Abstract
VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.
Highlights
A diverse array of cellular signaling pathways are regulated by small G proteins of the Ras GTPase superfamily that activate lipid kinases of the phosphatidylinositol-3-kinase (PI3K) family, thereby amplifying lipid kinase production of phosphatidylinositol (PIP) signaling lipids phosphorylated at the 3 position of the inositol headgroup [1,2,3,4,5,6,7,8,9,10,11,12]
The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A
The findings reveal that Rab5 drives activation of the VPS34CII lipid kinase and PI3P production via a dual mechanism characterized by nearly equivalent, major contributions from both membrane recruitment and allosteric activation
Summary
A diverse array of cellular signaling pathways are regulated by small G proteins of the Ras GTPase superfamily that activate lipid kinases of the phosphatidylinositol-3-kinase (PI3K) family, thereby amplifying lipid kinase production of phosphatidylinositol (PIP) signaling lipids phosphorylated at the 3 position of the inositol headgroup [1,2,3,4,5,6,7,8,9,10,11,12]. 2) the allosteric activation model postulates that the G-protein-kinase interaction increases the specific activity (turnover rate) of each membrane-bound kinase molecule by triggering a change in its conformation or docking geometry on the target membrane surface. These activation mechanisms are not mutually exclusive; in principle, G protein stimulation of net PI3K activity could employ either mechanism alone or both together. A previous singlemolecule analysis of H-Ras activation of PI3Ka, a class I PI3K, revealed that H-Ras association increases net PI3Ka lipid kinase activity and production of signaling lipid PI[3,4,5]P3 via the membrane recruitment mechanism, with no allosteric increase in the turnover rate of the membrane-bound kinase molecule [13]. Given the broad significance and diversity of G protein regulation of PI3K enzymes in normal cell function as well as many human disease states, further studies defining the range of G-proteinPI3K activation mechanisms may significantly advance both signaling biology and pharmaceutical development
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