Cryo-EM structure and biochemical analysis reveal the basis of the functional difference between human PI3KC3-C1 and -C2

Ma, Mingyu; Liu, Jun Jie; Li, Yan; Huang, Yuwei; Ta, Na; Chen, Yang; Fu, Hua; Ye, Ming-Da; Ding, Yue-He; Huang, Weijiao; Wang, Jia; Dong, Meng‐Qiu; Yu, Li; Wang, Hongwei

doi:10.1038/cr.2017.94

Cited by 46 publications

(53 citation statements)

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“…Recent studies of the class III PI3K complexes have highlighted the importance of their regulatory and accessory partners in membrane anchoring (Ma et al, 2017;Rostislavleva et al, 2015;Tan et al, 2016). The absence of known regulatory partners for PI3K-C2a and other class II family members, however, has limited our understanding of how this class of PI3Ks are recruited to the membrane, although it has been assumed that their unique PX-C2 modules will play an important role in this process (Campa et al, 2015;Falasca and Maffucci, 2007;Foster et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…The class III PI3K Vps34 is also localized via its accessory subunits as part of a multi-subunit complex including Vps15, Beclin-1, and ATG14L (Simonsen and Tooze, 2009). Recent structures of Vps34 complexes further revealed that membrane anchoring occurs via two arms of the complex between Beclin-1, ATG14L, and the kinase domain of Vps34 (Ma et al, 2017;Rostislavleva et al, 2015). In contrast, PI3K-C2a and other class II PI3Ks are generally considered to function as isolated enzymes, lacking known regulatory or accessory subunits to promote membrane targeting (Campa et al, 2015;Falasca and Maffucci, 2007;Foster et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α

et al. 2018

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Phosphorylation of phosphoinositides by the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2a is essential for many processes, including neuroexocytosis and formation of clathrin-coated vesicles. A defining feature of the class II PI3Ks is a C-terminal module composed of phox-homology (PX) and C2 membrane interacting domains; however, the mechanisms that control their specific cellular localization remain poorly understood. Here we report the crystal structure of the C2 domain of PI3K-C2a in complex with the phosphoinositide head-group mimic inositol hexaphosphate, revealing two distinct pockets for membrane binding. The C2 domain preferentially binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol (3,4,5)trisphosphate, and low-resolution structures of the combined PX-C2 module by small-angle X-ray scattering reveal a compact conformation in which cooperative lipid binding by each domain binding can occur. Finally, we demonstrate an unexpected role for calcium in perturbing the membrane interactions of the PX-C2 module, which we speculate may be important for regulating the activity of PI3K-C2a.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α

et al. 2018

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“…Cryo-EM Structure of the BECN1-MIT-Fusion Complex. Previous EM analyses of PI3KC3s have been limited by the dynamic character of these complexes and by their tendency to be in preferred orientations on EM grids (11,17,25,26). Even when preferred orientations can be reduced or eliminated, the dynamics of the catalytic right arm of the complex (27) has still limited analysis (14).…”

Section: Characterization Of Pi3kc3-c1 Containing a Nrbf2-mit-becn1 Fmentioning

confidence: 99%

Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I

Young

Goerdeler

Hurley

2019

Proc. Natl. Acad. Sci. U.S.A.

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Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

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“…Previous EM analyses of PI3KC3s have been limited by the dynamic character of these complexes and by their tendency to be in preferred orientations on EM grids (11,17,25,26).…”

Section: Cryo-em Structure Of the Becn1-mit-fusion Complexmentioning

confidence: 99%

Structural Pathway for Allosteric Activation of the Autophagic PI 3-Kinase Complex I

Young

Goerdeler

Hurley

2019

Preprint

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Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the pro-autophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryo-EM was used to visualize stepwise PI3KC3-C1 activating effects of binding the NRFB2 MIT domains. Binding of a single NRBF2 MIT domain to bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.Autophagy is a core cellular process, conserved throughout eukaryotes, which is the central recycling system for the removal of misfolded proteins, damaged organelles, and the recycling of nutrients in starvation. Autophagic dysfunction is implicated in many disease states, including neurodegeneration, immune disorders, cancer, and aging, among others (1). The class III phosphatidylinositol-3 kinase complexes (PI3KC3) I and II (PI3KC3-C1 and -C2), respectively, are essential for the initiation and expansion of autophagosomes (2-5). PI3KC3 generates the lipid phosphatidylinositol-3-phosphate, PI(3)P, which is recognized by the WIPI proteins. WIPIs in turn recruit the machinery that conjugates the autophagosomal marker LC3 to the expanding autophagosomal membrane (6). PI3KC3-C1 has been proposed to be a promising therapeutic target for autophagy activators (7) because the generation of PI(3)P is absolutely required for the recruitment of downstream autophagy proteins. There is considerable medical interest in selectively activating this pathway to promote human health and treat disease, yet there are no FDA approved pharmaceuticals that uniquely activate autophagy.PI3KC3-C1 consists of the lipid kinase VPS34, the putative serine/threonine protein kinase VPS15, the regulatory subunit BECN1, and the early autophagy-specific targeting subunit ATG14 (8,9). In PI3KC3-C2, ATG14 is replaced with UVRAG (10), while the other three subunits are preserved. The overall architecture of both PI3KC3-C1 and -C2 has the shape of the letter V (11, 12) (Fig. 1A). The long coiled-coils of BECN1 and ATG14 scaffold the left arm in the standard view, with the membrane binding BARA domain of BECN1 located at the outermost tip of the arm. PI3KC3-C2 is inhibited by Rubicon and the HIV-1 protein Nef (13-15), which regulate membrane docking by the tip of the left arm. The catalytic domains of the kinases VPS34 and VPS15 are at the tip of the right arm (11,12). PI3KC3 complexes are phosphoregulated by the Unc-51 like autophagy-activating kinase 1 (ULK1) complex (16),

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Cryo-EM structure and biochemical analysis reveal the basis of the functional difference between human PI3KC3-C1 and -C2

Cited by 46 publications

References 30 publications

Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α

Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α

Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I

Structural Pathway for Allosteric Activation of the Autophagic PI 3-Kinase Complex I

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