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.

Disrupted RabGAP function of the p85 subunit of phosphatidylinositol 3-kinase results in cell transformation.

Rab proteins regulate vesicle fusion events during the endocytosis, recycling, and degradation of activated receptor tyrosine kinases. The p85alpha subunit of phosphatidylinositol 3-kinase has GTPase-activating protein activity toward Rab5 and Rab4, an activity severely reduced by a single point mutation (p85-R274A). Expression of p85-R274A resulted in increased platelet-derived growth factor receptor (PDGFR) activation and downstream signaling (Akt and MAPK) and in decreased PDGFR degradation. We now report that the biological consequences of p85-R274A expression cause cellular transformation as determined by the following: aberrant morphological phenotype, loss of contact inhibition, growth in soft agar, and tumor formation in nude mice. Immunohistochemistry shows that the tumors contain activated PDGFR and high levels of activated Akt. Coexpression of a dominant negative Rab5-S34N mutant attenuated these transformed properties. Our results demonstrate that disruption of the RabGAP function of p85alpha due to a single point mutation (R274A) is sufficient to cause cellular transformation via a phosphatidylinositol 3-kinase-independent mechanism partially reversed by Rab5-S34N expression. This critical new role for p85 in the regulation of Rab function suggests a novel role for p85 in controlling receptor signaling and trafficking through its effects on Rab GTPases.