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 p85α 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 p85α 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. Rab proteins regulate vesicle fusion events during the endocytosis, recycling, and degradation of activated receptor tyrosine kinases. The p85α 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 p85α 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. Receptor tyrosine kinases (RTKs) 3The abbreviations used are: RTK, receptor tyrosine kinase; GAP, GTPase-activating protein; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; SH, Src homology. activate a variety of downstream signaling pathways, including the Ras/MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways important for cell proliferation and cell survival. Overexpression of wild-type or constitutively active mutant RTKs has been observed in several types of human cancers and can result in increased RTK kinase activity and the activation of signaling pathways promoting tumor formation (1Bache K.G. Slagsvold T. Stenmark H. EMBO J. 2004; 23: 2707-2712Crossref PubMed Scopus (173) Google Scholar). RTK overexpression can result from gene amplification, from increased gene expression, and more recently, from defects in receptor down-regulation (2Grandal M.V. Zandi R. Pedersen M.W. Willumsen B.M. van Deurs B. Poulsen H.S. Carcinogenesis. 2007; 28: 1408-1417Crossref PubMed Scopus (108) Google Scholar, 3Peschard P. Park M. Cancer Cell. 2003; 3: 519-523Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 4Thien C.B. Langdon W.Y. Nat. Rev. Mol. Cell Biol. 2001; 2: 294-307Crossref PubMed Scopus (523) Google Scholar, 5Dikic I. Giordano S. Curr. Opin. Cell Biol. 2003; 15: 128-135Crossref PubMed Scopus (286) Google Scholar). Rab proteins are small monomeric GTPases important in the regulation of RTK endocytosis, trafficking, and degradation pathways (reviewed in Refs. 6Stenmark H. Olkkonen V.M. Genome Biol. 2001; 2 (reviews3007.1–reviews3007.7)Crossref PubMed Google Scholar, 7Deneka M. Neeft M. van der Sluijs P. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 121-142Crossref PubMed Scopus (118) Google Scholar, 8Markgraf D.F. Peplowska K. Ungermann C. FEBS Lett. 2007; 581: 2125-2130Crossref PubMed Scopus (100) Google Scholar). Rab5 regulates vesicle fusion events during the endocytosis of activated RTKs, important for moving the receptor complex from the plasma membrane to the early/sorting endosome. RTKs are believed to continue their downstream signaling during endocytosis. Rab4 regulates vesicle trafficking from the early/sorting endosome back to the plasma membrane to return deactivated RTKs to the cell surface for further rounds of activation and signaling. Some fraction of receptors located in the early/sorting endosome are diverted to the late endosome and lysosome, where they are degraded via a mechanism that requires receptor monoubiquitination (9d'Azzo A. Bongiovanni A. Nastasi T. Traffic. 2005; 6: 429-441Crossref PubMed Scopus (202) Google Scholar, 10Sigismund S. Polo S. Di Fiore P.P. Curr. Top Microbiol. Immunol. 2004; 286: 149-185Crossref PubMed Scopus (136) Google Scholar, 11Haglund K. Di Fiore P.P. Dikic I. Trends Biochem. Sci. 2003; 28: 598-603Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 12Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 13Marmor M.D. Yarden Y. Oncogene. 2004; 23: 2057-2070Crossref PubMed Scopus (324) Google Scholar). Because Rab5 and Rab4 regulate the trafficking of activated internalized RTK complexes through the endocytic pathway, they control the location, magnitude, and duration of receptor signaling (14Ceresa B.P. Histol. Histopathol. 2006; 21: 987-993PubMed Google Scholar, 15Di Fiore P.P. De Camilli P. Cell. 2001; 106: 1-4Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 16Miaczynska M. Pelkmans L. Zerial M. Curr. Opin. Cell Biol. 2004; 16: 400-406Crossref PubMed Scopus (459) Google Scholar). Recent studies in human disease, including cancer, have suggested that defects in the endocytic pathway can give rise to sustained RTK levels, sustained receptor activation, and downstream signaling driving aberrant cell proliferation, cell survival, and tumorigenesis (1Bache K.G. Slagsvold T. Stenmark H. EMBO J. 2004; 23: 2707-2712Crossref PubMed Scopus (173) Google Scholar, 17Clague M.J. Urbe S. J. Cell Sci. 2001; 114: 3075-3081Crossref PubMed Google Scholar). In particular, defects in receptor monoubiquitination have been linked to cancer (3Peschard P. Park M. Cancer Cell. 2003; 3: 519-523Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 18Waterman H. Katz M. Rubin C. Shtiegman K. Lavi S. Elson A. Jovin T. Yarden Y. EMBO J. 2002; 21: 303-313Crossref PubMed Scopus (226) Google Scholar). A recent report demonstrated that Tpr-Met, a chromosomal translocation product involving the kinase domain of the Met RTK, was not down-regulated efficiently because it was mislocalized to the cytoplasm and lacked the binding site for the Cbl ubiquitin-protein isopeptide ligase (19Mak H.H. Peschard P. Lin T. Naujokas M.A. Zuo D. Park M. Oncogene. 2007; 26: 7213-7221Crossref PubMed Scopus (37) Google Scholar). We used a mouse NIH 3T3 fibroblast cell model system to study receptor trafficking of the endogenously expressed PDGFRβ. This well characterized RTK has known downstream signaling pathways, including Ras/MAPK and PI3K/Akt, activated upon PDGF stimulation. We characterized the interaction of the p85α subunit of PI3K with different binding proteins and the functions of these protein complexes in receptor-mediated endocytosis (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 21Ignatiuk A. Quickfall J.P. Hawrysh A.D. Chamberlain M.D. Anderson D.H. J. Biol. Chem. 2006; 281: 5956-5964Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Previously, we reported that the BCR homology domain of p85 has GTPase-activating protein (GAP) activity toward Rab5 and Rab4 (but not Rab11) and enhanced intrinsic Rab GTPase activity by ∼1000-fold (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), switching the Rab to an inactive GDP-bound conformation. We generated a p85 mutant lacking RabGAP activity (p85-R274A), yet it retained normal ability to bind to activated PDGFRs as well as to bind to and regulate the p110 catalytic subunit of PI3K (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). NIH 3T3 cells stably expressing p85-R274A were assayed for changes in PDGFR activation, downstream cellular signaling, and PDGFR degradation (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The loss of p85 RabGAP activity increased the magnitude and duration of tyrosine-phosphorylated PDGFR and the resulting MAPK and Akt signaling in response to PDGF stimulation. Cells expressing p85-R274A also appeared to have a decreased rate of PDGFR degradation. This suggests that p85 RabGAP activity is important in preventing uncontrolled PDGFR signaling. Moreover, disruption of RabGAP function as a result of p85-R274A expression could have tumorigenic potential because of enhanced PDGFR signaling and diminished PDGFR down-regulation. To test this hypothesis, we have characterized the oncogenic potential of cells expressing the p85-R274A RabGAP mutant, determining changes in cell morphology, proliferation, contact inhibition, and anchorage-independent growth. Cells stably expressing the p85-R274A mutant showed all these characteristics of oncogenic transformation and also formed tumors in nude mice. Coexpression of the dominant negative Rab5-S34N mutant blunted these effects, consistent with the ability of p85-R274A to act at the level of Rab5 function. These results suggest that a subtle point mutation in p85 that disrupts its RabGAP activity is sufficient to convert it into an oncogene and that deregulation of Rab function can lead to cellular transformation. Plasmids and Cell Culture—The plasmids encoding FLAG-tagged wild-type p85 and p85-R274A have been described (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The insert for the hemagglutinin (HA) epitope-tagged HA-Rab5-S34N plasmid was generated by PCR amplification of the entire Rab5 coding sequence from the pCMV-Myc-Rab5-S34N plasmid (amino acids 1–215; Ser34 changed to Asn) obtained from Dr. Li (University of Oklahoma) (22Liu K. Li G. J. Biol. Chem. 1998; 273: 10087-10090Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) using primers that added a 5′-BamHI site and 3′-EcoRI site. The BamHI/EcoRI-digested insert was subcloned into the BglII/EcoRI-digested HA3 vector (23King T.R. Fang Y. Mahon E.S. Anderson D.H. J. Biol. Chem. 2000; 275: 36450-36456Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The resulting plasmid encoded the HA-Rab5-S34N protein containing three of the at the of The encoding HA-Rab5-S34N was with and into The resulting plasmid encoded HA-Rab5-S34N with or and also The entire HA-Rab5-S34N coding sequence was to that it the mutation of Ser34 to NIH 3T3 cells expressing and were generated and in containing as described (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). with HA-Rab5-S34N was by of cells with the plasmid encoding HA-Rab5-S34N and in and were for HA-Rab5-S34N expression using an Cells were and used for PDGF as (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). have been described D.H. Oncogene. 1998; 16: PubMed Scopus Google Scholar) and used the from PDGFR and The used were as Cell MAPK Akt and were used to the and using and were for the different cell were and to with the for are of a result from at three Cell and were using a using an The was the contact cells were on a and to for The was changed The of cells were with and with of the entire were with a The results are for three the anchorage-independent growth the cells were in soft as described D.H. Oncogene. 1998; 16: PubMed Scopus Google Scholar). Cells were in soft in and to of soft was on were as described for the cell of growth. The formed are reported as the of cells on were and the was A of was using to the of the The for the different results are proliferation was assayed using the Cell proliferation to the The cells were in for at in a The cells were for growth in containing or was with in of The results were using and the is with and nude were with cells NIH 3T3 or in the as described Li T. J. J. Immunol. 2001; PubMed Scopus Google Scholar). was by the and of the The tumor was determined by the The tumor was using and the is with were to by the of were and and the was as described Li T. J. J. Immunol. 2001; PubMed Scopus Google Scholar). The were used for PDGFR Akt Cell MAPK and The were with the using an to the using the or were with were with a using an at a of p85-R274A a in NIH 3T3 3T3 cell stably expressing FLAG-tagged wild-type p85 and a p85 mutant with a single point mutation to in the BCR homology domain that disrupts its RabGAP activity were used (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The expression levels of wild-type p85 and p85-R274A in the cell were as determined using is as a control Cells wild-type p85 and control NIH 3T3 cells a morphological by the cells appeared more and In cells formed on normal growth the loss of growth via cell contact not These with report of enhanced and sustained PDGFR activation and downstream signaling (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), to further the tumorigenic potential of these Cells a of and important of transformation observed in cell is the loss of contact cell were for loss of contact by the cells and determining cells to and Cells were and for with formation was by The cell in to the control NIH 3T3 and important in tumor formation is the ability of cells to To the ability of cells to cells were into soft cells were and The cell in soft agar, control cells and cells not These results demonstrate that p85-R274A expression is sufficient to cause cells to contact and to for of cellular transformation. We these results using different cell expressing that the effects observed were the result of p85 RabGAP function. were using p85-R274A Cells cells normal cells in growth and are To was a in the growth of cells in p85 RabGAP cells were in or with PDGF wild-type and cell in and PDGF with control NIH 3T3 This was determined using a cell proliferation in cell was Some of this to due to the of increased p85 expression or the cells showed of cell proliferation and PDGF This is consistent with the transformed of cells and with sustained activation of MAPK and Akt pathways in response to PDGF reported (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Because these pathways cell proliferation and cell survival, this could the enhanced growth we observed these p85-R274A Cells in the tumorigenic potential of the p85-R274A we determined these cells tumors in nude mice. We nude with control NIH 3T3 or cells and tumor with cells to tumors that in A and with NIH 3T3 cells of the with control cells with NIH 3T3 cells also not tumors not from p85-R274A were and with The tumor is the The also showed that the tumors a cell of a consistent with a tumor of fibroblast The tumors were to and well with events of the p85-R274A tumors demonstrated high levels of expression in tumor We also PDGFR expression in tumor and PDGFR was as using the to the receptor kinase of its activation We activation of MAPK and high levels of Akt activation in tumor downstream signaling of activated in as in cell the PDGFR and its downstream MAPK and Akt signaling pathways are activated in tumor were with to demonstrate an of and These results demonstrate the of p85-R274A expression is tumor formation with activated PDGFR signaling, via the Akt cell PDGFR and Cell of p85-R274A by of the p85-R274A mutant resulted in enhanced PDGFR activation, downstream signaling to the MAPK pathway (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and cellular transformation. We showed in that p85-R274A mutation intrinsic p85 RabGAP activity toward that expression of p85-R274A the regulation of Rab proteins in expression of p85-R274A p85 and Rab5 and Rab4 in their active promoting PDGFR endocytosis Receptor tyrosine kinase signaling to the Akt pathway is to from receptor activation of the pathway at the plasma In Ras/MAPK signaling has been to from signaling and continue during endocytosis and trafficking of the receptor to the early/sorting endosome B.P. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar, G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar, L. S. C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. S. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, Y. A. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, D.H. 2006; PubMed Scopus Google Scholar). PDGFR endocytosis using a Rab5 dominant negative mutant we to MAPK signaling to a Akt signaling. In expression of Rab5-S34N act of the p85-R274A RabGAP and sufficient to the endocytosis and of activated PDGFR To test this we determined of a dominant negative Rab5-S34N mutant, with binding to the effects of p85-R274A expression on PDGFR activation, signaling, and cell transformation. of PDGF PDGFR activated and expressed in of Rab5-S34N with p85-R274A reduced PDGFR activation and receptor levels of downstream including MAPK and Akt was also upon of using to their activated Coexpression with Rab5-S34N was sufficient to levels in cells as model These results that expression of a dominant negative Rab5-S34N mutant can the ability of p85-R274A expression to cause sustained PDGFR activation, expression, and signaling. To test this also the transformed of p85-R274A expression, the biological of Rab5-S34N expression on cell contact and anchorage-independent growth of cells was Coexpression of Rab5-S34N was of the transformed cell of cells to a more normal from tumorigenic showed that expression of Rab5-S34N a dominant negative to the oncogenic of p85-R274A expression, the of as well as the and of in soft and The ability of Rab5-S34N expression to the transformed of cells the that Rab5 of the p85-R274A RabGAP types of tumors have increased RTK levels that are to aberrant cell proliferation and cell (1Bache K.G. Slagsvold T. Stenmark H. EMBO J. 2004; 23: 2707-2712Crossref PubMed Scopus (173) Google Scholar, P. T. 2001; PubMed Scopus Google Scholar, S. J. A. A. A. 2006; Google Scholar). In high RTK expression is known to the result of gene amplification or deregulation of receptor P. T. 2001; PubMed Scopus Google Scholar). A cause for increased RTK levels has been suggested to result from defects in receptor-mediated endocytosis and degradation pathways (1Bache K.G. Slagsvold T. Stenmark H. EMBO J. 2004; 23: 2707-2712Crossref PubMed Scopus (173) Google Scholar). We have that p85α has intrinsic activity several small monomeric including Rab5 and Rab4 (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). These Rab GTPases critical in down-regulation and degradation of activated growth factor receptors via regulation of vesicle fusion during receptor endocytosis and We have also that expression of the p85-R274A mutant the rate of PDGFR degradation upon PDGF stimulation. these receptors are tyrosine-phosphorylated and cause sustained activation of MAPK and Akt signaling pathways, known to cell and (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The p85-R274A mutant protein was to its ability to bind to activated PDGFRs and to with the p110 catalytic subunit of PI3K, resulting in normal levels of PI3K activity (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). the p85-R274A its biological effects through a We have that cells have PDGFR trafficking consistent with Rab D. S. and D. H. are to further the mechanism of this PDGFR In this we have demonstrated the biological consequences of mutant p85-R274A expression and the with dominant negative Rab5-S34N We have that cells have several characteristics of including loss of contact for growth of and the ability to tumors in nude mice. cells the of transformed and we further demonstrated that a dominant negative Rab5-S34N mutant could transformed properties. Rab5 and are known Overexpression of wild-type or cells C. De A. S. D. M. Biochem. 2003; PubMed Scopus Google Scholar), a role for in is a small of that Rab proteins and of endocytosis can in are several endocytic proteins and that are known to their expression is or they are (reviewed in Refs. 17Clague M.J. Urbe S. J. Cell Sci. 2001; 114: 3075-3081Crossref PubMed Google and R. Dikic I. FEBS Lett. 2005; PubMed Scopus Google Scholar). is also that expression of Rab proteins is in is expressed in cancer D. C. M. G. 2006; PubMed Scopus Google Scholar), and the function of is not well it is known to the and plasma that it regulate trafficking these D. C. M. G. 2006; PubMed Scopus Google Scholar, A. C. Olkkonen V.M. P. A. K. Zerial M. J. Cell Sci. PubMed Google Scholar). The gene is in and D. Y. K. L. J. A. R. J. 2006; PubMed Scopus Google Scholar). The protein is in and protein kinase A to J. J. Cell Biol. 2002; PubMed Scopus Google Scholar). of the are in endocytic pathways, are in cancers of the and C. G. C. P. J. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J.P. A. K. J. K. D. Nat. 2004; PubMed Scopus Google Scholar). This further that in receptor endocytosis pathways to human These suggest that a novel of that receptor levels and cellular signaling by the endocytosis, recycling, and degradation of growth factor This is the of a subtle point mutation in p85α of cells into tumor cells by the regulation of PDGFR activation, signaling, and down-regulation. To are studies that have for p85α in human cancers or M. K. S. M. 2002; 16: PubMed Scopus Google Scholar, C. P. A. S. C. L. J.P. M.A. J. I. EMBO J. 1998; PubMed Scopus Google Scholar, M. G. 2004; PubMed Scopus Google Scholar, C. Cancer 2001; Google Scholar, G. R. M. T. H. S. J.P. 2000; PubMed Scopus Google Scholar). of p85α because of a acids or a mutation acids has been in C. P. A. S. C. L. J.P. M.A. J. I. EMBO J. 1998; PubMed Scopus Google Scholar) and human M. K. S. M. 2002; 16: PubMed Scopus Google Scholar) The in these p85α mutant proteins all or of the domain and results in deregulation of the PI3K p110 of p85α have also been on encoding the of the protein in cancer cells M. G. 2004; PubMed Scopus Google Scholar, C. Cancer 2001; Google Scholar). (amino acids were for in and 2 cancer cell C. Cancer 2001; Google Scholar). defects and were with an and three containing small and These are located a negative site p85α and were suggested to cause the observed increased PI3K activity C. Cancer 2001; Google Scholar). (amino acids of p85α were for in and a resulting in a p85α protein and increased PI3K activity M. G. 2004; PubMed Scopus Google Scholar). A study of reported with a mutation in p85α PI3K activity and a p85α sequence G. R. M. T. H. S. J.P. 2000; PubMed Scopus Google Scholar). Our new that a single point mutation (R274A) the RabGAP known as the BCR domain of p85α is sufficient for cell transformation. a more of p85α for in human cancers to p85α that its RabGAP activity are The ability of p85α to regulate Rab5 and Rab4 GTPases it to control receptor trafficking events during receptor-mediated endocytosis and degradation. A single point mutation in p85α it and cellular expression of this mutant high levels of PDGFR activation and signaling resulting in cell transformation. The p85-R274A mutation not its ability to bind and regulate PI3K p110 activity in response to PDGF (20Chamberlain M.D. Berry T.R. Pastor M.C. Anderson D.H. J. Biol. Chem. 2004; 279: 48607-48614Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), that the effects of this mutation are of PI3K These results suggest functions for the p85α protein in to its role in downstream PI3K p110 signaling. The ability of p85α to regulate endocytosis and receptor trafficking it to control the location, magnitude, and duration of PDGFR signaling. of the control of the GTPase of Rab5 and Rab4 by expression of p85-R274A the magnitude and duration of PDGFR signaling, resulting in cell transformation. We are to Dr. G. Li for We S. and G. for and Dr. P. for critical of the

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