Abstract
Rab5 is a small monomeric GTPase that mediates protein trafficking during endocytosis. Inactivation of Rab5 by GTP hydrolysis causes a conformational change that masks binding sites on its “switch regions” from downstream effectors. The p85 subunit of phosphatidylinositol 3-kinase (PI3K) is a GTPase activating protein (GAP) towards Rab5. Whereas p85 can bind with both Rab5-GTP and Rab5-GDP, the PI3K catalytic subunit p110β binds only Rab5-GTP, suggesting it interacts with the switch regions. Thus, the GAP functions of the catalytic arginine finger (from p85) and switch region stabilization (from p110β) may be provided by both proteins, acting together. To identify the Rab5 residues involved in binding p110β, residues in the Rab5 switch regions were mutated. A stabilized recombinant p110 protein, where the p85-iSH2 domain was fused to p110 (alpha or beta) was used in binding experiments. Eleven Rab5 mutants, including E80R and H83E, showed reduced p110β binding. The Rab5 binding site on p110β was also resolved through mutation of p110β in its Ras binding domain, and includes residues I234, E238 and Y244. This is a second region within p110β important for Rab5 binding. The Rab5-GTP:p110β interaction may be further elucidated through the characterization of these non-binding mutants in cells.
Highlights
The p110β subunit can regulate endocytosis and autophagy through its ability to bind to Rab[5], a small GTPase with a key role in protein trafficking[11]
Since p110β has been reported to bind to Rac[1] via its Ras-binding domain (RBD), we verified that the Myc-iSH2-p110β protein could bind to the activated form of Rac[1] (Fig. 1e)[17]
The p110β-I234A, p110β-E238R and p110β-Y244A mutants showed significantly reduced binding to Rab[5]. These results suggest that p110β residues I234, E238 and Y244, but not L232 or D239, within the RBD of p110β contribute to Rab[5] binding
Summary
The p110β subunit can regulate endocytosis and autophagy through its ability to bind to Rab[5], a small GTPase with a key role in protein trafficking[11]. The p85α subunit binds directly to Rab[5] and can downregulate Rab[5] activity via p85-encoded GAP activity[14] Consistent with this finding, knockdown of p85α resulted in increased Rab5-GTP levels[11]. This study set out to characterize the key residues within the p110β RBD that mediate binding to Rab[5] and the corresponding Rab[5] residues critical for the binding of p110β. Mutation of these key residues allowed the generation of non-binding mutants of p110β and Rab[5] that can be used to further study the role of this complex in the regulation of cell properties
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