The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

Buckles, Thomas; Ohashi, Yohei; Tremel, Shirley; McLaughlin, Stephen H.; Pardon, Els; Steyaert, Jan; Gordon, Moshe T.; Williams, Roger L.; Falke, Joseph J.

doi:10.1016/j.bpj.2020.10.028

Cited by 16 publications

(16 citation statements)

References 68 publications

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“…5d, e ). This change in conformation of complex II after associating with Rab5a on membranes is consistent with the recent, elegant study of VPS34 complex II by single-molecule kinetics that showed that Rab5a coupled to membranes both increases the density of complex II on membranes and increases the specific activity of complex II on Rab5a membranes relative to membranes without Rab5 54 . While it is clear that a significant component of the increase in activity of complex I on Rab1a-coupled membranes and complex II on Rab5a-coupled membranes is due to enhanced recruitment, the conformational changes in VPS34 seen in the cryo-ET suggest that activation involves multiple steps.…”

Section: Discussionsupporting

confidence: 90%

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

et al. 2021

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The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

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Section: Discussionsupporting

confidence: 90%

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

et al. 2021

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“…Here we have described how HDX-MS can be utilized to rapidly identify epitopes for nanobodies and stabilizing conformational changes induced upon binding. HDX-MS is a well-established technique for efficiently defining antibody-binding sites for biopharmaceuticals (Berkowitz et al, 2012) and has been used to define nanobody-binding sites (Buckles et al, 2020;Rostislavleva et al, 2015). The identification of the NB1-PIK3R5 nanobody, which stabilized the dynamic C terminus of the p101 subunit, allowed us to obtain a high-resolution map of the p110g-p101 complex by cryo-EM, which is described in depth in another article.…”

Section: Discussionmentioning

confidence: 99%

HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

et al. 2021

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There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kg, is a heterodimeric complex composed of a catalytic p110g subunit bound to a p101 or p84 regulatory subunit. PI3Kg is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G proteincoupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kg binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kg regulation and identifies sites that may be exploited for therapeutic development.

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“…Here we have described how HDX-MS can be utilized to rapidly identify epitopes for nanobodies and stabilizing conformational changes induced upon binding. HDX-MS is a well-established technique for efficiently defining antibody binding sites for biopharmaceuticals (Berkowitz et al, 2012), and has been used to define nanobody binding sites (Buckles et al, 2020; Rostislavleva et al, 2015). The identification of the NB1-PIK3R5 nanobody, which stabilized the dynamic C-terminus of the p101 subunit, allowed us to obtain a high resolution map of the p110 γ -p101 complex by cryo-EM, which is described in depth in another manuscript.…”

Section: Discussionmentioning

confidence: 99%

HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

Rathinaswamy

Fleming

Dalwadi

et al. 2021

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There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and GPCRs to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3K single chain camelid nanobodies using hydrogen deuterium exchange mass spectrometry (HDX-MS) for structural and biochemical studies. This allowed us to identify nanobodies that stimulated lipid kinase activity, blocked Ras activation and specifically inhibited p101-mediated GPCR activation. Overall, this reveals novel insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

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The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism

Cited by 16 publications

References 68 publications

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases

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