Systematic Evaluation of Anti-apoptotic Growth Factor Signaling in Vascular Smooth Muscle Cells

Peptide growth factors contribute to the pathogenesis of cardiovascular diseases by inducing a variety of cellular responses including anti-apoptotic effects. Several of the signaling molecules that are activated by growth factor receptors such as Src family kinases (Src), phosphatidylinositol 3′-kinase (PI3K), phospholipase Cγ (PLCγ), Ras, and SHP-2 were shown to mediate survival signals. We systematically investigated the relative contribution of each signaling molecule for growth factor-dependent cell survival in vascular smooth muscle cells (VSMC). Our approach was the use of mutated plateletderived growth factor (PDGF) β-receptors (βPDGFR) in which the tyrosine residues required for binding of each signaling molecule were individually mutated to phenylalanine. To bypass endogenous PDGFR in VSMC we used chimeric receptors (ChiRs), containing the extracellular domain of the macrophage colony-stimulating factor (M-CSF) receptor and the cytoplasmic domain of the wild type (WT) or mutated βPDGFR. Selective activation of the ChiR-WT with M-CSF significantly reduced apoptosis to the same extent as PDGF-BB in non-transfected cells. Deletion of the binding site for PI3K, but not for Src, RasGAP, SHP-2, or PLCγ, completely abolished the anti-apoptotic effect. Consistently, a ChiR mutant that only binds PI3K was fully able to mediate cell survival as efficiently as the ChiR-WT. Furthermore, the PDGF-dependent anti-apoptotic effect in non-transfected cells was completely abolished by the PI3K inhibitor wortmannin, whereas inhibitors of Src, PLCγ, ERK, or p38 MAP kinase had no effect. The exploration of downstream signaling events revealed that PDGF-BB activates the anti-apoptotic Akt signaling pathway in a PI3K-dependent manner. Moreover, Akt phosphorylates and thus inactivates the pro-apoptotic proteins BAD and Forkhead transcription factors (FKHR, FKHRL1). We conclude that growth factor-dependent cell survival in VSMC is mediated only by activation of the PI3K/Akt pathway, whereas all other receptor-associated signaling molecules do not play a significant role. Peptide growth factors contribute to the pathogenesis of cardiovascular diseases by inducing a variety of cellular responses including anti-apoptotic effects. Several of the signaling molecules that are activated by growth factor receptors such as Src family kinases (Src), phosphatidylinositol 3′-kinase (PI3K), phospholipase Cγ (PLCγ), Ras, and SHP-2 were shown to mediate survival signals. We systematically investigated the relative contribution of each signaling molecule for growth factor-dependent cell survival in vascular smooth muscle cells (VSMC). Our approach was the use of mutated plateletderived growth factor (PDGF) β-receptors (βPDGFR) in which the tyrosine residues required for binding of each signaling molecule were individually mutated to phenylalanine. To bypass endogenous PDGFR in VSMC we used chimeric receptors (ChiRs), containing the extracellular domain of the macrophage colony-stimulating factor (M-CSF) receptor and the cytoplasmic domain of the wild type (WT) or mutated βPDGFR. Selective activation of the ChiR-WT with M-CSF significantly reduced apoptosis to the same extent as PDGF-BB in non-transfected cells. Deletion of the binding site for PI3K, but not for Src, RasGAP, SHP-2, or PLCγ, completely abolished the anti-apoptotic effect. Consistently, a ChiR mutant that only binds PI3K was fully able to mediate cell survival as efficiently as the ChiR-WT. Furthermore, the PDGF-dependent anti-apoptotic effect in non-transfected cells was completely abolished by the PI3K inhibitor wortmannin, whereas inhibitors of Src, PLCγ, ERK, or p38 MAP kinase had no effect. The exploration of downstream signaling events revealed that PDGF-BB activates the anti-apoptotic Akt signaling pathway in a PI3K-dependent manner. Moreover, Akt phosphorylates and thus inactivates the pro-apoptotic proteins BAD and Forkhead transcription factors (FKHR, FKHRL1). We conclude that growth factor-dependent cell survival in VSMC is mediated only by activation of the PI3K/Akt pathway, whereas all other receptor-associated signaling molecules do not play a significant role. The pathogenesis of vascular diseases involves an abnormal accumulation of lipids, inflammatory cells, and vascular smooth muscle cells (VSMC) 1The abbreviations used are: VSMC, vascular smooth muscle cells; SH, Src homology; Src, Src family kinases; PI3K, phosphatidyl inositol-3 kinase; PLCγ, phospholipase Cγ1; RasGAP, GTPase activating protein of Ras; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; STAT, signal transducer and activator of transcription; GSK3β, glycogen synthase kinase-3β, CSF1R, colony-stimulating factor 1 receptor; ChiR, chimeric CSF1R/PDGFR mutants; M-CSF, macrophage CSF; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; MAP, mitogen-activated protein; ERK, extracellular signal-related kinase. within the intimal layer of the vessel wall. Recent studies have demonstrated that in addition to cell proliferation and migration, dysregulated apoptosis plays a prominent role in the pathogenesis and progression of atherosclerosis (1.Stoneman V.E. Bennett M.R. Clin. Sci. (Lond.). 2004; 1: 1Google Scholar). Peptide growth factors such as platelet-derived growth factor (PDGF) and insulin-like growth factor-I are potent inhibitors of cell death. These growth factors exert their biological responses via activation of transmembrane receptor tyrosine kinases. Upon ligand binding, receptor tyrosine kinases autophosphorylate on tyrosine residues and subsequently recruit and activate SH2 domain-containing cytoplasmic signaling molecules such as Src family kinases (Src), the GTPase activating protein of Ras (RasGAP), the phosphotyrosine phosphatase SHP-2, phosphatidylinositol 3′-kinase (PI3K), and phospholipase Cγ1 (PLCγ) (2.Heldin C.H. Westermark B. Physiol. Rev. 1999; 79: 1283-1316Crossref PubMed Scopus (1978) Google Scholar). Recent studies indicated that all of these signaling molecules are implicated in survival signaling and therefore may contribute to growth factor-mediated cell survival. In PDGF-stimulated cells, Src family kinases are required for activation of STAT (signal transducer and activator of transcription) 3 (3.Cirri P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. Biophys. Res. Commun. 1997; 239: 493-497Crossref PubMed Scopus (56) Google Scholar, 4.Wang Y.Z. Wharton W. Garcia R. Kraker A. Jove R. Pledger W.J. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (100) Google Scholar), which protects fibroblasts from apoptosis and antagonizes the pro-apoptotic effects of activated STAT1 (5.Shen Y. Devgan G. Darnell Jr., J.E. Bromberg J.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1543-1548Crossref PubMed Scopus (233) Google Scholar). In addition, Src was shown to mediate anti-apoptotic effects by activating PI3K (6.Schlessinger J. Cell. 2000; 100: 293-296Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). PI3K mediates anti-apoptotic signals of several growth factors via activation of its downstream target Akt (7.Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1293) Google Scholar). The serine/threonine kinase Akt (8.Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1828) Google Scholar, 9.Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1667) Google Scholar) phosphorylates and thus inactivates several pro-apoptotic proteins such as BAD (10.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4946) Google Scholar), Forkhead transcription factors or caspase-9 (11.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 12.Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2735) Google Scholar). Further downstream mediators of this pathway include NFκB and glycogen synthase kinase-3β (GSK3β) (9.Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1667) Google Scholar, 13.Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar). Recently, SHP-2 was shown to be essential for insulin-like growth factor-I- or PDGF-mediated cell survival, as it suppresses caspase 3-dependent apoptosis in fibroblasts (14.Ivins Zito C. Kontaridis M.I. Fornaro M. Feng G.S. Bennett A.M. J. Cell. Physiol. 2004; 199: 227-236Crossref PubMed Scopus (85) Google Scholar). Another well characterized survival pathway is the Ras/Raf/extracellular-regulated kinase (ERK) cascade, which leads to the phosphorylation of BAD (15.Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1680) Google Scholar, 16.Scheid M.P. Schubert K.M. Duronio V. J. Biol. Chem. 1999; 274: 31108-31113Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Finally, PLCγ is required for insulin-like growth factor-I-mediated protection against apoptosis (17.Chattopadhyay A. Carpenter G. J. Cell Sci. 2002; 115: 2233-2239Crossref PubMed Google Scholar), as it mediates cell survival via protein kinase C-dependent phosphorylation of Bcl-2 proteins such as Bcl-2 and BAD (18.Zhao M. Eaton J.W. Brunk U.T. FEBS Lett. 2001; 509: 405-412Crossref PubMed Scopus (57) Google Scholar, 19.Ito T. Deng X. Carr B. May J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. H. M.R. M.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of the studies investigated only of the growth signal and studies used which may or a which may cell survival by protein inhibitors as well as proteins the of the signaling molecule in cells, the their role for cellular the relative of Src, PLCγ, RasGAP, PI3K, and SHP-2 for growth factor-mediated cell survival are not from these we to systematically the relative contribution of each of the signaling molecules for growth factor-mediated cell survival in We the this receptor tyrosine which is for and binds each of the receptor-associated signal binding Our approach was to in which the binding for or of the receptor-associated signaling were individually by tyrosine to we PDGFR signaling in VSMC, which are in and in which PDGFR signaling is as these cells endogenous PDGFR and thus to to PDGFR in cells, it is to bypass endogenous βPDGFR. we used chimeric M-CSF receptor containing the extracellular domain of the CSF1R, which is not in VSMC, and the cytoplasmic domain of the βPDGFR. these signaling is with The use of this to activate signaling molecules that are to the and to the of the ChiR to VSMC against Cell were from by as J. R. Physiol. Rev. PubMed Scopus Google Scholar). were in a in with and and of ChiR ChiR was from of the and of the an site K. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in the are as A. A. Cooper J.A. M. Cell. Biol. PubMed Google Scholar, M. C. Kazlauskas A. Cell. Biol. PubMed Google Scholar, Kazlauskas A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, K. Kazlauskas A. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and were by and were in the Google Scholar), and the cell as S. J.A. C. Kazlauskas A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). were to were with and VSMC were or with PDGF-BB or M-CSF for indicated in the or of inhibitors as were with and in 1 were and the were to or The and the were with a the kinase of the βPDGFR. cells were on a and the proteins were to and to To the of signaling molecules with the activated receptor from or cells were to against the extracellular domain of the PLCγ, RasGAP, and To the Src and the Src was from or cells the and cells were to or BAD was with a BAD were to protein an and with proteins were in BAD cells were on a were to a BAD or a BAD To the of ChiR in VSMC and the phosphorylation of downstream signaling cell cells were on a and with and were against BAD and GSK3β, and The are of and of was by or cell was used to cell by the of cytoplasmic and were in containing and for in To the anti-apoptotic effect of PDGF-BB or M-CSF the cells were and subsequently for in the or of the cells were for in containing in the or of PDGF-BB or The cell were and apoptosis was the cell and M-CSF were from Cell The against the extracellular domain of the was from The were from and The against SHP-2 was from and the PLCγ was from The against the and the of PI3K were from Kazlauskas and The used for of Src was from and the Src family Src, and of Src, a was The and BAD were from Cell The BAD was from are as was by was of systematically the role of each of the signaling molecules PI3K, RasGAP, SHP-2, and for growth factor-mediated cell survival in a cell we a of ChiR in which the tyrosine residues required for the of each of the signaling were individually mutated to of was on the in which the tyrosine phosphorylation for binding of PI3K, RasGAP, SHP-2, and PLCγ were of receptors that have the tyrosine required for binding of of the proteins In this the were as their in the was shown to the tyrosine phosphorylation of the Kazlauskas A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). ChiR were in an receptor was which an of tyrosine is to the protein E. E. J. S. J. C.H. Cell. Biol. PubMed Google Scholar), which is in Ras activation via binding of Finally, we a to as which a to within the kinase domain and as a demonstrated in and all ChiR were and tyrosine and binding of the of ChiR VSMC the were and to against the extracellular domain of the M-CSF receptor or as a tyrosine phosphorylation and with signaling VSMC the ChiR-WT or the phosphorylation site were or with M-CSF for Cell were to with an the βPDGFR. were by with revealed that were of ChiR in all of the The receptor was and with a of of was against RasGAP, PLCγ, and SHP-2 to of these signaling molecules with the activated of and with The receptor with and no in kinase and was used as a were and and the were with an that Src family were with to of the ChiR or with to the of Src in each of the and tyrosine and binding of the of ChiR The was as and B. tyrosine phosphorylation and with signaling The are of To the of the receptor and binding of signaling were from or cells, and the were to against the extracellular domain of the and against each of the signal In addition, the receptor was and with an shown in and M-CSF the phosphotyrosine of all of the with the of the mutant The of receptor was not in to or phosphorylation may the of phosphorylation or the of the receptor to autophosphorylate is with of the S. J.A. C. Kazlauskas A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, ChiR-WT with Src, the of PI3K, RasGAP, SHP-2, and PLCγ in a manner. ChiR or binding of receptor-associated proteins was in receptor from non-transfected The of tyrosine reduced the binding of PLCγ, the mutant to with PI3K and the and not RasGAP, SHP-2, and Src M-CSF and the binding of the of of the PLCγ, RasGAP, and and the with PLCγ, RasGAP, and whereas of the other signal with these with of SHP-2 to the activated receptor and to all with the of the and that SHP-2 is able to with binding site tyrosine that binding or activation of PI3K SHP-2 binding to this or these A. C. U. U. C. C.H. K. M. A. Oncogene. 1999; PubMed Scopus Google Scholar) have that SHP-2 is to to tyrosine which is to the that PI3K binding may the of SHP-2 with tyrosine which as a in the biological was not and to recruit signaling molecules M-CSF These that the ChiR mutant in VSMC and to anti-apoptotic M-CSF PDGF-dependent Cell in that the activated ChiR-WT survival responses we the apoptosis of PDGF-stimulated non-transfected VSMC with that of VSMC or and to a and in cell The of with PDGF-BB significantly and cell by and Consistently, or of VSMC with a and in cell with cells. with M-CSF significantly reduced the of or apoptosis by and whereas it had no effect on cell survival in non-transfected VSMC of signaling by M-CSF in VSMC was to mediate responses to the same extent as PDGF-BB in non-transfected cells. of in the role of each of the signaling molecules for protection from we this in VSMC ChiR-WT or the mutant We apoptosis by of cells. of the revealed that of the binding site for PI3K but not for Src, RasGAP, SHP-2, or PLCγ, completely the anti-apoptotic effect Consistently, binding and activation of only PI3K activation of RasGAP, SHP-2, and PLCγ via the receptor was to mediate the anti-apoptotic effect. In all other were to mediate a significant anti-apoptotic effect To the that were to of we apoptosis was by to the a of PI3K binding completely abolished the anti-apoptotic effect mediated by the whereas of the site to the receptor fully this cellular of by in of receptor is the in addition to the binding of other signaling molecules may with these To the which we by the use of ChiR in a we all in PDGF-stimulated non-transfected VSMC in the of Consistently, of PI3K by completely the anti-apoptotic effect of PDGF-BB that this signaling is in anti-apoptotic whereas of Src kinases and p38 had no effect on PDGF-dependent signaling of PLCγ by PDGF-mediated cell survival but not apoptosis was by to cell survival by used in In that anti-apoptotic signaling by the is mediated by only of the receptor-associated signaling PI3K, whereas all other signal do not play a significant role. PI3K/Akt Cell via of BAD and Forkhead activation of Akt is to play a role in PI3K-dependent of we investigated the role of the receptor-associated proteins for Akt The phosphorylation of Akt was by a Akt in VSMC as well as in PDGF-stimulated non-transfected VSMC in the of Akt was and activated for several In addition to Akt its was by the to its We the contribution of each signaling molecule to Akt In cells, Akt was M-CSF with a and to a extent as in VSMC The receptor not efficiently activate whereas the tyrosine to all other had no effect on activation of In the cells, Akt activation was completely abolished of the site activation of whereas the and Akt activation to a The that these are able to Akt may be by binding of PI3K to these the is PI3K efficiently binds to the activated that Akt is activated in to PI3K binding to the and was with to the PDGF-stimulated endogenous of PI3K, but not of Src, PLCγ, or phosphorylation of Akt and These that only PI3K is able to efficiently activate the anti-apoptotic Akt pathway and that the mediates its effect via the PI3K/Akt survival To the signaling downstream of we the of a of pro-apoptotic To this we the pro-apoptotic protein BAD from or cells by the of its phosphorylation which is the phosphorylation a PDGF-BB a and BAD phosphorylation effect was on in VSMC the receptor not mediate phosphorylation of BAD whereas cells In addition, of PI3K with the PDGF-dependent BAD phosphorylation whereas other inhibitors had no Finally, the pro-apoptotic transcription factors of the Forkhead family and of the same phosphorylation as Forkhead transcription factors were and thus by that were of binding PI3K in to M-CSF and PI3K was not In the we have systematically investigated the relative of the signaling that are to the activated for protection against apoptosis in receptor and we that PDGF-dependent cell survival is mediated by only of the signaling the other receptor-associated signaling molecules such as Src, PLCγ, RasGAP, and SHP-2 were shown to mediate survival their contribution to growth anti-apoptotic effects is not significant with PI3K/Akt In addition, we characterized the signaling downstream from which of pro-apoptotic BAD and Forkhead transcription factors and The of ChiR was used to the signaling that contribute to cell survival in a cell that endogenous PDGFR, To endogenous PDGFR, the cytoplasmic of mutated were via binding of M-CSF to the extracellular this we were able to activate in cells and the activation of signaling their The of the and of chimeric as well as the use of inhibitors revealed that the signaling only PI3K is for protection against Deletion of the in the receptor completely abolished the anti-apoptotic whereas of the to the receptor was fully to cell survival. These that binding and activation of PI3K to the is and for of Consistently, of PI3K with to a of cell survival in non-transfected cells. In of all other binding in the ChiR as well as of Src, PLCγ, or MAP kinases not protection against of the PI3K/Akt pathway shown to apoptosis in a variety of cell A. P. S. J. J. 1997; PubMed Scopus Google Scholar, J. A. Tsichlis P.N. N. 1997; PubMed Scopus Google Scholar, K. A. Chan T.O. Tsichlis P.N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. A. M.J. Cell. Biol. 1997; PubMed Scopus Google Scholar). The of downstream signaling events revealed that Akt and the Akt Forkhead transcription and were only PI3K was Upon phosphorylation pro-apoptotic proteins such as Forkhead transcription and are and in the to (11.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, J. H. Yang E. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). these that growth protection from apoptosis is mediated via PI3K, and the PI3K-dependent Akt signaling pathway to of pro-apoptotic proteins is for cellular survival. on that Src, RasGAP, SHP-2, and PLCγ all mediate survival signals (3.Cirri P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. Biophys. Res. Commun. 1997; 239: 493-497Crossref PubMed Scopus (56) Google Scholar, 4.Wang Y.Z. Wharton W. Garcia R. Kraker A. Jove R. Pledger W.J. Oncogene. 2000; 19: 2075-2085Crossref PubMed Scopus (100) Google Scholar, Y. Devgan G. Darnell Jr., J.E. Bromberg J.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1543-1548Crossref PubMed Scopus (233) Google Scholar, Zito C. Kontaridis M.I. Fornaro M. Feng G.S. Bennett A.M. J. Cell. Physiol. 2004; 199: 227-236Crossref PubMed Scopus (85) Google Scholar, A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1680) Google Scholar, 16.Scheid M.P. Schubert K.M. Duronio V. J. Biol. Chem. 1999; 274: 31108-31113Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, A. Carpenter G. J. Cell Sci. 2002; 115: 2233-2239Crossref PubMed Google Scholar, M. Eaton J.W. Brunk U.T. FEBS Lett. 2001; 509: 405-412Crossref PubMed Scopus (57) Google Scholar, 19.Ito T. Deng X. Carr B. May J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. H. M.R. M.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), it that of significantly to growth factor-mediated cell survival in The these studies and may be by the that these signaling exert anti-apoptotic effects by to PI3K/Akt activation by inducing survival as indicated by studies (14.Ivins Zito C. Kontaridis M.I. Fornaro M. Feng G.S. Bennett A.M. J. Cell. 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In we completely anti-apoptotic effects of activated ChiR mutant was able to activate a ChiR which mediated a of the ChiR-WT in which the site is in the not or apoptosis that is not able to mediate anti-apoptotic effects in the of other signaling is that or is of survival effects. apoptosis factor have only effect on VSMC apoptosis M. P. Biol. PubMed Scopus Google Scholar) we used to was used as a of which by and activation of receptors J. Proc. 1999; Full Text PDF PubMed Scopus Google Scholar) and is used to apoptosis in cell To that were to apoptosis we used a which activates the pathway to apoptosis in may a of are from vascular such as which the progression of these P. J. 1995; Google Scholar, M. S. J. 1995; 91: PubMed Scopus Google Scholar). a of is its to activate signaling PLCγ Deng F. Y. Biochem. J. 2002; PubMed Scopus Google Scholar). we with apoptosis that growth factor-dependent activation of PI3K/Akt In the of the signaling that are on in a PDGF-stimulated cell that only signaling to the PI3K, for cell survival, whereas all other receptor-associated signaling molecules do not play a significant role. PI3K is able to efficiently activate the anti-apoptotic Akt signaling pathway and mediates its survival effects via of pro-apoptotic proteins including BAD and Forkhead transcription We the of We Kazlauskas and for and