PKB Activation Research Articles

Fish oil supplementation suppresses resistance exercise and feeding-induced increases in anabolic signaling without affecting myofibrillar protein synthesis in young men.

Fish oil (FO) supplementation potentiates muscle protein synthesis (MPS) in response to a hyperaminoacidemic–hyperinsulinemic infusion. Whether FO supplementation potentiates MPS in response to protein ingestion or when protein ingestion is combined with resistance exercise (RE) remains unknown. In a randomized, parallel group design, 20 healthy males were randomized to receive 5 g/day of either FO or coconut oil control (CO) for 8 weeks. After supplementation, participants performed a bout of unilateral RE followed by ingestion of 30 g of whey protein. Skeletal muscle biopsies were obtained before and after supplementation for assessment of muscle lipid composition and relevant protein kinase activities. Infusion of l‐[ring‐13C6] phenylalanine was used to measure basal myofibrillar MPS at rest (REST), in a nonexercised leg following protein ingestion (FED) and following RE and protein ingestion (FEDEX). MPS was significantly elevated above REST during FEDEX in both the FO and CO groups, but there was no effect of supplementation. There was a significant increase in MPS in both groups above REST during FED but no effect of supplementation. Supplementation significantly decreased panPKB activity at REST in the FO group but not the CO group. There was a significant increase from REST at post‐RE for PKB and AMPK α2 activity in the CO group but not in the FO group. In FEDEX, there was a significant increase in p70S6K1 activity from REST at 3 h in the CO group only. These data highlight that 8 weeks of FO supplementation alters kinase signaling activity in response to RE plus protein ingestion without influencing MPS.

SMILE Is an Insulin-Inducible Transcriptional Corepressor of Hepatic Gluconeogenic Gene Programs

Insulin is the major hormone that regulates hepatic glucose metabolism by repressing gluconeogenic enzyme-encoding genes, and the counteracting glucagon/protein kinase (PKA)–inducible coactivating peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) signaling pathway is also well characterized in hepatic glucose metabolism (1–3). Until now, however, the regulation of the insulin/protein kinase B (PKB)/Akt–inducible corepressor signaling pathway has remained largely unknown. Previously, it was believed that insulin suppression of gluconeogenesis was largely mediated through PKB activity via direct phosphorylation and dephosphorylation mechanisms (4). However, in this issue of Diabetes , Lee et al. (5) dissect the role of the small heterodimer partner–interacting leucine zipper protein (SMILE) in insulin-mediated hepatic glucose metabolism. SMILE is a member of the CREB/ATF family of basic-region leucine zipper (bZIP) transcription factors and has been reported to function as a corepressor of nuclear receptor superfamily genes, including estrogen-related receptor γ (ERRγ), glucocorticoid receptor (GR), hepatocyte nuclear factor 4α (HNF4α), and cAMP-responsive element–binding protein H (CREBH) (6–8). In fact, ERRγ, GR, HNF4α, and CREBH have all been implicated in upregulating gluconeogenic gene expression (9–12). Lee et al. (5) show that SMILE is an insulin-inducible corepressor …

Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.

Sa1987 Lipid Phosphatase SHIP2 Functions As Oncogene in Colorectal Cancer by Regulating PKB Activation

Sa1987 Lipid Phosphatase SHIP2 Functions As Oncogene in Colorectal Cancer by Regulating PKB Activation

Fine-tuning the intensity of the PKB/Akt signal enables diverse physiological responses

The PI3K/PDK1/PKB signaling pathway plays essential roles in regulating neuronal survival, differentiation and plasticity in response to neurotrophic factors, neurotransmitters and ion channels. Both PDK1 and PKB can interact at the plasma membrane with a phosphoinositide synthesized by PI3K, the second messenger PtdIns(3,4,5)P3, enabling PDK1 to phosphorylate and activate PKB. In the PDK1 K465E knock-in mice expressing a mutant form of PDK1 incapable of phosphoinositide binding, activation of PKB was markedly affected, but not totally abolished. It has been recently proposed that in the absence of PtdIns(3,4,5)P3 binding, PDK1 can still moderately activate PKB due to a docking site-mediated interaction of these 2 kinases. A recent report has uncovered that in the PDK1 K465E mice neurons, a PKB signal threshold was sufficient to support neuronal survival responses, whereas neuritogenesis, neuronal polarization and axon outgrowth were severely impaired. We propose here that the low-efficiency mechanism of PKB activation observed in the PDK1 K465E mice might represent the ancestral mechanism responsible for the essential functions of this pathway, while the phosphoinositide-dependent activation should be considered an evolutionary innovation that enabled the acquisition of novel functions.

Activation of PKB and ERK, but not PI3K, is involved in fucosylated chondroitin sulphate from Acaudina molpadioides induced glucose uptake

Fucosylated chondroitin sulphate from sea cucumber can mitigate hyperglycaemia. However, the possible mechanism is unclear. We investigated the effects of fucosylated chondroitin sulphate from Acaudina molpadioides (Am-CHS) on glucose uptake and its mechanism using TNF-α-induced 3T3-L1 insulin resistant adipocytes. Results showed that Am-CHS could significantly increase glucose uptake and GLUT4 translocation, and phosphorylated PI3K, PKB, and ERK. Am-CHS reversed MK2206-induced reduction in glucose uptake, GLUT4 translocation, and PKB phosphorylation. Am-CHS antagonized U0126-induced decrease in glucose uptake, GLUT4 translocation, and ERK1/2 phosphorylation. Am-CHS inhibited wortmannin-induced reduction in glucose uptake and GLUT4 translocation reduction. Wortmannin-restrained PKB and ERK1/2 phosphorylation was antagonized by Am-CHS, but the lowered PI3K phosphorylation remained unchanged. Am-CHS also lowered blood glucose, increased m-GLUT4, p-PKB (Ser473), p-ERK1/2, p-PI3K p85, and p-IRS-1 (Tyr612) expressions, and decreased p-PKB (Ser308) expression in adipose tissues of insulin resistant mice. These indicate that PKB and ERK activation are involved in Am-CHS-triggered glucose, but not PI3K.

The hexosamine biosynthesis pathway and O-GlcNAcylation maintain insulin-stimulated PI3K-PKB phosphorylation and tumour cell growth after short-term glucose deprivation.

Glucose provides an essential nutrient source that supports glycolysis and the hexosamine biosynthesis pathway (HBP) to maintain tumour cell growth and survival. Here we investigated if short-term glucose deprivation specifically modulates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) cell survival pathway. Insulin-stimulated PKB activation was strongly abrogated in the absence of extracellular glucose as a consequence of the loss of insulin-stimulated PI3K activation and short-term glucose deprivation inhibited subsequent tumour cell growth. Loss of insulin-stimulated PKB signalling and cell growth was rescued by extracellular glucosamine and increased flux through the HBP. Disruption of O-GlcNAc transferase activity, a terminal step in the HBP, implicated O-GlcNAcylation in PKB signalling and cell growth. Glycogenolysis is known to support cell survival during glucose deprivation, and in A549 lung cancer cells its inhibition attenuates PKB activation which is rescued by increased flux through the HBP. Our studies show that rerouting of glycolytic metabolites to the HBP under glucose-restricted conditions maintains PI3K/PKB signalling enabling cell survival and proliferation.

Insulin-sensitizing and insulin-mimetic activities of Sarcopoterium spinosum extract

Ethnopharmacological relevanceSarcopoterium spinosum is an abundant plant in Israel, used by Bedouin medicinal practitioners for the treatment of diabetes. In our previous study we validated the anti-diabetic activity of Sarcopoterium spinosum. The aim of this study was to further clarify its mechanism of action. Materials and methodsIn-vivo studies were performed on KK-a/y mice given the extract for 6 weeks. Insulin tolerance test was performed, and relative pancreatic islets area was measured. Mechanisms of action were investigated in L6 myotubes using protein array, Western blot analysis and confocal microscopy. Glucose uptake assays were performed in 3T3-L1 adipocytes. ResultsSarcopoterium spinosum extract reduced fasting blood glucose and improved insulin sensitivity in treated mice. Hypertrophic islets were detected in diabetic, but not in Sarcopoterium spinosum-treated mice. Sarcopoterium spinosum phosphorylated PTEN on ser380 and thr382/383, which are known inhibitory sites. PKB was not phosphorylated by Sarcopoterium spinosum, however, translocation of PKB from cytoplasm to the membrane and nucleus was detected. Target proteins of PKB were regulated by Sarcopoterium spinosum; GSK3β was phosphorylated and cytosolic localization of FoxO was increased. Glucose uptake was increased in a PI3K and AMPK-independent mechanism. ConclusionsWe suggest that Sarcopoterium spinosum inhibited PTEN and activated PKB by a mechanism which is independent of ser473 and thr308 phosphorylation. Other post translation modifications might be involved and should be analyzed further in order to understand this unique PKB activation. Identifying the active molecules in the extract, may lead to the development of new agents for the treatment of insulin resistance.

REDD1 reduces the responsiveness of mTORC1 after resistance exercise and amino acid intake in skeletal muscle of elderly people (702.4)

The aim of the present experiment was to highlight the difference in mTORC1 regulation between young and elderly people after exercise and amino acid intake. Eight younger (23±1 y) and 8 older (70±7 y) healthy volunteers were recruited. After an overnight fast, a first biopsy sample was taken in the vastus lateralis muscle. Then, the subjects performed a one‐leg resistance exercise consisting in 10 sets of 10 leg extensions at 70% of their 3‐RM (repetition‐maximum) previously measured. Immediately after the exercise completion, the subjects drank a beverage containing 30 g of whey protein hydrolyzate whose 2.5 g was leucine. Thirty minutes after the beverage intake, a second biopsy was taken in the exercised leg and a third one in the contralateral leg. In both groups, feeding increased the S6K1 phosphorylation and resistance exercise potentiated this effect. However, the range of these increases was about the half in elderly as compared to the young subjects confirming a reduced responsiveness of mTORC1 in elderly people. Feeding doubled the phosphorylation of PKB (Ser473) in both groups. Resistance exercise reinforced this effect in young but not in elderly people. The expression of REDD1 was systematically higher in elderly people. REDD1 was slightly and not significantly increased by exercise in young subjects whereas it was doubled in elderly suggesting that REDD1 is probably a major actor for the development of anabolic resistance during ageing. The phosphorylation state of PRAS40 (Thr246) increased after feeding but similarly in both groups. The phosphorylation state of AMPK (Thr172) was affected neither by feeding nor by exercise. All together, these results indicate that mTORC1 acts as a molecular integrator receiving positive and negative signals from its upstream regulators. The reduced responsiveness to anabolic stimuli observed in elderly people seems related to a higher REDD1 expression combined with a lower exercise‐induced activation of PKB.Grant Funding Source: Supported by the Walloon Region (Plan Marshall – Wagralim ‐ Geramino)

Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism

ObjectiveThe bacteria Staphylococcus aureus is part of the normal bacterial flora and produces a repertoire of enterotoxins which can cause food poisoning and toxic shock and might contribute to the pathogenesis of inflammatory diseases. These enterotoxins directly cross-link the T cell receptor with MHC class II, activating large amounts of T cells and are therefore called superantigens. It was recently discovered that the superantigen SEA binds to the cytokine receptor gp130. As obesity and type 2 diabetes are highly associated with inflammation of the adipose tissue and gp130 has been shown to play an important role in adipocytes, we wanted to investigate the effect of SEA on adipocyte signaling and function. Materials/methodsBinding of SEA to gp130 was examined using surface plasmon resonance in a cell free system. Effects of SEA on adipocyte signaling, insulin sensitivity and function were studied using western blotting and biological assays for lipolysis, lipogenesis and glucose uptake. ResultsWe demonstrate that SEA binds to gp130 with a medium affinity. Furthermore, SEA induces phosphorylation of a key downstream target, STAT3, in adipocytes. SEA also inhibits insulin-induced activation of PKB and PKB downstream signaling which was associated with reduced basal and insulin induced glucose uptake, reduced lipogenesis as well as reduced ability of insulin to inhibit lipolysis. ConclusionsSEA inhibits insulin signaling as well as insulin biological responses in adipocytes supporting that bacterial infection might contribute to the development of insulin resistance and type 2 diabetes.

A PIP5 Kinase Essential for Efficient Chemotactic Signaling

SummaryIn neutrophils and Dictyostelium, chemoattractant gradients generate directed cell migration by eliciting signaling events that bias intrinsic motility and favor the production and retention of upgradient pseudopods [1, 2]. Phosphoinositides are actively regulated during chemotaxis in these cells, most iconically in the production of PI(3,4,5)P3 gradients within the plasma membrane [3, 4]. Although it is now known that PI(3,4,5)P3 signaling is nonessential for gradient sensing [5, 6], the role of the related phosphoinositide PI(4,5)P2 is little understood, despite its clear importance in many cell biological processes [7]. We describe here a PIP5 kinase, PikI, which produces PI(4,5)P2 and is essential for efficient chemotaxis of Dictyostelium cells. Without PikI, PI(4,5)P2 levels are reduced by 90%, and while pikI− cells move at normal speeds, they are highly disorientated in cAMP gradients. Following chemotactic stimulation, Ras is efficiently activated in pikI− cells, yet Ras-dependent responses (including activation of PKB) are severely impaired. PikI is phosphorylated by PKB [8], and in vitro studies of a phosphomimic mutant suggest that this phosphorylation increases PikI activity. We propose that adequate PI(4,5)P2 levels are required to couple activated Ras to its downstream effectors and that these levels are regulated by PikI, making it a crucial player in gradient sensing.

Short-term hypoxia induces a selective death of GABAergic neurons

It is known that brief episodes of hypoxia protect neurons from death caused by global ischemia and hypoxia (hypoxic preconditioning). At the same time, brief hypoxia may cause a phenomenon of posthypoxic hyperexcitability during reoxygenation, which can lead to the death of separate neurons due to their individual differences. In this work we compare the effects of short-term hypoxia on the initiation of preconditioning and posthypoxic hyperexcitability in two populations of neurons: inhibitory GABAergic neurons and excitatory glutamatergic neurons. Preconditioning effect was evaluated according to the suppression of the NMDA-receptor activity. The phenomenon of posthypoxic hyperexcitability was estimated by the appearance of spontaneous synchronized Ca2+ spikes in the neuronal network during reoxygenation after each episode of hypoxia. It is shown that the preconditioning effect occurs only in glutamatergic neurons. In the GABAergic neurons the effect of preconditioning was not observed. The activity of NMDA receptors in these neurons was not suppressed but increased after each episode of hypoxia. At the moment of posthypoxic synchronous Ca2+-spike generation, a global increase of the cytoplasmic Ca2+ concentration occurred in a few of GABAergic neurons, followed by the apoptotic death of these cells. The anti-inflammatory cytokine, interleukin-10 (IL-10) prevented the development of posthypoxic hyperexcitability, inhibiting spontaneous synchronous Ca2+ spike, and protected GABAergic neurons from the death, restoring the preconditioning effect in them. PI3-kinase inhibitors wortmannin and LY294002 prevented the IL-10 protective effect abolishing the inhibiting effect of IL-10 on the generation of the Ca2+ synchronous spike. These findings point out to the leading role of GABAergic neurons in the development of posthypoxic hyperexcitability. We suggest that the reason for posthypoxic hyperexcitability in the network is a weakening of the inhibiting effect of GABAergic neurons. Activation of different signaling pathways leading to activation of PKB- and PKG-dependent phosphorylation in the neurons of this type represents a possible strategy to protect neurons from death during hypoxia.

Astrocytic phosphorylation of PDK1 on Tyr9 following an excitotoxic lesion in the mouse hippocampus

3-phosphoinositide-dependent kinase-1 (PDK1) is suggested to play important roles in the regulation of synaptic plasticity and neuronal cell survival in the mature CNS. Although few studies have investigated the roles of PDK1, little is known about PDK1 changes in glial cells under neuropathological conditions. In current report, phosphorylation of PDK1 was monitored specially on tyrosine residues, following the induction of an excitotoxic lesion in rat brain by using kainic acid administration. In injured hippocampal CA3 region, Tyr9 phosphorylation of PDK1 was increased from 4h until 3 day post-injection. Double immunohistochemistry further evaluated that these phosphorylated forms of PDK1 were localized in astrocytes not other cells. Overexpression of unphosphorylatable mutant, PDK1-Y9F leads to inhibit Protein kinase B (PKB/Akt) activation and cAMP responsive element binding protein (CREB) phosphorylation. In conclusion, our results suggested for the first time that tyrosine phosphorylation of PDK1 is required for PKB and CREB activation in KA-mediated excitotoxic lesion in mouse brain.

Platelet-derived growth factor-D induces expression of cyclooxygenase-2 in rat mesangial cells through activation of PI3K/PKB and PKCs

Platelet-derived growth factor (PDGF)-D is suggested to be a key factor in the development of several renal pathologies, including mesangioproliferative glomerulonephritis. Cyclooxygenase (COX)-2 is a protein involved in the biosynthesis of inflammatory prostaglandins. In this study, we investigated the effect of PDGF-D on the regulation of COX-2 expression in rat mesangial cells (RMCs). Treatment with PDGF-D induced COX-2 at both the protein and mRNA levels in RMCs, suggesting that the PDGF-D-mediated induction of COX-2 is due to COX-2 transcriptional upregulation. PDGF-D treatment also led to a rapid but transient activation of PKB and extracellular signal regulated kinase (ERK)-1/2. Activities of JNK-1/2 and p38 MAPK, however, were not influenced by PDGF-D in RMCs. Markedly, pharmacological inhibition studies showed that pretreatment with LY294002 (a PI3K/PKB inhibitor) or GF109203X (a pan-PKC inhibitor) suppressed the PDGF-D-induced expression of COX-2 protein and mRNA, while pretreatment with PD98059 (an ERK-1/2 inhibitor) or PP1 (an Src inhibitor) had no effect on it. These findings collectively demonstrate for the first time that PDGF-D induces COX-2 by transcriptional upregulation in RMCs and the induction is largely related to PI3K/PKB and PKCs activities.

PtdIns5 P is an oxidative stress‐induced second messenger that regulates PKB activation

Oxidative stress initiates signaling pathways, which protect from stress-induced cellular damage, initiate apoptosis, or drive cells into senescence or into tumorigenesis. Oxidative stress regulates the activity of the cell survival factor PKB, through the regulation of PtdIns(3,4,5)P₃ synthesis. Whether oxidative stress regulates other phosphoinositides to control PKB activation is not clear. Here we show that PtdIns5P is a redox-regulated second messenger. In response to hydrogen peroxide (H₂O₂), we measured an increase in PtdIns5P in cells derived from human osteosarcoma, U2OS (5-fold); breast tumors, MDA-MB-468 (2-fold); and fibrosarcoma, HT1080 (3-fold); and in p53-null murine embryonic fibroblasts (8-fold). In U2OS cells, the increase in H₂O₂-dependent PtdIns5P did not require mTOR, PDK1, PKB, ERK, and p38 signaling or PIKfyve, a lipid kinase that increases PtdIns5P in response to osmotic and oncogenic signaling. A reduction in H₂O₂-induced PtdIns5P levels by the overexpression of PIP4K revealed its role in PKB activation. Suppression of H₂O₂-induced PtdIns5P generation reduced PKB activation and, surprisingly, reduced cell sensitivity to growth inhibition by H₂O₂. These data suggest that inhibition of PIP4K signaling might be useful as a novel strategy to increase the susceptibility of tumor cells to therapeutics that function through increased oxidative stress.

Ubiquitination of the tomato cell death suppressor Adi3 by the RING E3 ubiquitin ligase AdBiL

Programmed cell death (PCD) is an organized process by which organisms selectively remove cells according to developmental needs or in response to biotic or abiotic stress. Despite recent efforts to understand mechanisms by which cell death takes place in plants, several gaps remain in our understanding of the molecular elements involved. The tomato PCD suppressor Adi3 is an AGC kinase that shares functional homology with the mammalian inhibitor of apoptosis PKB. Regulation of PKB stability, cell localization, and activation state is achieved through post-translational modifications such as ubiquitination. In an effort to understand the regulation of Adi3 function, we studied its interaction with the E3 ubiquitin ligase AdBiL. Using in vitro ubiquitination assays we show that AdBiL is an active E3 ubiquitin ligase using the E2 ubiquitin ligase UBC8 to ubiquitinate Adi3. Adi3 is also degraded in a proteasome-dependent manner. Our data draws additional parallels between Adi3 and PKB to support the functional relationship between these two PCD regulators.

Daydreamer, a Ras effector and GSK-3 substrate, is important for directional sensing and cell motility

How independent signaling pathways are integrated to holistically control a biological process is not well understood. We have identified Daydreamer (DydA), a new member of the Mig10/RIAM/lamellipodin (MRL) family of adaptor proteins that localizes to the leading edge of the cell. DydA is a putative Ras effector that is required for cell polarization and directional movement during chemotaxis. dydA(-) cells exhibit elevated F-actin and assembled myosin II (MyoII), increased and extended phosphoinositide-3-kinase (PI3K) activity, and extended phosphorylation of the activation loop of PKB and PKBR1, suggesting that DydA is involved in the negative regulation of these pathways. DydA is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is required for some, but not all, of DydA's functions, including the proper regulation of PKB and PKBR1 and MyoII assembly. gskA(-) cells exhibit very strong chemotactic phenotypes, as previously described, but exhibit an increased rate of random motility. gskA(-) cells have a reduced MyoII response and a reduced level of phosphatidylinositol (3,4,5)-triphosphate production, but a highly extended recruitment of PI3K to the plasma membrane and highly extended kinetics of PKB and PKBR1 activation. Our results demonstrate that GSK-3 function is essential for chemotaxis, regulating multiple substrates, and that one of these effectors, DydA, plays a key function in the dynamic regulation of chemotaxis.

Abstract 316: Effect of Different Substrates on Functional Performance and Kinase Activation During Reperfusion After Ischemia in Hearts from Obese Insulin Resistant Rats

Insulin resistance decreases glucose uptake, alters lipid metabolism and impairs PKB-dependent signaling in its target tissues. In view of the myocardial dependence on glycolysis during energy deficiency, insulin resistance may exacerbate the harmful effects of ischaemia. We hypothesize that obesity-induced insulin resistance affects functional recovery and activation of PKB, JNK, ERK and PTEN during early reperfusion. Methods: Insulin resistance was induced by feeding rats a high calorie diet for 16 weeks. Hearts from diet rats (D) and age-matched controls (C) were subjected to (i) 15 min global ischaemia and different reperfusion times for Western blot and measurement of functional recovery, or (ii) 35 min regional ischaemia and 2 hours reperfusion for infarct size determination (IS) using tetrazolium. Substrates were glucose (G) (10mM) or glucose (10mM) plus fatty acid (FA) (1.2mM palmitate /3%BSA) (G+FA). N=4-8/group. Results: Regardless of the substrate, functional recovery during reperfusion of hearts from D was higher than those of C (% recovery of total work: D+G/C+G, 69.1±3.4/50.7±4.5, D+G+FA/C+G+FA: 105.4±3.6/92.7±3.9), associated with a reduction in IS (D+G/C+G 29.7±2.8/43.5±2.5, p<0.05; D+G+FA/C+G+FA 26.7±3.2/36.6±4, p<0.05). When perfused with glucose hearts from D showed increased activation of PKB and JNK at different times during reperfusion (D+G/C+G: 10 min PKBs473, 1.41±0.11/1.00±0.06; JNKp54, 1.80±0.15/1.00±0.13; JNKp46, 1.59±0.11/1.00±0.08). Addition of FA caused further activation of the kinases (e.g. D+G+FA/D+G: 5 min ERKp44, 1.54±0.11/1.00±0.11, ERKp42, 1.46±0.13/1.00±0.07; 10 min ERKp44, 1.83±0.19/1.00±0.05, ERKp42, 1.27±0.10/1.00±0.05; 30 min PKBs473, 2.91±0.35/1.00±0.09) and inhibition of PTEN (1.79±0.11/1.00±0.10). Conclusion: Contrary to expectations, hearts from D rats showed an increased tolerance to ischaemia/reperfusion (I/R) injury which was further improved by fatty acids in the perfusate. The pattern of PKB, JNK, ERK and PTEN phosphorylation during reperfusion is substrate dependent and significantly higher in hearts from D rats. With glucose as substrate activation of PKB and JNK during reperfusion was higher in hearts from D rats while FA further improved activation of ERK and PKB.

Prodigiosin down‐regulates SKP2 to induce p27KIP1 stabilization and antiproliferation in human lung adenocarcinoma cells

High levels of SKP2 are a poor prognostic factor in multiple human cancers and mostly correlate with low p27(KIP1) levels. Prodigiosin is a bacterial tripyrrole pigment with strong pro-apoptotic activity. Induction of cell cycle blockade underlies one of its anticancer actions but the mechanisms involved are unclear. The aim of this study was to explore the role of the SKP2-p27(KIP1) axis in prodigiosin's cytostatic effect on human lung adenocarcinoma cells. Prodigiosin's effects on cell cycle progression and long-term cell proliferation of human lung adenocarcinoma cells were characterized by flow cytometry and colony formation assay, respectively. Real-time RT-PCR and promoter activity analyses were performed for assessing transcriptional control, while cycloheximide chase analysis evaluated protein stability. Immunoblotting was employed for mechanistic study. Prodigiosin increased p27(KIP1) expression mainly by stabilizing p27(KIP1) through transcriptional repression of SKP2. Importantly, SKP2 overexpression or p27(KIP1) depletion restored the colony forming capacity of prodigiosin-treated cells. Furthermore, prodigiosin induced PKB dephosphorylation, leading to PKB inhibition as revealed by decreased serine 9 phosphorylation of GSK-3β. Constitutive PKB activation reduced prodigiosin-induced SKP2 repression. Prodigiosin also down-regulated E2F1 (mediates PI3K/PKB-induced SKP2 transcription), but E2F1 overexpression failed to restore SKP2 expression in prodigiosin-treated cells. Transcriptional repression of SKP2 and the consequent accumulation of p27(KIP1) are essential for prodigiosin's antiproliferative action. Mechanistically, prodigiosin induces PKB inhibition to down-regulate SKP2 in a GSK-3β- and E2F1-independent manner. Our findings further implicate the potential for developing prodigiosin as a novel class of SKP2-targeting anticancer agent.

GPCR activation of Ras and PI3Kc in neutrophils depends on PLCb2/b3 and the RasGEF RasGRP4.

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.