| 1993 |
Yeast VPS34 encodes a phosphatidylinositol 3-kinase (PI3K) required for vacuolar protein sorting; vps34 deletion strains lack detectable PI3K activity and exhibit severe vacuolar protein sorting defects, and overexpression of Vps34p increases PI3K activity specifically precipitated with anti-Vps34p antisera. |
Gene deletion, overexpression, in vitro PI3K assay, immunoprecipitation |
Science |
High |
8385367
|
| 1990 |
VPS34 protein (875 aa, ~95 kDa) lacks signal sequence or transmembrane domains; it is found partly in a particulate fraction solubilized by urea but not Triton X-100, indicating membrane association via protein–protein interactions; Vps34p-null cells show defects in vacuolar protein sorting and vacuole segregation to daughter cells. |
Gene cloning/sequencing, immunoprecipitation, cell fractionation, fluorescence microscopy of vacuoles |
Molecular and cellular biology |
High |
2247081
|
| 2001 |
Two distinct yeast Vps34 PI3K complexes exist: Complex I (Vps15p/Vps30p/Apg14p/Vps34p) required for autophagy, and Complex II (Vps15p/Vps30p/Vps38p/Vps34p) required for CPY vacuolar protein sorting; Vps30p functions as a shared subunit of both. |
Co-immunoprecipitation, pull-down, mass spectrometry identification of subunits, phenotypic analysis of deletion mutants |
The Journal of cell biology |
High |
11157979
|
| 2005 |
hVps34 is a nutrient-regulated lipid kinase required for activation of S6K1 and mTOR signaling; hVps34 is inhibited by amino acid or glucose starvation and by AMPK activation; it acts upstream of mTOR, as hVps34 knockdown inhibits phosphorylation of both S6K1 and 4EBP1. |
siRNA knockdown, anti-hVps34 antibody microinjection, FYVE-domain PI3P sequestration, overexpression, kinase activity assays |
The Journal of biological chemistry |
High |
16049009
|
| 2006 |
Beclin 1 co-immunoprecipitates with hVps34 in glioblastoma cells; siRNA depletion of Beclin 1 specifically blunts the autophagic response to nutrient deprivation or ceramide without affecting EGF receptor post-endocytic sorting, cathepsin D TGN-to-lysosome trafficking, or early endosomal EEA1 association, demonstrating that Beclin 1 selectively directs hVps34 into the autophagic rather than general trafficking pathway. |
Co-immunoprecipitation, siRNA knockdown, autophagy assays, EGFR degradation assay, fluid-phase endocytosis |
Journal of cell science |
High |
16390869
|
| 2006 |
hVps34 siRNA knockdown causes accumulation of enlarged LAMP1-positive late endosomes depleted of PI(3)P, impairs inward vesiculation of multivesicular bodies, slows cathepsin D maturation, and delays EGFR degradation, but does not block early endocytic uptake or TGN-to-late-endosome cathepsin D traffic, identifying hVps34 as specifically required for PI(3)P generation in late endosomes/MVBs. |
siRNA knockdown, electron microscopy, GFP-2×FYVE PI(3)P probe, EGFR degradation assay, cathepsin D processing assay |
Journal of cell science |
High |
16522686
|
| 2006 |
Gpa1 (Gα subunit) is present at endosomes and directly interacts with both Vps34 and Vps15 to stimulate PI3P production; Vps15 resembles a Gβ subunit (seven-WD repeat) and binds GDP-Gpa1, revealing a preformed effector–Gβ-like assembly at the endosome. |
Genetic epistasis screen (~5000 deletion strains), direct protein interaction assay, PI3P production measurement at endosomes |
Cell |
High |
16839886
|
| 2008 |
Amino acids trigger a rise in intracellular Ca2+ that activates hVps34 via direct binding of Ca2+/calmodulin (CaM) to an evolutionarily conserved motif in hVps34; this CaM binding is required for hVps34 lipid kinase activity and for subsequent mTOR Complex 1 activation. |
Ca2+ imaging, direct CaM-binding assay, lipid kinase activity assay, CaM-binding motif mutagenesis |
Cell metabolism |
High |
18460336
|
| 2009 |
hVps15 is required for hVps34 lipid kinase activity in mammalian cells; co-expression with hVps15 markedly increases hVps34 activity, and Beclin 1/UVRAG activation of hVps34 requires hVps15 co-expression; hVps34 activity in cells is independent of Ca2+/CaM (chelation of Ca2+ or CaM has no effect on hVps34 activity in vitro or in cells). |
In vitro lipid kinase assay, co-expression, BAPTA/AM treatment, EDTA/EGTA wash, W7 CaM inhibitor |
The Biochemical journal |
High |
18957027
|
| 2003 |
hVPS34 and its adaptor p150 colocalize with Rab7 on late endosomes; hVPS34 PI3K activity is dependent on nucleotide cycling of Rab7; total cellular PI3P levels are modulated by Rab7 expression, identifying Rab7 as a regulator of late endosomal hVPS34 function. |
Co-immunoprecipitation, colocalization imaging, PI3K activity assay, Rab7 nucleotide-state mutants |
Traffic |
Medium |
14617358
|
| 2002 |
Rab5-GTP promotes endosomal localization of hVps34/p150; the p150 HEAT and WD40 domains are required for Rab5 binding; p150 is required for EEA1 endosomal targeting (recombinant p150 fragments displace EEA1); however, Rab5 does not significantly recruit hVps34/p150 from cytosol to membrane, indicating Rab5 regulates localization rather than membrane recruitment. |
Dominant-active Rab5 expression, recombinant fragment competition, subcellular fractionation, immunofluorescence colocalization |
Traffic |
Medium |
12010460
|
| 2003 |
M. tuberculosis virulence toxin lipoarabinomannan (LAM) blocks phagosome maturation by inhibiting a Ca2+/calmodulin–hVPS34 cascade; Ca2+ and calmodulin are required for hVPS34-mediated PI3P production on phagosomes in vivo, and LAM from virulent (but not avirulent) mycobacteria blocks cytosolic Ca2+ rise to prevent PI3P generation. |
In vitro PI3P production assay on liposomes, in vivo phagosomal PI3P imaging, Ca2+ chelation, calmodulin inhibition |
The Journal of experimental medicine |
Medium |
12925680
|
| 2013 |
ULK1 phosphorylates Beclin 1 at Ser14 following amino acid starvation or mTOR inhibition, enhancing the lipid kinase activity of the ATG14L-containing VPS34 complex; this phosphorylation is required for full autophagic induction in mammals and C. elegans. |
In vitro kinase assay, site-directed mutagenesis (S14A), VPS34 complex immunoprecipitation and lipid kinase assay, C. elegans genetic rescue |
Nature cell biology |
High |
23685627
|
| 2013 |
MTORC1 directly phosphorylates ATG14 to inhibit the ATG14-containing PIK3C3 complex specifically; MTORC1 inactivation by nutrient starvation selectively activates the ATG14-containing (autophagy-specific) PIK3C3 complex without affecting UVRAG-containing PIK3C3 complexes. |
In vitro kinase assay (MTORC1 phosphorylation of ATG14), lipid kinase assay of immunopurified complexes, starvation/rapamycin treatment |
Autophagy |
High |
24013218
|
| 2013 |
AMPK directly phosphorylates PIK3C3/VPS34 and BECN1 to differentially regulate PIK3C3 complexes in response to energy starvation: AMPK inhibits non-autophagic PIK3C3 complexes while activating pro-autophagic (ATG14-containing) complexes via Beclin 1 phosphorylation. |
In vitro kinase assay, complex-specific lipid kinase assay, phosphorylation site mapping |
Autophagy |
Medium |
23669030
|
| 2013 |
Acetylated Hsp70 binds the Beclin 1–Vps34 complex upon autophagy-inducing stress and recruits the E3 SUMO ligase KAP1, which SUMOylates Vps34 at Lys840, increasing Vps34 lipid kinase activity; Hsp70 knockdown abolishes Beclin 1–Vps34 complex formation and prevents autophagosome formation. |
Co-immunoprecipitation, Vps34 SUMO modification assay, lipid kinase assay, siRNA knockdown, autophagosome formation assay in Hsp70 KO MEFs |
Proceedings of the National Academy of Sciences of the United States of America |
Medium |
23569248
|
| 2014 |
VPS34-IN1 (25 nM IC50 in vitro) is a highly selective Vps34 inhibitor; its administration rapidly disperses PI(3)P from endosomal membranes within 1 minute; Vps34-produced PI(3)P at endosomes controls SGK3 activation by enabling PDK1-site and hydrophobic-motif phosphorylation via SGK3's PX domain; a second pool of PI(3)P from class I PI3K→PI(3,4,5)P3→PI(3)P conversion also contributes to SGK3 activity. |
In vitro kinase selectivity panel (340 protein kinases, 25 lipid kinases), PI(3)P probe imaging, SGK3 phosphorylation assays, PX-domain mutation |
The Biochemical journal |
High |
25177796
|
| 2014 |
SAR405 (KD 1.5 nM) selectively inhibits Vps34 kinase activity; its unique binding mode within the ATP-binding cleft explains selectivity; Vps34 inhibition by SAR405 disrupts late endosome–lysosome compartments and prevents autophagy. |
Biophysical binding assay (KD measurement), crystal structure of inhibitor-Vps34 complex, cell-based autophagy and vesicle trafficking assays |
Nature chemical biology |
High |
25326666
|
| 2015 |
Crystal structure of the 385-kDa endosomal Vps34 complex II (PIK3C3-CII: Vps34/Vps15/Beclin1/UVRAG) at 4.4 Å reveals a Y-shaped assembly centered on the Vps34 C2 domain; Vps15 kinase domain engages the Vps34 activation loop to regulate its activity; HDX-MS identifies a Vps30/Beclin1 loop critical for complex II activity on giant liposomes but not for complex I. |
X-ray crystallography (4.4 Å), hydrogen-deuterium exchange mass spectrometry, liposome-based lipid kinase assay |
Science |
High |
26450213
|
| 2015 |
mTOR directly phosphorylates UVRAG at Ser550 and Ser571 to activate the VPS34-UVRAG complex; this activation generates a lysosomal PI(3)P pool required for autolysosomal tubulation and lysosome reformation (ALR); loss of these phosphorylation sites reduces VPS34 lipid kinase activity and causes massive cell death due to impaired ALR. |
In vitro mTOR kinase assay, phosphomutant UVRAG constructs, VPS34 lipid kinase assay, lysosomal tubulation imaging, cell survival assays |
The EMBO journal |
High |
26139536
|
| 2017 |
VPS34 is specifically acetylated by p300 at K771 (reducing affinity for PI substrate) and K29 (hindering VPS34–Beclin 1 complex formation); p300 inhibition induces VPS34 deacetylation and autophagy even in AMPK−/−, TSC2−/−, or ULK1−/− cells, establishing p300-dependent acetylation as a direct control of VPS34 activity independent of upstream kinases. |
Acetyltransferase assay, acetylation site mutagenesis, in vitro lipid kinase assay, autophagy induction in triple-knockout MEFs, liver fasting model |
Molecular cell |
High |
28844862
|
| 2017 |
EM and crosslinking mass spectrometry reveal five conformational substates of PI3KC3-C1; in one substate the VPS34 catalytic domain is dislodged from the complex while remaining tethered by an intrinsically disordered linker; a 'leashed' construct that prevents dislodging blocks enzyme activity in vitro and autophagy induction in yeast, identifying catalytic-domain dislodging as an allosteric switch regulated by VPS15. |
Electron microscopy, crosslinking mass spectrometry, in vitro lipid kinase assay, yeast genetic autophagy assay |
Molecular cell |
High |
28757208
|
| 2021 |
Cryo-electron tomography of complex II on Rab5a-GTP-decorated vesicles shows Rab5a-GTP recruits and activates complex II by binding between the VPS34 C2 domain and VPS15 WD40 domain, releasing the VPS34 kinase domain from VPS15-mediated inhibition into a catalysis-competent position; Rab1a specifically recruits and activates autophagy complex I (not complex II) via the same VPS34 interface but in a distinct manner. |
Electron cryotomography, hydrogen-deuterium exchange mass spectrometry, Rab GTPase mutant binding assays, in vitro lipid kinase assay on vesicles |
Nature communications |
High |
33692360
|
| 2021 |
ULK1/2 phosphorylates VPS15 at six sites including the major site Ser861; mutation of these sites reduces autophagosome formation in cells and VPS34 lipid kinase activity in vitro, establishing VPS15 as a ULK substrate that links ULK activity to VPS34 complex regulation. |
Phosphoproteomics in Ulk1/2 DKO MEFs, in vitro VPS34 lipid kinase assay with phosphomutant VPS15, autophagosome formation assays |
The EMBO journal |
High |
34121209
|
| 2010 |
The Rubicon RUN domain directly interacts with the hVps34 catalytic subunit and contributes to efficient inhibition of PI3KC3 lipid kinase activity; a RUN domain deletion mutant fails to rescue autophagy deficiency in Rubicon-depleted cells. |
Co-immunoprecipitation, in vitro PI3K lipid kinase assay, deletion mutagenesis, siRNA complementation assay |
The Journal of biological chemistry |
Medium |
21062745
|
| 2007 |
The myotubularin PI3-phosphatase MTM1 directly binds the hVPS15/hVPS34 complex via the WD40 domain of hVPS15; overexpression of catalytically active (but not dead) MTM1 depletes endosomal PI(3)P; the hVPS15/hVPS34 complex forms mutually exclusive complexes with Rab5, Rab7, or MTM1, suggesting Rab GTPases and MTM1 act as molecular switches controlling PI(3)P synthesis and degradation. |
Co-immunoprecipitation, PI(3)P level measurement, catalytic-dead mutant controls, domain-mapping pulldown |
Traffic |
Medium |
17651088
|
| 2013 |
Conditional deletion of Pik3c3 in differentiated sensory neurons causes rapid neurodegeneration; large-diameter myelinated neurons accumulate enlarged vacuoles and ubiquitin aggregates while small-diameter neurons activate a non-canonical PIK3C3-independent LC3-positive autophagosome pathway still dependent on ATG7; Pik3c3/Atg7 double-mutant analysis shows the unconventional pathway requires ATG7. |
Conditional knockout mice (Cre-lox), electron microscopy, immunohistochemistry, Atg7 conditional KO comparison, double-mutant analysis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
20439739
|
| 2011 |
Conditional deletion of Vps34 in T lymphocytes severely reduces T cell numbers; Vps34-deficient T cells show increased death and reduced IL-7Rα surface expression despite intact autophagy; intracellular analysis shows mislocalization of EEA1, HRS, and Vps36, preventing IL-7Rα retromer-pathway recycling to the cell surface. |
Conditional KO mice, flow cytometry, intracellular trafficking assays, endosomal marker localization |
Journal of immunology |
Medium |
22021616
|
| 2014 |
PIK3C3 generates PI(3)P that recruits ankyrin-B (AnkB) via PtdIns(3)P binding; AnkB bridges the dynactin subunit p62 and PI(3)P-enriched organelle membranes to promote dynein-mediated fast axonal transport of synaptic vesicles, mitochondria, endosomes, and lysosomes; loss of PIK3C3 or AnkB impairs retrograde organelle transport and shortens axon tracts. |
AnkB knockout, PIK3C3 loss-of-function, dynactin membrane association assay, live-cell organelle transport imaging in hippocampal neurons, 3D-STORM |
The Journal of cell biology |
Medium |
25533844
|
| 2015 |
FBXL20, an F-box protein whose expression is induced by p53-dependent transcription after DNA damage, ubiquitinates Vps34 (via SCF-Skp1-Cullin1 complex) for proteasomal degradation; CDK-mediated phosphorylation of Vps34 provides the signal for FBXL20-mediated ubiquitination, leading to inhibition of autophagy and receptor endocytosis. |
Ubiquitination assay, proteasome inhibitor treatment, CDK phosphorylation assay, FBXL20 overexpression/knockdown, autophagy and receptor endocytosis assays |
Genes & development |
Medium |
25593308
|
| 2016 |
Vps34 PI(3)P on late endosomes recruits the Rab7-GAP Armus (TBC1D2) via Armus's PH domain binding to PI(3)P; in Vps34-deficient cells, Armus fails to localize to late endosomes, causing Rab7-GTP accumulation, enlarged late endosomes, impaired intraluminal vesicle formation, and defective EGFR degradation; Rab7 silencing or Armus overexpression rescues vacuolization. |
Vps34−/− MEFs, protein-lipid overlay and liposome-binding assays, Rab7-GTP pull-down, EGFR degradation assay, rescue experiments |
Journal of cell science |
High |
27793976
|
| 2017 |
VPS34 promotes K63-linked ubiquitination of VPS34 by UBC-13/UEV-1/CHN-1 in C. elegans (ortholog), which stabilizes VPS34 protein; loss of this ubiquitination reduces VPS34 levels and impairs phagosome PI(3)P generation and maturation; UBE3C/TRABID reciprocally regulate K29/K48-branched ubiquitination of VPS34 targeting it to proteasomal degradation in mammals. |
In vitro ubiquitination assay, C. elegans genetics, VPS34 protein level measurement, phagosome maturation assay |
The Journal of cell biology / Nature communications |
Medium |
29092895 33637724
|
| 2018 |
ULK1 O-GlcNAcylation at Thr754 (by OGT, following PP1-mediated dephosphorylation of adjacent mTOR site Ser757 and AMPK phosphorylation) is required for ULK1 to bind and phosphorylate ATG14L, which in turn activates VPS34 lipid kinase activity for PI(3)P production and phagophore formation. |
O-GlcNAc modification mapping, ULK1 phosphomutants, ATG14L binding assay, VPS34 lipid kinase assay, autophagy flux assays |
Cell reports |
Medium |
30517873
|
| 2020 |
Membrane physicochemical properties (degree of lipid unsaturation, negative charge, and curvature) strongly modulate VPS34 complex activity; the BATS domain of ATG14L makes autophagy complex I more active than endocytic complex II on membranes; the Beclin1 BARA domain membrane-interacting loops are critical for complex II but have minor roles for complex I. |
In vitro lipid kinase assays with defined liposome compositions, HDX-MS of complexes on membranes |
eLife |
High |
32602837
|
| 2021 |
AHCYL1 senses intracellular S-adenosyl-L-homocysteine (SAH); SAH binding to the AHCYL1 C-terminus promotes binding of the AHCYL1 N-terminus to the PIK3C3 catalytic domain, inhibiting PIK3C3 and suppressing autophagy in an mTORC1-independent manner. |
Co-immunoprecipitation, domain-mapping pulldown, SAH-binding assay, PIK3C3 kinase activity assay, autophagy flux assay, in vivo validation |
Autophagy |
Medium |
33993848
|
| 2023 |
ULK1 phosphorylates LDHA at Ser196 under nutrient deprivation, increasing lactate production; lactate then lactylates Vps34 at Lys356 and Lys781 (mediated by acetyltransferase KAT5/TIP60); Vps34 lactylation enhances its association with Beclin1, Atg14L, and UVRAG and increases Vps34 lipid kinase activity to promote autophagy and endolysosomal trafficking. |
Lactylation site mapping, KAT5 acetyltransferase assay, co-immunoprecipitation of Vps34 complexes, VPS34 lipid kinase assay, autophagy flux assay |
Science advances |
Medium |
37267363
|
| 2014 |
NRBF2 is a specific subunit of Vps34 Complex I (with Vps34, Vps15, Beclin-1, ATG14L) but not Complex II; NRBF2 directly interacts with the Vps15 WD40 domain; NRBF2 knockdown inhibits starvation-induced autophagosome formation (GFP-LC3 puncta, LC3-II lipidation) and increases p62, establishing NRBF2 as a positive regulator of autophagy within Complex I. |
Co-immunoprecipitation, direct binding assay, siRNA knockdown, autophagy flux assays |
The Biochemical journal |
Medium |
24785657
|
| 2016 |
Atg38/NRBF2 uses its MIT domain to bridge Atg14 and Vps30 coiled-coil I regions within Complex I; the Atg38 C-terminal domain mediates homodimerization (2.2 Å crystal structure) and phagophore assembly site localization; one Atg38 homodimer engages a single Complex I, whereas human NRBF2 homodimer can bridge two Complex I assemblies. |
HDX-MS, X-ray crystallography (2.2 Å), electron microscopy, yeast genetics |
Autophagy |
High |
27630019
|
| 2016 |
Vps34-generated PI(3)P recruits Armus (Rab7-GAP) and is required for Rab7 inactivation during late endosome maturation; separately, C. elegans VPS-34 recruits TBC-2 (Rab5-GAP) via PI(3)P binding to inactivate RAB-5 and ensure directionality of endosome maturation. |
Vps34 KO MEFs, Rab7-GTP assay, C. elegans VPS-34 genetics, TBC-2 endosome localization, PH domain PI(3)P binding |
Journal of cell science |
Medium |
28455411
|
| 2022 |
VPS34 generates PI(3)P that serves as substrate for PIKfyve to produce PI(3,5)P2; this VPS34→PIKfyve phosphoinositide cascade positively regulates the Retriever/WASH/CCC recycling pathway; PIKfyve inhibition displaces Retriever and CCC from endosomes; VPS34-dependent PI(3)P is required for initial SNX17 recruitment in this recycling pathway. |
VPS34 and PIKfyve inhibitors, endogenous colocalization, PI(3)P/PI(3,5)P2 assays, cargo recycling assays (integrin surface levels) |
eLife |
Medium |
35040777
|
| 2011 |
Pik3c3 null embryos are lethal between E7.5 and E8.5 with failure of mesoderm formation and severely reduced cell proliferation; mTOR signaling is drastically reduced in null embryos, suggesting PIK3C3-dependent mTOR activation is a major contributor to early embryonic cell proliferation. |
Pik3c3 null mouse generation, embryo morphology, BrdU proliferation assay, mTOR signaling western blot, blastocyst culture |
PloS one |
Medium |
21283715
|
| 2024 |
The pro-oxidant menadione sodium bisulfite (MSB) inhibits VPS34 lipid kinase activity through oxidation of key cysteine residues, disrupting endosome identity and sorting; in a myotubular myopathy (MTM1-loss) model, dietary MSB improved muscle histology, function, and extended lifespan, consistent with VPS34 being the pathogenic kinase when its phosphatase antagonist MTM1 is absent. |
VPS34 kinase activity assay under oxidative conditions, cysteine oxidation mapping, MTM1-deficient mouse dietary treatment, muscle histology and functional assays |
Science |
Medium |
39446948
|
| 2015 |
Disruption of the Beclin 1–ATG14L protein–protein interaction (required for VPS34 Complex I formation and localization but not Complex II) selectively inhibits autophagy without disrupting Beclin 1–UVRAG interaction or vesicle trafficking, demonstrating that Complex I and Complex II have separable functions accessible through their unique subunit interfaces. |
NanoBRET cellular PPI assay, VPS34 Complex I/II immunoprecipitation, autophagy assays, transferrin recycling assay |
Journal of the American Chemical Society |
Medium |
32320221
|