scholarly journals pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels

2017 ◽  
Vol 217 (1) ◽  
pp. 329-346 ◽  
Author(s):  
Amriya Naufer ◽  
Victoria E.B. Hipolito ◽  
Suriakarthiga Ganesan ◽  
Akriti Prashar ◽  
Vanina Zaremberg ◽  
...  
Keyword(s):  
Filamentous Bacteria ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Cellular Processes ◽  
Ph Dependent ◽  
Cellular Pathways ◽  
Luminal Ph ◽  
Phagocytic Cups ◽  
Phosphatidylinositol 3

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3)P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes.

scholarly journals Class II phosphatidylinositol 3-kinase isoforms in vesicular trafficking

2021 ◽  
Author(s):  
Kazuaki Yoshioka
Keyword(s):  
Class Ii ◽  
Vesicular Trafficking ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Cellular Processes ◽  
Endosomal Signaling ◽  
Lipid Kinases ◽  
Reported Data ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinases (PI3Ks) are critical regulators of many cellular processes including cell survival, proliferation, migration, cytoskeletal reorganization, and intracellular vesicular trafficking. They are a family of lipid kinases that phosphorylate membrane phosphoinositide lipids at the 3′ position of their inositol rings, and in mammals they are divided into three classes. The role of the class III PI3K Vps34 is well-established, but recent evidence suggests the physiological significance of class II PI3K isoforms in vesicular trafficking. This review focuses on the recently discovered functions of the distinct PI3K-C2α and PI3K-C2β class II PI3K isoforms in clathrin-mediated endocytosis and consequent endosomal signaling, and discusses recently reported data on class II PI3K isoforms in different physiological contexts in comparison with class I and III isoforms.

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

2008 ◽  
Vol 19 (12) ◽  
pp. 5360-5372 ◽  
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.

Immunocytochemical Detection of Phosphatidylinositol 3-kinase Activation by Insulin and Leptin

2003 ◽  
Vol 51 (3) ◽  
pp. 275-283 ◽  
Author(s):  
Kevin D. Niswender ◽  
Byron Gallis ◽  
James E. Blevins ◽  
Marshall A. Corson ◽  
Michael W. Schwartz ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Serine Phosphorylation ◽  
Phosphatidylinositol 3 Kinase ◽  
Aortic Endothelial Cells ◽  
Downstream Signaling ◽  
Cellular Processes ◽  
Pi3k Activity ◽  
Pi3k Activation ◽  
Hepatic Portal ◽  
Phosphatidylinositol 3

Intracellular signaling mediated by phosphatidylinositol 3-kinase (PI3K) is important for a number of cellular processes and is stimulated by a variety of hormones, including insulin and leptin. A histochemical method for assessment of PI3K signaling would be an important advance in identifying specific cells in histologically complex organs that are regulated by growth factors and peptide hormones. However, current methods for detecting PI3K activity require either homogenization of the tissue or cells or the ability to transfect probes that bind to phosphatidylinositol 3,4,5 trisphosphate (PIP3), the reaction product of PI3K catalysis. Here we report the validation of an immunocytochemical method to detect changes in PI3K activity, using a recently developed monoclonal antibody to PIP3, in paraformaldehyde-fixed bovine aortic endothelial cells (BAECs) in culture and in hepatocytes of intact rat liver. Treatment with either insulin or leptin increased BAEC PIP3 immunoreactivity, and these effects were blocked by pretreatment with PI3K inhibitors. Furthermore, infusion of insulin into the hepatic portal vein of fasted rats caused an increase of PIP3 immunostaining in hepatocytes that was associated with increased serine phosphorylation of the downstream signaling molecule protein kinase B/Akt (PKB/Akt). We conclude that immunocytochemical PIP3 staining can detect changes in PI3K activation induced by insulin and leptin in cell culture and intact liver.

scholarly journals Ca2+-regulated Pool of Phosphatidylinositol-3-phosphate Produced by Phosphatidylinositol 3-Kinase C2α on Neurosecretory Vesicles

2008 ◽  
Vol 19 (12) ◽  
pp. 5593-5603 ◽  
Author(s):  
Peter J. Wen ◽  
Shona L. Osborne ◽  
Isabel C. Morrow ◽  
Robert G. Parton ◽  
Jan Domin ◽  
...  
Keyword(s):  
Critical Role ◽  
Neurosecretory Cells ◽  
Secretory Vesicles ◽  
Endosomal Trafficking ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Product ◽  
Phosphatidylinositol 3

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2α and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2α knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2α knockdown and expression of PI3K-C2α catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca2+. We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2α is indeed regulated by Ca2+. We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2α occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca2+ in vitro and in living neurosecretory cells.

scholarly journals Differential Association of Phosphatidylinositol 3-Kinase, SHIP-1, and PTEN with Forming Phagosomes

2007 ◽  
Vol 18 (7) ◽  
pp. 2463-2472 ◽  
Author(s):  
Lynn A. Kamen ◽  
Jonathan Levinsohn ◽  
Joel A. Swanson
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Leading Edge ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Inositol Phosphatase ◽  
Lipid Phosphatases ◽  
Phagocytic Cups ◽  
Src Homology 2 Domain ◽  
Phosphatidylinositol 3

In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] regulate Fcγ receptor-mediated phagocytosis. Inhibition of phosphatidylinositol 3-kinase (PI3K) or overexpression of the lipid phosphatases phosphatase and tensin homologue (PTEN) and Src homology 2 domain-containing inositol phosphatase (SHIP-1), which hydrolyze PI(3,4,5)P3 to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], respectively, inhibit phagocytosis in macrophages. To examine how these enzymes regulate phagosome formation, the distributions of yellow fluorescent protein (YFP) chimeras of enzymes and pleckstrin homology (PH) domains specific for their substrates and products were analyzed quantitatively. PTEN-YFP did not localize to phagosomes, suggesting that PTEN regulates phagocytosis globally within the macrophage. SHIP1-YFP and p85-YFP were recruited to forming phagosomes. SHIP1-YFP sequestered to the leading edge and dissociated from phagocytic cups earlier than did p85-cyan fluorescent protein, indicating that SHIP-1 inhibitory activities are restricted to the early stages of phagocytosis. PH domain chimeras indicated that early during phagocytosis, PI(3,4,5)P3 was slightly more abundant than PI(3,4)P2 at the leading edge of the forming cup. These results support a model in which phagosomal PI3K generates PI(3,4,5)P3 necessary for later stages of phagocytosis, PTEN determines whether those late stages can occur, and SHIP-1 regulates when and where they occur by transiently suppressing PI(3,4,5)P3-dependent activities necessary for completion of phagocytosis.

Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage

2011 ◽  
Vol 75 (5) ◽  
pp. 940-950 ◽  
Author(s):  
Myung Rae Park ◽  
Mukesh Kumar Gupta ◽  
Hye Ran Lee ◽  
Ziban Chandra Das ◽  
Sang Jun Uhm ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Meiotic Progression ◽  
Phosphatidylinositol 3

scholarly journals The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase.

1995 ◽  
Vol 15 (8) ◽  
pp. 4453-4465 ◽  
Author(s):  
S Pons ◽  
T Asano ◽  
E Glasheen ◽  
M Miralpeix ◽  
Y Zhang ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Regulatory Subunit ◽  
Stable Complex ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Cellular Processes ◽  
Src Homology 2 ◽  
Src Homology ◽  
And Function ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.

scholarly journals Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

2016 ◽  
Vol 113 (29) ◽  
pp. 8224-8229 ◽  
Author(s):  
Lindsey N. Young ◽  
Kelvin Cho ◽  
Rosalie Lawrence ◽  
Roberto Zoncu ◽  
James H. Hurley
Keyword(s):  
Complex I ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Negative Stain ◽  
Binding Factor ◽  
Hydrogen Deuterium ◽  
Negative Stain Electron Microscopy ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen–deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.

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