The p85α Subunit of Phosphatidylinositol 3′-Kinase Binds to and Stimulates the GTPase Activity of Rab Proteins

Abstract

Rab5 and Rab4 are small monomeric GTPases localized on early endosomes and function in vesicle fusion events. These Rab proteins regulate the endocytosis and recycling or degradation of activated receptor tyrosine kinases such as the platelet-derived growth factor receptor (PDGFR). The p85␣ subunit of phosphatidylinositol 3-kinase contains a BH domain with sequence homology to GTPase activating proteins (GAPs), but has not previously been shown to possess GAP activity. In this report, we demonstrate that p85␣ has GAP activity toward Rab5, Rab4, Cdc42, Rac1 and to a lesser extent Rab6, with little GAP activity toward Rab11. Purified recombinant Rab5 and p85␣ can bind directly to each other and not surprisingly, the p85␣-encoded GAP activity is present in the BH domain. Because p85␣ stays bound to the PDGFR during receptor endocytosis, p85␣ will also be localized to the same early endosomal compartment as Rab5 and Rab4. Taken together, the physical co-localization and the ability of p85␣ to preferentially stimulate the down-regulation of Rab5 and Rab4 GTPases suggests that p85␣ regulates how long Rab5 and Rab4 remain in their GTP-bound active state. Cells expressing BH domain mutants of p85 show a reduced rate of PDGFR degradation as compared with wild type p85 expressing cells. These cells also show sustained activation of the mitogen-activated protein kinase and Akt pathways. Thus, the p85␣ protein may play a role in the down-regulation of activated receptors through its temporal control of the GTPase cycles of Rab5 and Rab4.Down-regulation of signal transduction pathways activated by receptor tyrosine kinases, such as the PDGFR, 1 includes endocytosis of the activated receptor complex (1, 2). Receptormediated endocytosis involves multiple vesicle fusion events that effectively deliver the receptor-signaling complex to the early endosome. This complex is then disassembled and the receptor is either recycled back to the plasma membrane or sorted to the late endosome and lysosome for degradation (3, 4). Rab5 is a small monomeric GTPase involved in early endosomal fusion events such as the fusion of clathrin-coated vesicles (containing activated receptors undergoing endocytosis) with the early/sorting endosomes (reviewed in Refs. 5 and 6). GDP-bound Rab5 is inactive and bound to a guanine dissociation inhibitor (GDI) protein in the cytosol. GTP-bound Rab5 is active and localized on the cytoplasmic face of early/sorting endosomes where it is involved in binding specific effector proteins such as the early-endosomal autoantigen 1 (EEA1) (7,8). EEA1 is a cytosolic protein that is recruited to early endosomal membranes by binding to Rab5-GTP and the lipid product of the class III PI3K p150/hVPS34, phosphatidylinositol 3Ј-phosphate (7, 9, 10). EEA1 is a core component required for early endosomal fusion events (7,8,11,12). The half-life of phosphatidylinositol 3Ј-phosphate, and the duration of Rab5-GTP have been suggested to influence the rate and extent of the endosome fusion reaction (13). The low intrinsic GTPase act...

Insulin-stimulated Interaction between Insulin Receptor Substrate 1 and p85α and Activation of Protein Kinase B/Akt Require Rab5

“…siRNAs against Rab5 Attenuate Akt Activation by Insulin without Affecting MAPK Activity-Previous studies suggested that Rab5 might interact with PI3Ks and thus mediate insulin signaling (34,35). To determine whether specific isoforms of Rab5 affect insulin signaling, siRNAs directed against Rab5a, -5b, and -5c were synthesized.…”

“…Rab5 has been shown to interact with two types of PI3K (34) and to regulate phosphoinositide turnover in the endocytic pathway (29). More recent research showed that p85␣ binds to both GDP and GTP forms of Rab5A and is a GTPaseactivating Protein for Rab5A (35)(36)(37). Our results suggest that Rab5 might recruit p85␣ to IRS1 that is localized to and phosphorylated at the cytoskeleton-membrane interface and may possibly activate a subpopulation of PI3K molecules, which are required for insulin-stimulated PKB/Akt activation.…”

“…More recent research showed that p85␣ binds to both GDP and GTP forms of Rab5A and that it functions as a GTPase-activating Protein for Rab5A (35)(36)(37). Identification of PI4-, and PI5-phosphatases as novel Rab5 effectors reinforced the involvement of Rab5A in phosphoinositide turnover in the endocytic pathway (29).…”

Caspase 8 Promotes Peripheral Localization and Activation of Rab5

“…The caspase 8 mutants C360A and Y380F were previously reported and cloned into pEGFP-N2 vector (13). Bovine FLAG-tagged wild type p85␣, p85␣/R274A (mutant defective for Rab-GAP activity), and p85␣/R368A/R649A (double mutant ⌬R, defective in both SH2 domains via point mutations) were previously described (8).…”

Phosphatidylinositol 3-Kinase and Rab5 GTPase Inversely Regulate the Smad Anchor for Receptor Activation (SARA) Protein Independently of Transforming Growth Factor-β1

“…The p85␣ subunit of PI3K has been shown to bind directly to the early endosomal protein, Rab5, and to inactivate it through GAP activity toward Rab5 (18). Therefore, inhibition of PI3K could result in increased activity of Rab5.…”