Kegg Pathway: Phosphatidylinositol signaling system

Pancreas. 2009 Nov 16;
Hui H, Tang YG, Zhu L, Khoury N, Hui Z, Wang KY, Perfetti R, Go VL

OBJECTIVES:: That glucagonlike peptide-1 (GLP-1) induces differentiation of primate embryonic stem (ES) cells into insulin-producing cells has been reported by several groups and also confirmed with our observations. METHODS:: To further elucidate the process in detail and the signaling pathways involved in this differentiation, we induced human ES cells HUES1 differentiated into insulin secretion cells by GLP-1 treatment. RESULTS:: A time-dependent pattern of down expression of the stem cell markers (human telomerase reverse transcriptase and octamer-4), and the appearance of multiple beta-cell-specific proteins (insulin, glucokinase, glucose transporter, type 2, and islet duodenal homeobox 1) and hedgehog signal molecules (Indian hedgehog, sonic hedgehog, and hedgehog receptor, patched) have been identified. Cotreatment with hedgehog signal inhibitor cytopamine was able to block this differentiation, providing evidence of the involvement of the hedgehog signaling pathway in GLP-1-induced differentiation. We also observed increased transcripts of the transcription factors of activator protein 1, serum response element-1, DNA-binding transcription factors, and cAMP response element in GLP-1-induced ES cell differentiation. Inhibition profile by its specific inhibitors indicated that the cyclic adenosine monophosphate and Phosphatidylinositol-3-kinase pathways, but not the mitogen-activated protein kinase pathway, were required for the induced differentiation of ES cells. CONCLUSIONS:: These data support that GLP-1 directs human ES cell differentiation into insulin-producing cells via hedgehog, cyclic adenosine monophosphate, and Phosphatidylinositol-3-kinase pathways.

Clin Chim Acta. 2009 Nov 11;
Banudevi S, Senthilkumar K, Sharmila G, Arunkumar R, Vijayababu R, Arunakaran J

BACKGROUND: Prostate cancer is one of the most frequently diagnosed cancers in men. Progression of these tumors is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. Among these, insulin-like growth factors (IGFs) signaling pathway contributes a major role. In this study, we examined the effect of zinc on insulin-like growth factors signaling in prostate cancer cells. METHODS: Human androgen-independent prostatic carcinoma (PC-3) cells were treated with different concentrations of zinc (20-100micromol/l) for 24 and 48h. Cell viability was performed by 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Insulin-like growth factor binding protein-3 (IGFBP-3), insulin-like growth factor-I receptor (IGF-IR), insulin receptor substrates-1 (IRS-1) and IRS-2, Phosphatidylinositol-3 kinase (PI-3 K), protein kinase B or Akt, phosphorylated Akt (p-Akt), extracellular regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), and cyclin D1 protein levels were assessed by Western blot analysis. Apoptosis was confirmed by 4',6'-diaminido-2-phenylindole dihydrochloride (DAPI) staining, and mitochondrial membrane potential was performed using rhodamine-123 staining method. RESULTS: Zinc significantly reduces the cell viability of PC-3 cells. It decreases the protein levels of IGF-IR, IRS-1, and IRS-2 and increases the level of IGFBP-3. Zinc reduces the levels of PI-3 K, Akt, ERK1/2, and cyclin Dl. Loss of mitochondrial membrane potential and apoptotic cell death were also observed in zinc-treated cells. CONCLUSION: This study suggests that zinc decreases the survival of androgen-independent prostate cancer cells by modulating the expression of IGF system components and its signaling molecules. Thus, zinc may be qualified as a potential agent for the treatment of prostate cancer.

Biochem Biophys Res Commun. 2009 Nov 5;
Poon HY, Stone JC

In leukocytes, diacylglycerol (DAG) regulates the small GTPase Ras through the agency of Ras guanyl releasing proteins (RasGRPs). Ras is thought to regulate Phosphatidylinositol 3-kinase (PI3K). Therefore, DAG signaling is hypothesized to impact PI3K activity. The DAG analogue "pico" was used to activate RasGRPs in leukocyte-derived cell lines. PI3K signaling was monitored using antibodies that recognize the activated form of the PI3K-regulated protein kinase Akt. Diverse responses were documented. Some cell lines exhibit a DAG analogue-stimulated increase in phospho-Akt but this response proceeded even when Ras activation was blocked. In some Epstein-Barr virus-associated malignant cell lines and transformed B cells, high basal phospho-Akt was decreased by DAG analogue treatment. The pan-PKC inhibitor Bisindolymaleimide I blocked this effect. Basal phospho-Akt was also decreased by treatment with Go6976, an inhibitor of conventional protein kinase C and protein kinase D. While the proposed RasGRP-Ras-PI3K-Akt signaling axis may operate in some situations, our results indicate that alternative links between DAG targets such as protein kinases and the PI3K signaling system are more prominent.

Exp Mol Med. 2009 Nov 4;
Kim Y, Shanta SR, Zhou LH, Kim KP

Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of Phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We, also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases.

J Physiol. 2009 Oct 19;
Ishibashi H, Nakahata Y, Eto K, Nabekura J

Locus coeruleus (LC) noradrenergic neurons are implicated in a variety of functions including the regulation of vigilance and the modulation of sensory processing. Thyrotropin-releasing hormone (TRH) is an endogenous neuropeptide that induces a variety of behavioral changes including arousal and antinociception. In the present study, we explored whether the activity of LC noradrenergic neurons is modulated by TRH. Using current-clamp recording from isolated rat LC neurons, we found that TRH increased the firing rate of spontaneous action potentials. The TRH action was mimicked by TRH analogues including taltirelin and TRH-gly. In voltage-clamp recording at a holding potential of -50 mV, TRH produced an inward current associated with a decrease in the membrane K(+) conductance. This current was inhibited by the TRH receptor antagonist chlordiazepoxide. Following inhibition of the pH-sensitive K(+) conductance by extracellular acidification, the TRH response was fully inhibited. The TRH-induced current was also inhibited by the phospholipase C (PLC) inhibitor U-73122, but not by the protein kinase C inhibitor chelerythrine nor by chelation of intracellular Ca(2+) by BAPTA. The recovery from the facilitatory action of TRH on the spike frequency was markedly inhibited by high concentration of wortmannin. These results suggest that TRH activates LC noradrenergic neurons by decreasing an acid-sensitive K(+) conductance via PLC-mediated hydrolysis of Phosphatidylinositol 4,5-bisphosphate. The present findings demonstrate that TRH activates LC neurons and characterize the underlying signaling mechanisms. The action of TRH on LC neurons may influence a variety of CNS functions related to the noradrenergic system which include arousal and analgesia.

J Pharmacol Exp Ther. 2009 Oct 14;
Onali P, Dedoni S, Olianas MC

Tricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their pharmacological activity at opioid receptors has not yet been elucidated. In the present study, we investigated the actions of amoxapine, amitriptyline, nortriptyline, desipramine and imipramine at distinct cloned and native opioid receptors. In Chinese hamster ovary (CHO) cells expressing delta-opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at micromolar concentrations with amoxapine displaying the highest potency and efficacy. Amoxapine and amitriptyline inhibited cyclic AMP formation and induced the phosphorylation of signaling molecules along the extracellular signal-regulated kinase 1/2 (ERK1/2) and Phosphatidylinositol-3 kinase pathways. Amoxapine also activated delta-opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHO cells expressing kappa-opioid receptors (CHO/KOR), TCAs, but not amoxapine, exhibited higher receptor affinity and more potent stimulation of [(35)S]GTPgammaS binding than in CHO/DOR, and effectively inhibited cyclic AMP accumulation. Amitriptyline regulated ERK1/2 phosphorylation and activity in CHO/KOR and C6 glioma cells endogenously expressing kappa-opioid receptors and this effect was attenuated by the kappa-opioid antagonist nor-binaltorphimine. In rat nucleus accumbens, amitriptyline slightly inhibited adenylyl cyclase activity and counteracted the inhibitory effect of the full kappa agonist (-)-U-50,488. At the cloned mu-opioid receptor, TCAs showed low affinity and no significant agonist activity. These results demonstrate that TCAs differentially regulate opioid receptors with a preferential agonist activity on either delta or kappa subtypes and suggest that this property may contribute to their therapeutic and/or side effects.

Climacteric. 2009; 12 Suppl 1: 6-11
Simoncini T

Estrogen exerts pleiotropic functions on the cardiovascular system through binding to estrogen receptors (ERs). Traditionally, ERs have been recognized as transcription factors regulating the expression of target genes. In the past decades, however, numerous studies have revealed rapid actions of estrogen in different systems, especially in non-reproductive tissues such as the cardiovascular system. At this level, estrogen triggers rapid vasodilatation, exerts anti-inflammatory effects, regulates vascular cell growth and migration, and confers protection to cardiomyocytes. These so-called 'extranuclear actions' do not require gene expression or protein synthesis and are independent of the nuclear localization of ERs. Indeed, some of these actions are elicited by ERs residing at or near the plasma membrane. Through complex interactions with membrane-associated signaling molecules such as ion channels, G proteins and the tyrosine kinase c-Src, liganded extranuclear ERs lead to the activation of downstream cascades such as mitogen-activated protein kinase (MAPK) and Phosphatidylinositol 3-OH kinase (PI3K). These cascades are responsible for important cardiovascular actions of estrogen, for instance, the activation of nitric oxide synthesis or the remodeling of the endothelial actin cytoskeleton. Moreover, these cascades play crucial roles in regulating the expression of target proteins implicated in cell proliferation, apoptosis, differentiation, movement and homeostasis. Recent advancements in the characterization of the molecular basis of the extranuclear signaling of estrogen help us to understand the biological functions of estrogen and would be beneficial in elucidating current controversies on estrogen's clinical efficacy in the cardiovascular system.

Life Sci. 2009 Nov 18; 85(21-22): 719-31
Sheikpranbabu S, Haribalaganesh R, Banumathi E, Sirishkumar N, Lee KJ, Gurunathan S

AIMS: The purpose of this study was to investigate the effect of pigment epithelium-derived factor (PEDF) on the signaling cascade in porcine retinal endothelial cells (PRECs) related to angiogenesis induced by advanced glycation end-products (AGEs). MAIN METHODS: Endothelial cells were isolated from porcine retina by the enzymatic method. Immunocytochemistry was performed to confirm the identity of PRECs. The effect of AGEs and PEDF on cell viability was determined by the MTT assay. An in vitro wound-scratch assay was performed to study the migration of ECs, and in vitro tube formation was assessed by the on-gel assay system using an extracellular matrix. Inhibitor assays were carried out using LY294002, a Phosphatidylinositol 3-kinase (PI3K) inhibitor, and Akt inhibitor VIII. PI3K/Akt activity was assessed by transient transfection and western blot analysis. Induction of apoptosis by PEDF was determined by caspase-3 colorimetric assay and DNA fragmentation analysis. KEY FINDINGS: Treatment of PRECs with AGE-bovine serum albumin (AGE-BSA) significantly increased the cell proliferation, migration and tube formation compared to non-glycated BSA. AGE-BSA mediates cell survival via the PI3K/Akt/FKHR-dependent pathway as evidenced by transient transfection and western blot analyses. Furthermore, PEDF significantly inhibited the proliferation, migration and tube formation, both in the presence and absence of AGE-BSA in PRECs. PEDF inactivated the AGE-BSA-induced PI3K/Akt/FKHR activity and induced apoptosis via caspase-3. SIGNIFICANCE: The results reveal that PEDF inhibits AGE-BSA-induced PI3K/Akt/FKHR signaling in PRECs. Thus, PEDF has potent anti-angiogenic effects against AGE-induced angiogenesis and is suggested to be a promising molecule for the treatment of diabetic retinopathy.

Exp Cell Res. 2009 Oct 2;
Watanabe-Takano H, Takano K, Keduka E, Endo T

The small GTPase M-Ras is highly expressed in the central nervous system and plays essential roles in neuronal differentiation. However, its other cellular and physiological functions remain to be elucidated. Here, we clarify the novel functions of M-Ras in osteogenesis. M-Ras was prominently expressed in developing mouse bones particularly in osteoblasts and hypertrophic chondrocytes. Its expression was elevated in C3H/10T1/2 (10T1/2) mesenchymal cells and in MC3T3-E1 preosteoblasts during differentiation into osteoblasts. Treatment of C2C12 skeletal muscle myoblasts with bone morphogenetic protein-2 (BMP-2) to bring about transdifferentiation into osteoblasts also induced M-Ras mRNA and protein expression. Moreover, the BMP-2 treatment activated the M-Ras protein. Stable expression of the constitutively active M-Ras(G22V) in 10T1/2 cells facilitated osteoblast differentiation. M-Ras(G22V) also induced transdifferentiation of C2C12 cells into osteoblasts. In contrast, knockdown of endogenous M-Ras by RNAi interfered with osteoblast differentiation in 10T1/2 and MC3T3-E1 cells. Osteoblast differentiation in M-Ras(G22V)-expressing C2C12 cells was inhibited by treatment with inhibitors of p38 MAP kinase (MAPK) and c-Jun N-terminal kinase (JNK) but not by inhibitors of MAPK and ERK kinase (MEK) or Phosphatidylinositol 3-kinase. These results imply that M-Ras, induced and activated by BMP-2 signaling, participates in the osteoblastic determination, differentiation, and transdifferentiation under p38 MAPK and JNK regulation.

Horm Behav. 2009 Sep 30;
Stolzenberg DS, Zhang KY, Luskin K, Ranker L, Bress J, Numan M

A body of evidence supports the idea that the mesolimbic dopamine (DA) system modulates the natural increase in responsiveness female rats show toward offspring (biological or foster) at birth. In the absence of the full hormonal changes associated with pregnancy and birth, female rats do not show immediate responsiveness toward foster offspring. Activation of the mesolimbic DA system can produce an immediate onset of maternal behavior in these females. For example, female rats that are hysterectomized and ovariectomized on day 15 of pregnancy (15HO) and presented with pups 48 hours later normally show maternal behavior after 2-3 days of pup exposure, but will show maternal behavior on day 0 of testing after microinjection of the DA D(1) receptor agonist, SKF 38393, into the nucleus accumbens (NA) at the time of pup presentation. DA D(1) receptor stimulation is known to activate cAMP intracellular signaling cascades via its stimulation of adenylyl cyclase (AC). However, some DA D(1) receptors are also linked to phospholipase C (PLC) and are capable of activating Phosphatidylinositol signaling cascades. SKF 38393 stimulates both types of D(1) receptors. Here we provide evidence that the facilitatory effects of DA D(1) receptor stimulation in the NA on maternal behavior are mediated by AC-linked DA D(1) receptors. By examining the effects of intra-NA application of SKF 83822, a drug which selectively binds DA D(1)-AC receptors, or SKF 83959, a drug which selectively activates D(1)-PLC-linked receptors, we find that only SKF 83822 facilitates maternal behavior onset.

J Cell Biol. 2009 Sep 21; 186(6): 773-82
Simonsen A, Tooze SA

Autophagy or "self-eating" is a highly conserved pathway that enables cells to degrade pieces of themselves in autolysosomes to enable their survival in times of stress, including nutrient deprivation. The formation of these degradative compartments requires cytosolic proteins, some of which are autophagy specific, as well as intracellular organelles, such as the ER and Golgi, and the endosome-lysosome system. Here we discuss the cross talk between autophagy and intracellular compartments, highlighting recent exciting data about the role and regulation of the Vps34 class III Phosphatidylinositol (PI) 3-kinase in autophagy.

J Immunol. 2009 Oct 15; 183(8): 5261-9
Ennaciri J, Girard D

Although Syk has been reported to be associated with IL-2R(alpha)and IL-15R(alpha) in some hematopoietic cells, its association has never been investigated in the IL-4/IL-4R system. In this study, we demonstrate for the first time that Syk is constitutively associated with IL-4R(alpha)in human polymorphonuclear neutrophils (PMNs) and that IL-4 stimulation increases the amount of Syk associated with IL-4R(alpha). Moreover, upon IL-4 treatment, a pool of Syk associated with IL-4R(alpha) is phosphorylated. We also report that such association is not unique to PMNs because Syk associates with IL-4R(alpha) in Raji and in PBMC cells. Stimulation of PMNs by IL-4 increased the amount of Syk associated with PLC-gamma2, pAkt, and alpha-tubulin. Pretreatment of cells with the Syk-selective inhibitor piceatannol or Syk inhibitor II, significantly inhibited the ability of IL-4 to enhance phagocytosis and cell adhesion and to delay apoptosis, and these results correlate with the ability of piceatannol to reduce Syk activation and its association with IL-4R(alpha). Down-regulation of Syk by antisense techniques demonstrates the importance of Syk in the antiapoptotic effect of IL-4. We conclude that association of Syk to IL-4R(alpha) is of biological significance and that IL-4R(alpha) is a new candidate to be added to the few cytokine receptor components which associate with Syk.

Ann N Y Acad Sci. 2009 Sep; 1173: 470-7
Raychaudhuri SK, Raychaudhuri SP

A contributing role of nerve growth factor (NGF)-mediated neuroimmunologic mechanisms has provided a new dimension in the understanding of various cutaneous and systemic inflammatory diseases. Recent evidence implicates NGF as a key mediator of inflammation and pain. NGF influences an inflammatory reaction by regulating neuropeptides, angiogenesis, cell trafficking molecules, and T cell activation. All of these functions of NGF are relevant in maintenance or initiation of the critical biologic events in various rheumatologic conditions. The recognition of a pathologic role of NGF and its receptor system has provided an attractive opportunity to develop a novel class of therapeutics for inflammatory diseases and chronic pain syndromes. In this chapter we will discuss the role of NGF and its receptor system in psoriatic disease, inflammatory arthritis, and arthritic pain of osteoarthritis.

Am J Physiol Cell Physiol. 2009 Nov; 297(5): C1146-56
Nagl F, Schönhofer K, Seidler B, Mages J, Allescher HD, Schmid RM, Schneider G, Saur D

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) acts as a neurotransmitter and intracellular signaling molecule in the central and peripheral nervous system. NO regulates multiple processes like neuronal development, plasticity, and differentiation and is a mediator of neurotoxicity. The nNOS gene is highly complex with 12 alternative first exons, exon 1a-1l, transcribed from distinct promoters, leading to nNOS variants with different 5'-untranslated regions. Transcriptional control of the nNOS gene is not understood in detail. To investigate regulation of nNOS gene expression by retinoic acid (RA), we used the human neuroblastoma cell line TGW-nu-I as a model system. We show that RA induces nNOS transcription in a protein synthesis-dependent fashion. We identify the Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the atypical orphan nuclear receptor DAX1 (NR0B1) as critical mediators involved in RA-induced nNOS gene transcription. RA treatment increases DAX1 expression via PI3K/Akt signaling. Upregulation of DAX1 expression in turn induces nNOS transcription in response to RA. These results identify nNOS as a target gene of a novel RA/PI3K/Akt/DAX1-dependent pathway in human neuroblastoma cells and stress the functional importance of the transcriptional regulator DAX1 for nNOS gene expression in response to RA treatment.

Neoplasia. 2009 Sep; 11(9): 882-8
Witte HT, Jeibmann A, Klämbt C, Paulus W

Glioblastoma is the most common and most malignant intrinsic human brain tumor, characterized by extensive invasion and proliferation of glial (astrocytic) tumor cells, frequent activation of tyrosine kinase receptor signaling pathways, relative resistance to chemotherapy and radiotherapy, and poor prognosis. Using the Gal4-UAS system, we have produced glioma models in Drosophila by overexpressing homologs of human tyrosine kinase receptors under control of the glia-specific promoter reversed polarity (repo). Glial overexpression of activated epidermal growth factor receptor (EGFR) resulted in enhanced proliferation and migration of larval glial cells with increased numbers in the eye imaginal disc, diffuse tumor-like enlargement of the optic stalk, and marked ectopic invasion of glial cells along the optic nerve. Glial overexpression of the downstream kinase PI3K showed similar pathology. Overexpression of activated pvr (platelet-derived growth factor receptor/vascular endothelial growth factor receptor homolog) led to migration of glial cells along the optic nerve, whereas expression of activated htl (fibroblast growth factor receptor 1 homolog) and INR (insulin receptor) showed markedly elevated numbers of glial cells in the optic stalk. The EGFR/Phosphatidylinositol 3-phosphate kinase (PI3K) phenotype was partly reverted by the administration of the EGFR tyrosine kinase inhibitor gefitinib and completely rescued by the PI3K inhibitor wortmannin and the Akt inhibitor triciribine. We suggest that Drosophila models will be useful for deciphering signaling cascades underlying abnormal behavior of glioma cells for genetic screens to reveal interacting genes involved in gliomagenesis and for experimental therapy approaches.

Cell Signal. 2009 Feb 5;
Namkoong S, Kim CK, Cho YL, Kim JH, Lee H, Ha KS, Choe J, Kim PH, Won MH, Kwon YG, Shim EB, Kim YM

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.

Commun Integr Biol. 2009 Mar; 2(2): 107-9
Urwyler S, Brombacher E, Hilbi H

The Gram-negative opportunistic pathogen Legionella pneumophila replicates in phagocytes within a specific compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is a complex process requiring the bacterial Icm/Dot type IV secretion system and more than 100 translocated effector proteins, which putatively subvert cellular signaling and vesicle trafficking pathways. Phosphoinositide (PI) glycerolipids are pivotal regulators of signal transduction and membrane dynamics in eukaryotes. Recently, a number of Icm/Dot substrates were found to anchor to the LCV membrane by binding to PIs. One of these effectors, SidC, specifically interacts with Phosphatidylinositol-4 phosphate [PtdIns(4)P]. Using an antibody against SidC and magnetic beads coupled to a secondary antibody, intact LCVs were purified by immuno-magnetic separation, followed by density centrifugation. This purification strategy is in principle applicable to any pathogen vacuole that carries specific markers. The LCV proteome determined by LC-MS/MS revealed 566 host proteins, including novel components of the endosomal pathway, as well as the early and late secretory trafficking pathways. Thus, LCV formation is a robust process that involves many (functionally redundant) Icm/Dot substrates, as well as the interaction with different host cell vesicle trafficking pathways.

Mol Cell Biol. 2009 Nov; 29(21): 5710-7
El-Naggar S, Liu Y, Dean DC

Atk can be activated by two independent phosphorylation events. Growth factor-dependent phosphorylation of threonine 308 (Akt-308) by Phosphatidylinositol 3-kinase-dependent PDK1 leads to activation of mammalian target of rapamycin (mTor) complex 1 (TORC1) and stimulation of protein synthesis. Phosphorylation on serine 473 (Akt-473) is catalyzed by mTor in a second complex (TORC2), and Akt-473 phosphorylates Foxo3a to inhibit apoptosis. Accumulation of both phosphorylated forms of Akt is frequent in cancer, and TORC2 activity is required for progression to prostate cancer with Pten mutation. Here, we link Akt-473 to the Rb1 pathway and show that mTor is overexpressed with loss of the Rb1 family pathway. This leads to constitutive Akt-473 and, in turn, phosphorylation of Foxo3a and resistance to cell adhesion-dependent apoptosis (anoikis). Additionally, Akt-473 accumulation blocks c-Raf activation, thereby preventing downstream Erk activation. This block cannot be overcome by constitutively active Ras, and it also prevents induction of the Arf tumor suppressor by Ras. These studies link inactivation of the Rb1 pathway, a hallmark of cancer, to accumulation of Akt-473, resistance to anoikis, and a block in c-Raf/Erk activation.

Free Radic Biol Med. 2009 Nov 15; 47(10): 1486-93
Archuleta TL, Lemieux AM, Saengsirisuwan V, Teachey MK, Lindborg KA, Kim JS, Henriksen EJ

Oxidative stress is characterized as an imbalance between the cellular production of oxidants and the cellular antioxidant defenses and contributes to the development of numerous cardiovascular and metabolic disorders, including hypertension and insulin resistance. The effects of prolonged oxidant stress in vitro on the insulin-dependent glucose transport system in mammalian skeletal muscle are not well understood. This study examined the in vitro effects of low-level oxidant stress (60-90 microM, H(2)O(2)) for 4 h on insulin-stimulated (5 mU/ml) glucose transport activity (2-deoxyglucose uptake) and on protein expression of critical insulin signaling factors (insulin receptor (IR), IR substrates IRS-1 and IRS-2, Phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3)) in isolated soleus muscle of lean Zucker rats. This oxidant stress exposure caused significant (50%, p<0.05) decreases in insulin-stimulated glucose transport activity that were associated with selective loss of IRS-1 (59%) and IRS-2 (33%) proteins, increased (64%) relative IRS-1 Ser(307) phosphorylation, and decreased phosphorylation of Akt Ser(473) (50%) and GSK-3beta Ser(9) (43%). Moreover, enhanced (37%) phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was observed. Selective inhibition of p38 MAPK (10 microM A304000) prevented a significant portion (29%) of the oxidant stress-induced loss of IRS-1 (but not IRS-2) protein and allowed partial recovery of the impaired insulin-stimulated glucose transport activity. These results indicate that in vitro oxidative stress in mammalian skeletal muscle leads to substantial insulin resistance of distal insulin signaling and glucose transport activity, associated with a selective loss of IRS-1 protein, in part due to a p38 MAPK-dependent mechanism.

Curr Biol. 2009 Sep 15; 19(17): 1463-6
Buus R, Faronato M, Hammond DE, Urbé S, Clague MJ

The scattering response of epithelial cells to activation of the Met receptor tyrosine kinase represents one facet of an "invasive growth" program [1, 2]. It is a complex event that incorporates loss of cell-cell adhesion, morphological changes, and cell motility. Ubiquitination is a reversible posttranslational modification that may target proteins for degradation or coordinate signal transduction pathways [3, 4]. There are approximately 79 active deubiquitinating enzymes (DUBs) predicted in the human genome [5, 6]. Here, via a small interfering RNA (siRNA) library approach, we have identified 12 DUBs that are necessary for aspects of the hepatocyte growth factor (HGF)-dependent scattering response of A549 cells. Different phenotypes are evident that range from full loss of scattering, similar to receptor knockdown (e.g., USP30, USP33, USP47), to loss of cell-cell contacts even in the absence of HGF but defective motility (e.g., USP3, ATXN3L). The knockdowns do not incur defective receptor, Phosphatidylinositol 3-kinase, or MAP kinase activation. Our data suggest widespread involvement of the ubiquitin system at multiple stages of the Met activation response, implying significant crosstalk with phosphorylation-based transduction pathways. Development of small-molecule inhibitors of particular DUBs may offer a therapeutic approach to contain metastasis.