Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas

Abstract

Class IA PI3Ks are involved in the generation of the key lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP₃), and inappropriate activation of this pathway is implicated in a multitude of human diseases, including cancer, inflammation, and primary immunodeficiencies. Class IA PI3Ks are activated downstream of the Ras superfamily of GTPases, and Ras-PI3K interaction plays a key role in promoting tumor formation and maintenance in Ras-driven tumors. Investigating the detailed molecular events in the Ras-PI3K interaction has been challenging because it occurs on a membrane surface. Here, using maleimide-functionalized lipid vesicles, we successfully generated membrane-resident HRas and evaluated its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen-deuterium exchange MS. We screened all class IA PI3K isoforms and found that HRas activates both p110α and p110δ isoforms but does not activate p110β. The p110α and p110δ activation by Ras was synergistic with activation by a soluble phosphopeptide derived from receptor tyrosine kinases. Hydrogen-deuterium exchange MS revealed that membrane-resident HRas, but not soluble HRas, enhances conformational changes associated with membrane binding by increasing membrane recruitment of both p110α and p110δ. Together, these results afford detailed molecular insight into the Ras-PI3K signaling complex, provide a framework for screening Ras inhibitors, and shed light on the isoform specificity of Ras-PI3K interactions in a native membrane context.

Keywords: Ras protein; hydrogen exchange mass spectrometry; lipid signaling; lipid–protein interaction; phosphatidylinositide 3-kinase (PI 3-kinase); phosphoinositide.

Figure 1.

Schematic of protein constructs and generation of HRas-coupled membranes. A, domain architecture of class IA. Class IA PI3Ks are obligate heterodimers consisting of both a catalytic (p110) and regulatory (p85) subunits. The p110 subunit is composed of an ABD, RBD, C2 domain, helical domain, and kinase domain. The p85 subunit is composed of a Src homology 3 domain (SH3), a Bar cluster region homology domain (BH), two proline-rich regions, and two Src homology 2 domains (nSH2 and cSH2) separated by a coiled-coil inter-SH2 domain (iSH2). The p110 and p85 subunits of PI3Ks interact via an iSH2–ABD interaction, and the p85 subunit makes many important inhibitory contacts with p110 (i–iii). The cSH2-kinase domain inhibitory contact (iii) only occurs with p110β and p110δ but not p110α. HRas is composed of a G domain, which is involved in GTP binding, and a hypervariable region, which is subject to differential lipidation at three C-terminal cysteines. Our HRas lipid modification construct contains two substitution mutations (G12V and C118S) and ends at cysteine 181, which is subject to maleimide coupling. B, gel filtration traces of HRas-coupled membranes and uncoupled HRas with membranes and associated SDS-PAGE of fractions. HRas-coupled membranes are clearly separated from uncoupled HRas. Gel filtration was carried out on a Superdex^TM 200 5/150 GL Increase column (GE Healthcare). mAU, milli absorption units.

Figure 2.

PI3Kα and PI3Kδ are not activated by soluble HRas in a basal or phosphopeptide-stimulated state. A, schematic of conditions tested in the kinase assays. PI3Kα or PI3Kδ was added to maleimide-quenched plasma membrane-mimicking vesicles in the presence or absence of sHRas. B, specific activities of both PI3Kα and PI3Kδ under four different conditions: basal (vesicles only) or in the presence of soluble HRas, RTK-derived phosphopeptide (1 μm), or both soluble HRas and phosphopeptide. Assays measured the production of ADP in the presence of 1–1000 nm PI3K, 100 μm ATP, 0.45 mg/ml 5% PIP₂/10% PE-MCC/15% PC/30% PS/40% PE vesicles, and 5 μm soluble HRas. Kinase assays were performed in triplicate (representative assay, error shown as S.D., n = 3); p values greater than 0.05 are shown as not significant (NS).

Figure 3.

PI3Kα and PI3Kδ, but not PI3Kβ, are activated by HRas-coupled vesicles in a basal or phosphopeptide-stimulated state. A, schematic of kinase assay conditions. PI3Ks were added to either HRas-coupled (GTPγS- or GDP-loaded) or uncoupled maleimide-functionalized plasma membrane–mimicking vesicles. B, PI3K activation mediated by HRas-coupled vesicles is dependent on bound nucleotide. Shown are specific activities of PI3Kα in the presence of phosphopeptide and in the presence or absence of HRas loaded with GDP or GTPγS. C, specific activities of PI3Kα, PI3Kβ, and PI3Kδ under four different conditions: basal (vesicles only) and in the presence of GTPγS-loaded HRas-coupled vesicles, RTK-derived phosphopeptide, or both HRas-coupled vesicles and phosphopeptide. Assays measured the production of ADP in the presence of 1–1000 nm PI3K, 100 μm ATP, 0.45 mg/ml 5% PIP₂/10% PE-MCC/15% PC/30% PS/40% PE vesicles, and 1.3 μm membrane-bound HRas. Kinase assays were performed in triplicate (error shown as S.D., n = 3). p values greater than 0.05 are shown as not significant (NS).

Figure 4.

HDX-MS reveals the mechanism of PI3Kα activation by membrane-coupled HRas. A, peptides in p110α and p85α that showed deuterium exchange differences greater than 5% and 0.4 Da between vesicles and HRas-coupled vesicle conditions are mapped on the modeled structure of PI3Kα (Protein Data Bank (PDB) codes 3HHM (p110α, nSH2-iSH2), 2Y3A (cSH2), and 1HE8 (HRas)). The nSH2 is shown disconnected from p110α to represent the phosphopeptide-bound state. B, schematics of PI3Kα in its Tyr(P)-activated state with mapped changes in deuterium exchange associated with HRas-coupled vesicles relative to the vesicles-only condition. C, time course of deuterium incorporation for peptides in both p110α and p85α showing differences in percent deuteration between the shown conditions (error shown as S.D., n = 3). Concentrations in parentheses refer to HRas, membrane was present at 0.2 mg/ml, and Tyr(P) was present at 5 μm.

Figure 5.

HDX-MS reveals the mechanism of PI3Kδ activation by membrane-coupled HRas. A, peptides in p110δ and p85α that showed differences greater than 5% and 0.4 Da between vesicles and HRas-coupled vesicles conditions are mapped on the modeled structure of PI3Kδ (PDB codes 5DXU (p110δ, iSH2), 3HHM (nSH2), 2Y3A (cSH2), and 1HE8 (HRas)). The nSH2 and cSH2 are shown disconnected from p110δ to represent the phosphopeptide-bound state. B, schematics of PI3Kδ in its Tyr(P)-activated state with mapped changes in deuterium exchange associated with HRas-coupled vesicles relative to the vesicles-only condition. C, time course of deuterium incorporation for peptides in both p110δ and p85α showing differences in percent deuteration between the shown conditions (error shown as S.D., n = 3). Concentrations in parentheses refer to HRas, membrane was present at 0.2 mg/ml, and Tyr(P) was present at 5 μm.

Figure 6.

Depiction of PI3Kα and PI3Kδ activation by phosphorylated RTKs and Ras GTPases. Each individual input partially stimulates PI3K activity via membrane localization or the disruption of important inhibitory contacts, and all inputs are required for full activation. Ras proteins are activated by guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP.