Single-Molecule Study Reveals How Receptor and Ras Synergistically Activate PI3Kα and PIP3 Signaling

Abstract

Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.

Defining how oncogenic and developmental mutations ofPIK3R1alter the regulation of class IA phosphoinositide 3-kinases

“…To determine if either p110α or p110δ bound to Q572* represented a fully activated form we tested the activation by lipidated HRas, which activates p110α and p110δ through increased membrane recruitment. We found that lipidated HRas activated both p110α and p110δ bound to Q572* similar to the activation seen with wildtype p85α ( Fig 1E) (Buckles et al, 2017;Siempelkamp et al, 2017).…”

“…This is consistent with previous results showing that the combination of the activating point mutation in the C2 domain of p110α and the Q572* truncation does not lead to additive activation ). However, the complex of Q572* with p110α does not represent a fully activated form, as kinase activity can still be stimulated by the presence of lipidated HRas similar to wild type p110α/p85α (Buckles et al, 2017;Siempelkamp et al, 2017).…”

Molecular Mechanisms of Human Disease Mediated by Oncogenic and Primary Immunodeficiency Mutations in Class IA Phosphoinositide 3-Kinases

“…The PI3K isoforms are differentially activated downstream of Ras superfamily members ( 39 , 41 ), with p110α and p110δ being activated downstream of Ras family GTPases, and p110β being activated downstream of Rho family GTPases. Ras activates PI3K through enhanced membrane interaction, with Ras activation being strongly synergistic with activation downstream of phosphorylated receptors ( 42 , 43 ). Mutant p110α deficient in its ability to be activated by Ras leads to decreased oncogenic transformation, tumor maintenance, and angiogenesis downstream of mutant Ras ( 44 – 46 ).…”

Inhibitory effect of platinum (II) complex of curcumin on non-small cell lung cancer via the PI3K-AKT pathway

“…The central substrate is a product of P13K, i.e. phosphatidylinositol 3,4,5 triphosphate (PIP3) [26]. Cyclins and Bcl-2 proteins correlate closely with cell cycles and apoptosis, respectively [27].…”