Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity

Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation.

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Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex

Nature Cell Biology ◽

10.1038/ncb1854 ◽

2009 ◽

Vol 11 (4) ◽

pp. 468-476 ◽

Cited By ~ 585

Author(s):

Yun Zhong ◽

Qing Jun Wang ◽

Xianting Li ◽

Ying Yan ◽

Jonathan M. Backer ◽

...

Keyword(s):

Beclin 1 ◽

Phosphatidylinositol 3 Kinase ◽

Autophagic Activity ◽

Phosphatidylinositol 3

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Regulation of Hepatic Insulin-Like Growth Factor-Binding Protein-1 Gene Expression by Insulin: Central Role for Mammalian Target of Rapamycin Independent of Forkhead Box O Proteins

Endocrinology ◽

10.1210/en.2005-0902 ◽

2006 ◽

Vol 147 (5) ◽

pp. 2383-2391 ◽

Cited By ~ 16

Author(s):

Catherine Mounier ◽

Victor Dumas ◽

Barry I. Posner

Keyword(s):

Gene Expression ◽

Rat Liver ◽

Binding Protein ◽

Mammalian Target Of Rapamycin ◽

Pi3 Kinase ◽

Target Of Rapamycin ◽

Mrna Levels ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Inhibition ◽

Phosphatidylinositol 3

The expression of IGF-binding protein-1 (IGFBP-1) is induced in rat liver by dexamethasone and glucagon and is completely inhibited by 100 nm insulin. Various studies have implicated phosphatidylinositol 3-kinase, protein kinase B (Akt), phosphorylation of the transcription factors forkhead in rhabdomyosarcoma 1 (Foxo1)/Foxo3, and the mammalian target of rapamycin (mTOR) in insulin’s effect. In this study we examined insulin regulation of IGFBP-1 in both subconfluent and confluent hepatocytes. In subconfluent hepatocytes, insulin inhibition of IGFBP-1 mRNA levels was blocked by inhibiting PI3 kinase activation, and there was a corresponding inhibition of Foxo1/Foxo3 phosphorylation. In these same cells, inhibition of the insulin effect by rapamycin occurred in the presence of insulin-induced Foxo1/Foxo3 phosphorylation. In confluent hepatocytes, insulin could not activate the phosphatidylinositol 3-kinase (PI3 kinase)-Akt-Foxo1/Foxo3 pathway, but still inhibited IGFBP-1 gene expression in an mTOR-dependent manner. In subconfluent hepatocytes, the serine/threonine phosphatase inhibitor okadaic acid (100 nm) partially inhibited IGFBP-1 gene expression by 40%, but did not produce phosphorylation of either Akt or Foxo proteins. In contrast, 1 nm insulin inhibited the IGFBP-1 mRNA level by 40% and correspondingly activated Akt and Foxo1/Foxo3 phosphorylation to a level comparable to that observed with 100 nm insulin. These results suggest a potential role for a serine/threonine phosphatase(s) in the regulation of IGFBP-1 gene transcription, which is not downstream of mTOR and is independent of Akt. In conclusion, we have found that in rat liver, insulin inhibition of IGFBP-1 mRNA levels can occur in the absence of the phosphorylation of Foxo1/Foxo3, whereas activation of the mTOR pathway is both necessary and sufficient.

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c-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances Mitogenic and Survival Signals of Interleukin-4 Receptor by Linking With the Phosphatidylinositol 3′-Kinase Pathway

Blood ◽

10.1182/blood.v91.1.46.46_46_53 ◽

1998 ◽

Vol 91 (1) ◽

pp. 46-53 ◽

Cited By ~ 7

Author(s):

Hiroo Ueno ◽

Ko Sasaki ◽

Hiroaki Honda ◽

Tetsuya Nakamoto ◽

Tetsuya Yamagata ◽

...

Keyword(s):

Specific Inhibitor ◽

Pi3 Kinase ◽

Interleukin 4 ◽

Phosphatidylinositol 3 Kinase ◽

Proliferation And Differentiation ◽

Interleukin 4 Receptor ◽

Pi3 Kinase Pathway ◽

Kinase Pathway ◽

Phosphatidylinositol 3 ◽

Survival Signals

Interleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3′ (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.

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Both Src-Dependent and -Independent Mechanisms Mediate Phosphatidylinositol 3-Kinase Regulation of Colony-Stimulating Factor 1-Activated Mitogen-Activated Protein Kinases in Myeloid Progenitors

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6779-6798.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6779-6798 ◽

Cited By ~ 57

Author(s):

Angel W.-M. Lee ◽

David J. States

Keyword(s):

Kinase Inhibitors ◽

Pi3 Kinase ◽

Dominant Negative ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Phosphatidylinositol 3 Kinase ◽

Stimulating Factor ◽

In Cells ◽

Phosphatidylinositol 3 ◽

Myeloid Progenitors

ABSTRACT Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (ΔKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or ΔKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing ΔKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or ΔKI receptors. However, only in ΔKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.

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Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.4131 ◽

1998 ◽

Vol 18 (7) ◽

pp. 4131-4140 ◽

Cited By ~ 154

Author(s):

Christopher D. Kontos ◽

Thomas P. Stauffer ◽

Wen-Pin Yang ◽

John D. York ◽

Liwen Huang ◽

...

Keyword(s):

Fluorescent Protein ◽

Vascular Development ◽

Pi3 Kinase ◽

Pleckstrin Homology Domain ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Embryonic Vascular Development ◽

Phosphatidylinositol 3

ABSTRACT Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

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Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0080 ◽

2008 ◽

Vol 19 (12) ◽

pp. 5360-5372 ◽

Cited By ~ 715

Author(s):

Eisuke Itakura ◽

Chieko Kishi ◽

Kinji Inoue ◽

Noboru Mizushima

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Coiled Coil ◽

Pi3 Kinase ◽

Beclin 1 ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Interacting Protein ◽

Vacuolar Protein ◽

Phosphatidylinositol 3

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.

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Induction of Monocyte Chemotactic Protein 1 in Colonic Epithelial Cells by Entamoeba histolytica Is Mediated via the Phosphatidylinositol 3-Kinase/p65 Pathway

Infection and Immunity ◽

10.1128/iai.01442-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1765-1770 ◽

Cited By ~ 16

Author(s):

Srinivas J. Kammanadiminti ◽

Indranil Dey ◽

Kris Chadee

Keyword(s):

Epithelial Cells ◽

Entamoeba Histolytica ◽

Pi3 Kinase ◽

Classical Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Colonic Epithelial Cells ◽

Monocyte Chemotactic Protein ◽

Mrna Induction ◽

Chemotactic Protein ◽

Phosphatidylinositol 3

ABSTRACT The role intestinal epithelial cells play in the pathogenesis of amebic colitis is poorly understood. Herein, we demonstrate that secreted and soluble ameba (Entamoeba histolytica) proteins (SAP) induce expression of the chemoattractant monocyte chemotactic protein (MCP) in the colonic epithelial cell lines Caco-2, T84, and LS174T. MCP-1 mRNA induction was both dose and time dependent, with peak induction occurring at 8 h and with 100 μg/ml of SAP. Significant increase in MCP-1 protein expression was observed after 12 h. SAP failed to activate any of the mitogen-activated protein kinase pathways or IκB kinase activity. Moreover, inhibiting the classical pathway of NF-κB activation did not affect SAP-induced MCP-1 expression. Instead, we find that SAP-induced MCP-1 expression is dependent on posttranslational modification of the NFκB p65 subunit. SAP induced phosphorylation of p65 and enhanced NF-κB transcriptional activity, which are phosphatidylinositol 3-kinase (PI3 kinase) dependent. Treatment with PI3 kinase inhibitor LY290004 significantly abrogated the activation of Akt, p65, and MCP-1 mRNA induction. We conclude that colonic epithelial cells play a role in the initiation of inflammation by secreting chemokines in response to soluble ameba components.

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Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6661 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6661-6666 ◽

Cited By ~ 44

Author(s):

A J Muslin ◽

A Klippel ◽

L T Williams

Keyword(s):

Oocyte Maturation ◽

Kinase Activity ◽

Sh2 Domain ◽

Pi3 Kinase ◽

Meiotic Maturation ◽

Xenopus Laevis Oocytes ◽

Phosphatidylinositol 3 Kinase ◽

Kinase Activation ◽

Phosphatidylinositol 3

In somatic cells, phosphatidylinositol 3-kinase (PI3 kinase) is a critical intermediary in growth factor-induced mitogenesis. We have examined the role of this enzyme in meiotic maturation of Xenopus laevis oocytes. PI3 kinase activity was present in immunoprecipitates of the p85 subunit of PI3 kinase from immature oocytes and markedly increased following progesterone stimulation. Injection of bacterially expressed protein corresponding to the C-terminal SH2 domain of p85 (SH2-C) inhibited progesterone-induced PI3 kinase activation and meiotic maturation. Injection of protein corresponding to the N-terminal SH2 domain or the SH3 domain of p85 did not inhibit PI3 kinase activation or maturation. SH2-C did not inhibit oocyte maturation induced by c-mos RNA injection. In addition, radiolabelled SH2-C was used to probe oocyte lysates, revealing that a novel 200-kDa protein bound to SH2-C. This protein may be an important mediator of progesterone-induced lipid metabolism in oocytes.

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Insulin can block apoptosis by decreasing oxidative stress via phosphatidylinositol 3-kinase- and extracellular signal-regulated protein kinase-dependent signaling pathways in HepG2 cells

Acta Endocrinologica ◽

10.1530/eje.0.1480147 ◽

2003 ◽

pp. 147-155 ◽

Cited By ~ 52

Author(s):

S Kang ◽

J Song ◽

H Kang ◽

S Kim ◽

Y Lee ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Kinase ◽

Signaling Pathways ◽

Hepg2 Cells ◽

Caspase 3 ◽

Pi3 Kinase ◽

Phosphatidylinositol 3 Kinase ◽

Extracellular Signal ◽

Apoptotic Activity ◽

Phosphatidylinositol 3

OBJECTIVE: Insulin has well-known activities in controlling energy metabolism, cellular proliferation and biosynthesis of functional molecules to maintain a biological homeostasis. Recently, several studies have suggested that insulin may protect cells from apoptosis in different cell lines; however, little is known about the nature of its anti-apoptotic activity. In many clinical disorders, including type 2 diabetes mellitus, oxidative stress and the production of reactive oxygen species (ROS) is increased. With these facts as a background, we examined here whether insulin protects HepG2 cells from apoptosis by decreasing oxidative stress and, if so, which signaling steps are involved in this process. METHODS: Intracellular DNA content, the degree of nuclear condensation or poly(ADP-ribose) polymerase hydrolysis was measured to verify the occurrence of apoptotic events. Caspase-3 activity and ROS accumulation within cells were also measured. Western blot analysis was performed to identify signaling molecules activated in response to insulin. RESULTS: Serum starvation resulted in a marked accumulation of ROS, activation of caspase-3, and subsequent apoptotic cell death which were, in turn, markedly blocked by the addition of insulin. The anti-apoptotic activity of insulin was sensitive to blockade of two different signaling steps, activations of phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated protein kinase (ERK). CONCLUSION: Insulin exerts an anti-apoptotic activity by suppressing the excessive accumulation of ROS within cells through signaling pathways including stimulation of PI3 kinase and ERK in HepG2 cells.

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