociceptive stimuli to the head with membrane depolarization, increases in excitability, and depression of neuron responses to sensory stimulation during the short-term stage, with marked facilitation of responses during the long-term stage of sensitization. Acquisition of sensitization in the presence of polymyxin B resulted in partial suppression of responses to nociceptive stimuli. Changes in command neuron membrane excitability in these conditions, as well as changes in responses to tactile stimulation of the foot and chemical stimulation of the head, were similar to those seen in neurons of sensitized control animals. The inhibitor also had no effect on short-term depression of neuron responses induced by tactile stimulation of the head. In addition, acquisition of sensitization during administration of polymyxin B led to complete suppression of the facilitation of responses to tactile stimulation of the snail's head during the long-term stage of sensitization. It is suggested that in sensitized common snails, protein kinase C is involved in controlling the mechanisms of nociception and is also involved in the mechanisms of selective induction of plasticity in the synaptic inputs of command neurons, which are activated by tactile stimulation of the animal's head. Author Affiliation: (1) P. K. Anokhin Science Research Institute of Normal Physiology, Russian Academy of Medical Sciences, 6 Bolshaya Nikitskaya Street, 103009, Moscow, Russia Article History: Registration Date: 18/10/2004

The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.