Biology subjects

Bourguet, M.

Publications and source records attributed to Bourguet, M..

3 recordsLinked to original sources

bioRxiv2026

Structural basis for a phosphoinositide-driven mTORC2-AKT positive feedback loop

The mammalian target of rapamycin complex 2 (mTORC2) regulates metabolism, growth, survival and cytoskeletal organization, yet its activation mechanism is poorly understood. We show that mTORC2 is directly activated by membranes and our cryo-electron tomography structure of membrane-bound mTORC2 reveals the N-HEAT region of mTOR is at the major membrane interface. mTORC2 is further potently activated by a positive feedback loop involving reciprocal phosphorylation of mTORC2 and its substrate kinase AKT. Cryo-EM structures of dephosphorylated, autophosphorylated and AKT-phosphorylated mTORC2 reveal structural changes in the SIN1 subunit, regulating an autoinhibitory anchor. Reconstitution of the PDK1-AKT-mTORC2 hub on PIP3-containing membranes shows that PDK1/PIP3-dependent AKT activation drives SIN1-T86 phosphorylation, enabling mTORC2 to phosphorylate S473 of AKTs hydrophobic motif, establishing a PI3K-dependent, phosphorylation-driven positive feedback loop at the membrane.

biochemistry↗

bioRxiv2025

A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution

RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII, by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the Saccharomyces cerevisiae sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in S. cerevisiae VPS15 resulted in defective CPY sorting, loss of colocalization with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.

molecular biology↗

bioRxiv2025

Pervanadate-induced oxidation relieves autoinhibition of SRC protein tyrosine kinase

Dynamic regulation of protein tyrosine phosphorylation (pTyr) by phosphatases (PTPs) and kinases enables cells to sense and respond to environmental changes. The widely used chemical probe Pervanadate (PV) induces accumulation of high levels of pTyr in cells, an effect primarily attributed to its properties as a PTP inhibitor. This led to the assertion that PTPs are the master gatekeeper of intracellular pTyr homeostasis. Here, we use diverse approaches to reveal that PV disrupts cellular redox homeostasis and directly activates SRC family tyrosine kinases via oxidation of specific cysteine residues. Using mass spectrometry and biophysical approaches, we show that oxidation activates SRC by disrupting autoinhibition and altering phosphopeptide binding by its SH2 domain. We further establish that redox-sensitive cysteine residues are essential for SRC to promote cellular overgrowth. Our findings call for a re-evaluation of PV-based experiments and provide compelling evidence that oxidation is a crucial mechanism in controlling the oncogenic properties of SRC.

molecular biology↗