• ErbB3/HER3
• KSR1
• SHP2
• cancer
• dimerization
• immunodeficiency
• inflammatory diseases
• protein complexes
• pseudokinase

• Humans
• Animals
• Signal Transduction

• HU794/10-2, BR3662/4-1 Deutsche Forschungsgemeinschaft

• EP3 receptor
• GRK2
• PGE2
• PI3Kγ
• antinociception
• pain
• sensory neuron

• PI3K
• apoptosis
• macrophage
• regeneration
• zebrafish

The heterotrimeric G protein family plays essential roles during a varied array of cellular events; thus, its deregulation can seriously alter signaling events and the overall state of the cell. Heterotrimeric G-proteins have three subunits (α, β, γ) and are subdivided into four families, Gαi, Gα12/13, Gαq, and Gαs. These proteins cycle between an inactive Gα-GDP state and active Gα-GTP state, triggered canonically by the G-protein coupled receptor (GPCR) and by other accessory proteins receptors independent also known as AGS (Activators of G-protein Signaling). In this review, we summarize research data specific for the Gαi family. This family has the largest number of individual members, including Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, and Gαz, and constitutes the majority of G proteins α subunits expressed in a tissue or cell. Gαi was initially described by its inhibitory function on adenylyl cyclase activity, decreasing cAMP levels. Interestingly, today Gi family G-protein have been reported to be importantly involved in the immune system function. Here, we discuss the impact of Gαi on non-canonical effector proteins, such as c-Src, ERK1/2, phospholipase-C (PLC), and proteins from the Rho GTPase family members, all of them essential signaling pathways regulating a wide range of physiological processes.

• Fondo Nacional de Ciencia y Tecnología

• cancer
• cell migration
• endocytosis
• myopathy
• nutrient signaling
• phosphatidylinositol 3-kinase
• vesicle traffic

• G-proteins
• Gβγ
• PI3K (phosphatidylinositide 3-kinase)
• Ras
• class I PI3-kinases
• p110γ
• p87 (p84)

• Analogs
• Apoptosis
• CD36
• Carriers
• Chaperones
• Gene expression
• Lipid exchange
• Lipid transport proteins
• SR-BI
• Signal transduction
• Vitamin E
• “Mitocan”

• Humans
• Solubility
• Vitamin E Deficiency/drug therapy
• Vitamins/chemistry
• Vitamins/pharmacology
• alpha-Tocopherol/analogs & derivatives
• alpha-Tocopherol/chemistry
• alpha-Tocopherol/pharmacology

• Association
• Attention-deficit/hyperactivity disorder
• Genetic susceptibility
• Gene–environment interaction
• PIK3CG gene

Classically, Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in ERK activation following G-protein–coupled receptor (GPCR) activation. Here we show that PI3Kγ noncanonically regulates ERK phosphorylation in a kinase-independent mechanism, irrespective of the upstream signals. PI3Kγ sequesters PP2A, allowing sustained ERK function.

Classically Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in extracellular signal–regulated kinase (ERK) activation following G-protein coupled receptor (GPCR) activation. Knock-down of PI3Kγ unexpectedly resulted in loss of ERK activation to receptor tyrosine kinase agonists such as epidermal growth factor or insulin. Mouse embryonic fibroblasts (MEFs) or primary adult cardiac fibroblasts isolated from PI3Kγ knock-out mice (PI3KγKO) showed decreased insulin-stimulated ERK activation. However, expression of kinase-dead PI3Kγ resulted in rescue of insulin-stimulated ERK activation. Mechanistically, PI3Kγ sequesters protein phosphatase 2A (PP2A), disrupting ERK–PP2A interaction, as evidenced by increased ERK–PP2A interaction and associated PP2A activity in PI3KγKO MEFs, resulting in decreased ERK activation. Furthermore, β-blocker carvedilol-mediated β-arrestin-dependent ERK activation is significantly reduced in PI3KγKO MEF, suggesting accelerated dephosphorylation. Thus, instead of classically mediating the kinase arm, PI3Kγ inhibits PP2A by scaffolding and sequestering, playing a key parallel synergistic step in sustaining the function of ERK, a nodal enzyme in multiple cellular processes.

• CD36
• EPC-K1
• SEC14L2
• TOCOPHEROL
• TOCOPHERYL PHOSPHATE
• VITAMIN E
• cAMP

• Ascorbic Acid/analogs & derivatives
• Ascorbic Acid/pharmacology
• CD36 Antigens/metabolism
• Cell Line, Tumor
• Class Ib Phosphatidylinositol 3-Kinase/metabolism
• HEK293 Cells
• Humans
• Macrophages/drug effects
• Macrophages/metabolism
• Macrophages/microbiology
• Monocytes/drug effects
• Monocytes/metabolism
• Monocytes/microbiology
• Phagocytosis/drug effects
• Signal Transduction/drug effects
• Staphylococcus aureus/physiology
• THP-1 Cells/drug effects
• THP-1 Cells/metabolism
• Vascular Endothelial Growth Factor A/metabolism
• Vitamin E/analogs & derivatives
• Vitamin E/pharmacology
• alpha-Tocopherol/analogs & derivatives
• alpha-Tocopherol/pharmacology

• Adrenergic receptor
• Epidermal growth factor receptor
• Phosphatidylinositide 3-kinase
• Phosphorylation
• Src

• CREB
• catecholamine
• mouse model
• phosphodiesterases (PDEs)
• stereotactic surgery

• Calcium
• Cell Migration
• Extracellular Signal-regulated Kinase (ERK)
• NCX1
• Na+/K+-ATPase
• Sodium-Calcium Exchange

A large body of experimental and clinical data supports the notion that inflammation in acute pancreatitis has a crucial role in the pathogenesis of local and systemic damage and is a major determinant of clinical severity. Thus, research has recently focused on molecules that can regulate the inflammatory processes, such as phosphoinositide 3-kinases (PI3Ks), a family of lipid and protein kinases involved in intracellular signal transduction. Studies using genetic ablation or pharmacologic inhibitors of different PI3K isoforms, in particular the class I PI3Kδ and PI3Kγ, have contributed to a greater understanding of the roles of these kinases in the modulation of inflammatory and immune responses. Recent data suggest that PI3Ks are also involved in the pathogenesis of acute pancreatitis. Activation of the PI3K signaling pathway, and in particular of the class IB PI3Kγ isoform, has a significant role in those events which are necessary for the initiation of acute pancreatic injury, namely calcium signaling alteration, trypsinogen activation, and nuclear factor-κB transcription. Moreover, PI3Kγ is instrumental in modulating acinar cell apoptosis, and regulating local neutrophil infiltration and systemic inflammatory responses during the course of experimental acute pancreatitis. The availability of PI3K inhibitors selective for specific isoforms may provide new valuable therapeutic strategies to improve the clinical course of this disease. This article presents a brief summary of PI3K structure and function, and highlights recent advances that implicate PI3Ks in the pathogenesis of acute pancreatitis.

• Phosphoinositide 3-kinase
• Cell signaling
• Inflammation
• Pathogenesis
• Acute pancreatitis

• Acute Disease
• Animals
• Anti-Inflammatory Agents/therapeutic use
• Humans
• Isoenzymes
• Molecular Targeted Therapy
• Pancreas/drug effects
• Pancreas/enzymology
• Pancreas/pathology
• Pancreatitis/diagnosis
• Pancreatitis/drug therapy
• Pancreatitis/enzymology
• Phosphatidylinositol 3-Kinase/chemistry
• Phosphatidylinositol 3-Kinase/metabolism
• Phosphoinositide-3 Kinase Inhibitors
• Protein Conformation
• Protein Kinase Inhibitors/therapeutic use
• Signal Transduction
• Structure-Activity Relationship

Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KIIα in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KIIα with the ‘integral’ membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KIIα to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KIIα’s activity. We conclude from our results that PI4KIIα’s activity is regulated indirectly through changes in the membrane environment.

Type II PI4-kinase dysfunction is associated with diseases including cancer and Alzheimer's disease; however, the development of specific modulators has been hampered by a lack of structural information. Zhou et al. present the crystal structure of PI4KIIα in its ADP-bound form, providing insight into its regulation.

Mammals have genes coding for three PI5P4Ks (PtdIns5P 4-kinases), and these have different cellular localizations, tissue distributions and lipid kinase activities. We describe in the present paper a detailed molecular exploration of human PI5P4Ks α, β and γ, as well as their fly and worm homologues, to understand how and why these differences came to be. The intrinsic ATPase activities of the three isoforms are very similar, and we show that differences in their G-loop regions can account for much of their wide differences in lipid kinase activity. We have also undertaken an extensive in silico evolutionary study of the PI5P4K family, and show experimentally that the single PI5P4K homologues from Caenorhabditis elegans and Drosophila melanogaster are as widely different in activity as the most divergent mammalian isoforms. Finally we show that the close association of PI5P4Ks α and γ is a true heterodimerization, and not a higher oligomer association of homodimers. We reveal that structural modelling is consistent with this and with the apparently random heterodimerization that we had earlier observed between PI5P4Kα and PI5P4Kβ [Wang, Bond, Letcher, Richardson, Lilley, Irvine and Clarke (2010), Biochem. J. 430, 215–221]. Overall the molecular diversity of mammalian PI5P4Ks explains much of their properties and behaviour, but their physiological functionality remains elusive.

• Animals
• Carcinogenesis/genetics
• Cations, Divalent/metabolism
• Class Ia Phosphatidylinositol 3-Kinase/genetics
• Class Ia Phosphatidylinositol 3-Kinase/metabolism
• Enzyme Inhibitors/pharmacology
• Humans
• Isoenzymes/antagonists & inhibitors
• Isoenzymes/genetics
• Isoenzymes/metabolism
• Mutation
• Phosphoinositide-3 Kinase Inhibitors
• Phosphorylation/genetics
• Sf9 Cells
• Spodoptera
• Up-Regulation/genetics

• Dictyostelium
• Macropinocytosis
• PI3-kinase signalling
• PtdIns(3,4,5)P3
• Ras

• Animals
• Cell Communication/physiology
• Gene Expression Regulation/physiology
• Molecular Structure
• Phosphatidylinositols/chemistry
• Phosphatidylinositols/metabolism

• Intramural NIH HHS

The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

Hepatitis C virus (HCV) infections affect about 170 million people worldwide and often result in severe chronic liver disease. HCV is a positive-strand RNA virus inducing massive rearrangements of intracellular membranes to generate the sites of genome replication, designated the membranous web. The complex biogenesis of the membranous web is still poorly understood, but requires the concerted action of several viral nonstructural proteins and cellular factors. Recently, we and others identified the lipid kinase phosphatidylinositol-4 kinase III alpha (PI4KIIIα), catalyzing the synthesis of phosphatidylinositol 4-phosphate (PI4P), as an essential host factor involved in the formation of the membranous web. In this study, we characterized the virus-host interaction in greater detail using a genetic approach. We identified a highly conserved region in the viral phosphoprotein NS5A crucial for the interaction with PI4KIIIα. Surprisingly, we found that PI4KIIIα, despite being a lipid kinase, appeared to regulate the phosphorylation status of NS5A, thus contributing to viral replication. Our results furthermore suggest that the morphology of the membranous web is regulated by NS5A phosphorylation, providing novel insights into the complex regulation of viral RNA replication.

The protein kinase activity of PI3K phosphorylates specific serine residues in growth factor receptors to promote cell survival; these events are constitutively activated in some leukemias.

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.

The ability of cells to survive in the absence of proliferation (cell division), differentiation (cell maturation) or activation allows tissues to maintain cell populations that are poised for rapid responses to damage, infections, or other physiological demands. While this “survival-only” response is fundamental to all physiological processes, the underlying mechanisms are not understood. Many growth factors are potent regulators of cell survival through their ability to bind specific cell surface receptors, which in turn activate specialized enzymes called kinases. Phosphoinositide 3-kinase (PI3K) is a dual specificity kinase that is known to be involved in cell survival and malignant transformation, and it is able to phosphorylate both lipid and protein substrates. While the PI3K lipid kinase activity has been extensively studied, the functional significance of its protein kinase activity remains unclear. Here we show that PI3K protein kinase activity can directly phosphorylate growth factor receptors on human hematopoietic (blood) cells to promote a “survival-only” response. We further show that the protein kinase activity of PI3K can be hijacked to result in uncontrolled growth factor receptor phosphorylation and the deregulated survival of leukemic cells. Our studies provide the first evidence that the protein kinase activity of PI3K can control cell survival and that this activity may be deregulated in cancer.

• chromatin remodeling
• inositol phosphate
• nucleus
• phosphatidylinositol
• phosphoinositide
• plant
• signaling

• Autoimmunity/drug effects
• Autoimmunity/genetics
• Cell Transformation, Neoplastic/genetics
• Cell Transformation, Neoplastic/metabolism
• Cell Transformation, Neoplastic/pathology
• Diglycerides/metabolism
• Enzyme Inhibitors/pharmacology
• Eukaryotic Cells/enzymology
• Eukaryotic Cells/pathology
• Gene Expression Regulation, Neoplastic
• Humans
• Hypersensitivity/drug therapy
• Hypersensitivity/enzymology
• Hypersensitivity/genetics
• Hypersensitivity/pathology
• Inositol 1,4,5-Trisphosphate/metabolism
• Isoenzymes/antagonists & inhibitors
• Isoenzymes/classification
• Isoenzymes/genetics
• Isoenzymes/metabolism
• Neoplasms/drug therapy
• Neoplasms/enzymology
• Neoplasms/genetics
• Neoplasms/pathology
• Phosphatidylinositol 3-Kinases/classification
• Phosphatidylinositol 3-Kinases/genetics
• Phosphatidylinositol 3-Kinases/metabolism
• Phosphatidylinositol 4,5-Diphosphate/metabolism
• Phosphoinositide-3 Kinase Inhibitors
• Second Messenger Systems
• TOR Serine-Threonine Kinases/genetics
• TOR Serine-Threonine Kinases/metabolism

• Animals
• Evolution, Molecular
• Humans
• Phosphatidylinositol Phosphates/metabolism
• Phosphotransferases (Alcohol Group Acceptor)/classification
• Phosphotransferases (Alcohol Group Acceptor)/genetics
• Phosphotransferases (Alcohol Group Acceptor)/metabolism

• Wellcome Trust