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.

scholarly journals PtdIns(4,5)P2 signaling regulates ATG14 and autophagy

2016 ◽  
Vol 113 (39) ◽  
pp. 10896-10901 ◽  
Author(s):  
Xiaojun Tan ◽  
Narendra Thapa ◽  
Yihan Liao ◽  
Suyong Choi ◽  
Richard A. Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Functional Significance ◽  
Mammalian Cells ◽  
Beclin 1 ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Atg Proteins ◽  
Late Endosomes ◽  
Irradiation Resistance ◽  
Phosphatidylinositol 3

Autophagy is a regulated self-digestion pathway with fundamental roles in cell homeostasis and diseases. Autophagy is regulated by coordinated actions of a series of autophagy-related (ATG) proteins. The Barkor/ATG14(L)–VPS34 (a class III phosphatidylinositol 3-kinase) complex and its product phosphatidylinositol 3-phosphate [PtdIns(3)P] play key roles in autophagy initiation. ATG14 contains a C-terminal Barkor/ATG14(L) autophagosome-targeting sequence (BATS) domain that senses the curvature of PtdIns(3)P-containing membrane. The BATS domain also strongly binds PtdIns(4,5)P2, but the functional significance has been unclear. Here we show that ATG14 specifically interacts with type Iγ PIP kinase isoform 5 (PIPKIγi5), an enzyme that generates PtdIns(4,5)P2 in mammalian cells. Autophagosomes have associated PIPKIγi5 and PtdIns(4,5)P2 that are colocalized with late endosomes and the endoplasmic reticulum. PtdIns(4,5)P2 generation at these sites requires PIPKIγi5. Loss of PIPKIγi5 results in a loss of ATG14, UV irradiation resistance-associated gene, and Beclin 1 and a block of autophagy. PtdIns(4,5)P2 binding to the ATG14–BATS domain regulates ATG14 interaction with VPS34 and Beclin 1, and thus plays a key role in ATG14 complex assembly and autophagy initiation. This study identifies an unexpected role for PtdIns(4,5)P2 signaling in the regulation of ATG14 complex and autophagy.

scholarly journals Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L

2010 ◽  
Vol 190 (4) ◽  
pp. 511-521 ◽  
Author(s):  
Kohichi Matsunaga ◽  
Eiji Morita ◽  
Tatsuya Saitoh ◽  
Shizuo Akira ◽  
Nicholas T. Ktistakis ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Pi3 Kinase ◽  
Class Iii ◽  
Autophagosome Formation ◽  
Membranous Structure ◽  
Er Targeting ◽  
Endoplasmic Reticulum Targeting ◽  
Phosphatidylinositol 3

Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

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.

scholarly journals Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1

2011 ◽  
Vol 441 (1) ◽  
pp. 399-406 ◽  
Author(s):  
Harald W. Platta ◽  
Hilde Abrahamsen ◽  
Sigrid B. Thoresen ◽  
Harald Stenmark
Keyword(s):  
Tumour Suppressor ◽  
Beclin 1 ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Interacting Protein ◽  
Polyubiquitin Chains ◽  
Protein Levels ◽  
Regulatory Link ◽  
A Subunit ◽  
The Stability

Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.

scholarly journals hVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells

2009 ◽  
Vol 417 (3) ◽  
pp. 747-755 ◽  
Author(s):  
Ying Yan ◽  
Rory J. Flinn ◽  
Haiyan Wu ◽  
Rachel S. Schnur ◽  
Jonathan M. Backer
Keyword(s):  
Mammalian Cells ◽  
Specific Activity ◽  
Gene Activation ◽  
Nutrient Sensing ◽  
Beclin 1 ◽  
Class Iii ◽  
Acetoxymethyl Ester ◽  
Phosphoinositide 3 Kinase ◽  
Vacuolar Protein

The mammalian Class III PI3K (phosphoinositide 3-kinase), hVps34 [mammalian Vps (vacuolar protein sorting) 34 homologue], is an important regulator of vesicular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with a putative serine/threonine protein kinase, Vps15, which is required for Vps34p activity. The mammalian homologue of Vps15p, hVps15 (formerly called p150), also binds to hVps34, but its role in hVps34 signalling has not been evaluated. In the present study we have therefore compared the activity and regulation of hVps34 expressed without or with hVps15. We find that hVps34 has low specific activity when expressed alone; co-expression with hVps15 leads to a marked increase in activity. Notably, beclin-1/UVRAG (UV radiation resistance-associated gene) activation of hVps34 requires co-expression with hVps15; this may be explained by the observation that beclin-1/UVRAG expression increases hVps34/hVps15 binding. Regulation of hVps34 activity by nutrients also requires co-expression with hVps15. Finally, given a recent report that hVps34 activity requires Ca2+/CaM (calmodulin), we considered whether hVps15 might be involved in this regulation. Although hVps34 does bind CaM, we find its activity is not affected by treatment of cells with BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] or W7. Removal of CaM by EDTA or EGTA washes has no effect on hVps34 activity, and hVps34 activity in vitro is unaffected by Ca2+ chelation. The results of the present study show that, in mammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM.

The class III phosphatidylinositol 3-kinase Vps34 in Saccharomyces cerevisiae

2017 ◽  
Vol 398 (5-6) ◽  
pp. 677-685 ◽  
Author(s):  
Christina Reidick ◽  
Fahd Boutouja ◽  
Harald W. Platta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Role ◽  
Protein Sorting ◽  
Endocytic Trafficking ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

Abstract The class III phosphatidylinositol 3-kinase Vps34 (vacuolar protein sorting 34) catalyzes for the formation of the signaling lipid phosphatidylinositol-3-phopsphate, which is a central factor in the regulation of autophagy, endocytic trafficking and vesicular transport. In this article, we discuss the functional role of the lipid kinase Vps34 in Saccharomyces cerevisiae.

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