Multiple roles for the p85α isoform in the regulation and function of PI3K signalling and receptor trafficking.

The p85α protein is best known as the regulatory subunit of class 1A PI3Ks (phosphoinositide 3-kinases) through its interaction, stabilization and repression of p110-PI3K catalytic subunits. PI3Ks play multiple roles in the regulation of cell survival, signalling, proliferation, migration and vesicle trafficking. The present review will focus on p85α, with special emphasis on its important roles in the regulation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and Rab5 functions. The phosphatidylinositol-3-phosphatase PTEN directly counteracts PI3K signalling through dephosphorylation of PI3K lipid products. Thus the balance of p85α-p110 and p85α-PTEN complexes determines the signalling output of the PI3K/PTEN pathway, and under conditions of reduced p85α levels, the p85α-PTEN complex is selectively reduced, promoting PI3K signalling. Rab5 GTPases are important during the endocytosis, intracellular trafficking and degradation of activated receptor complexes. The p85α protein helps switch off Rab5, and if defective in this p85α function, results in sustained activated receptor tyrosine kinase signalling and cell transformation through disrupted receptor trafficking. The central role for p85α in the regulation of PTEN and Rab5 has widened the scope of p85α functions to include integration of PI3K activation (p110-mediated), deactivation (PTEN-mediated) and receptor trafficking/signalling (Rab5-mediated) functions, all with key roles in maintaining cellular homoeostasis.

Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is deregulated in many human diseases including cancer, diabetes, obesity, and autoimmunity. PI3K consists of a p110 catalytic protein and a p85alpha regulatory protein, required for the stabilization and localization of p110-PI3K activity. The p110-PI3K enzyme generates the key signaling lipid phosphatidylinositol 3,4,5-trisphosphate, which is dephosphorylated by the PI3-phosphatase PTEN. Here we show another function for the p85alpha regulatory protein: it binds directly to and enhances PTEN lipid phosphatase activity. We demonstrate that ectopically expressed FLAG-tagged p85 coimmunoprecipitates endogenous PTEN in an epidermal growth factor dependent manner. We also show epidermal growth factor dependent coimmunoprecipitation of endogenous p85 and PTEN proteins in HeLa cells. Thus p85 regulates both p110-PI3K and PTEN-phosphatase enzymes through direct interaction. This finding underscores the need for caution in analyzing PI3K activity because anti-p85 immunoprecipitations may contain both p85:p110-PI3K and p85:PTEN-phosphatase enzymes and thus measure net PI3K activity. We identify the N-terminal SH3-BH region of p85alpha, absent in the smaller p55alpha and p50alpha isoforms, as the region that mediates PTEN binding and regulation. Cellular expression of p85DeltaSH3-BH results in substantially increased magnitude and duration of pAkt levels in response to growth factor stimulation. The ability of p85 to bind and directly regulate both p110-PI3K and PTEN-PI3-phosphatase allows us to explain the paradoxical insulin signaling phenotypes observed in mice with reduced PI3K or PTEN proteins. This discovery will impact ongoing studies using therapeutics targeting the PI3K/PTEN/Akt pathway.

Deregulation of Rab5 and Rab4 proteins in p85R274A-expressing cells alters PDGFR trafficking.

Activated receptor tyrosine kinases recruit many signaling proteins to activate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K) consisting of a p85 regulatory protein and a p110 catalytic protein. We have recently shown the p85alpha protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a GAP-defective mutant, p85R274A, resulted in sustained levels of activated platelet-derived growth factor receptors (PDGFRs) and enhanced downstream signaling. In this report we have characterized Rab5- and Rab4-mediated PDGFR trafficking in cells expressing wild type p85 and GAP-defective mutant p85R274A. Wild type p85 overexpressing cells had slower PDGFR trafficking consistent with enhanced GAP activity deactivating Rab5 and Rab4 to block their vesicle trafficking functions. Mutant p85R274A expression increased the internalization rate of PDGFRs, a Rab5-dependent process, without preventing PDGFR ubiquitination. Immunofluorescence studies further demonstrated that p85R274A-expressing cells showed Rab5 accumulation at intracellular locations. Pull-down and FRAP (fluorescence recovery after photobleaching) experiments indicate this is likely membrane-associated Rab5-GTP, sustained due to decreased p85 GAP activity for the p85R274A mutant. These cells also had substantial amounts of activated PDGFRs in Rab4-positive recycling endosomes, a compartment that usually contains primarily deactivated/dephosphorylated receptors. Our results suggest that the PDGFR-associated GAP activity of p85 regulates both Rab5 and Rab4 functions in cells to influence the movement of activated PDGFR through endosomal compartments. Disruption of this regulation by p85R274A expression impacts PDGFR phosphorylation/dephosphorylation, degradation kinetics and downstream signaling by altering the time receptors spend in specific intracellular endosomal compartments. These results demonstrate that the p85alpha protein is an important regulator of Rab-mediated PDGFR trafficking, which significantly impacts receptor signaling and degradation.