Bcl-2, via Its BH4 Domain, Blocks Apoptotic Signaling Mediated by Mitochondrial Ras

Bcl-2 protects cells against Ras-mediated apoptosis; this protection coincides with its binding to Ras. However, the protection mechanism has remained enigmatic. Here, we demonstrate that, upon apoptotic stimulation, newly synthesized Bcl-2 redistributes to mitochondria, interacts there with activated Ras, and blocks Ras-mediated apoptotic signaling. We also show, by employing bcl-2 mutants, that the BH4 domain of Bcl-2 binds to Ras and regulates its anti-apoptotic activity. Experiments with a C-terminal-truncated Ras or a farnesyltransferase inhibitor demonstrate that the CAAX motif of Ras is essential for apoptotic signaling and Bcl-2 association. The results indicate a potential mechanism by which Bcl-2 protects cells against Ras-mediated apoptotic signaling. Bcl-2 protects cells against Ras-mediated apoptosis; this protection coincides with its binding to Ras. However, the protection mechanism has remained enigmatic. Here, we demonstrate that, upon apoptotic stimulation, newly synthesized Bcl-2 redistributes to mitochondria, interacts there with activated Ras, and blocks Ras-mediated apoptotic signaling. We also show, by employing bcl-2 mutants, that the BH4 domain of Bcl-2 binds to Ras and regulates its anti-apoptotic activity. Experiments with a C-terminal-truncated Ras or a farnesyltransferase inhibitor demonstrate that the CAAX motif of Ras is essential for apoptotic signaling and Bcl-2 association. The results indicate a potential mechanism by which Bcl-2 protects cells against Ras-mediated apoptotic signaling. Bcl-2 suppress apoptosis elicited by various pro-death stimuli (1Sentman C.L. Shutter J.R. Hockenbery D. Kanagawa O. Korsmeyer S.J. Cell. 1991; 67: 879-888Google Scholar, 2Strasser A. Harris A.W. Huang D.C. Krammer P.H. Cory S. EMBO J. 1995; 14: 6136-6147Google Scholar, 3Vaux D.L. Cory S. Adams J.M. Nature. 1988; 335: 440-442Google Scholar). Bcl-2 family members include anti- and pro-apoptotic factors that possess hydrophobic stretches of amino acids at the C termini that allow them to bind to intracellular membranes, including the membranes of ER 1The abbreviations used are: ER, endoplasmic reticulum; PKC, protein kinase C; Ab, antibody; wt, wild type; FTI, farnesyl transferase inhibitor; CAAX , motif whereA is an aliphatic residue and X is any amino acid; HMG, 1-O-hexadecyl-2-O-methyl-rac-glycerol; DISC, death-inducing signaling complex; BH, Bcl-2 homology; FADD, Fas-associated death domain and of mitochondria (4Gonzalez-Garcia M. Perez-Ballestero R. Ding L. Duan L. Biose L.H. Thompson C.B. Nunez G. Development. 1994; 120: 3033-3042Google Scholar, 5Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714Google Scholar, 6Yang T. Kozopas K. Craig R. J. Cell Biol. 1995; 128: 1173-1184Google Scholar). Bcl-2 and most of its homologs share four conserved regions: Bcl-2 homology domains (BH1–4), through which the proteins dimerize with each other or with other proteins (7Gross A. McDonnell J.M. Korsmeyer S.J. Gene Dev. 1999; 13: 1899-1911Google Scholar, 8Hunter J.J. Bond B.L. Parslow T.G. Mol. Cell. Biol. 1996; 16: 877-883Google Scholar, 9Reed C.J. Nature. 1997; 387: 773-776Google Scholar, 10Wang H.-G. Rapp U.R. Reed J.C. Cell. 1996; 15: 629-638Google Scholar, 11Yin X.-M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Google Scholar). The BH1 and BH2 domains of Bcl-2 are required for homodimerization with the pro-apoptotic Bcl-2 family member Bax. The BH3 domain of some members of the Bcl-2 family (BH3-only proteins, such as Bad, Bik, or Bid) interacts with other Bcl-2 proteins to initiate apoptosis (12David C. Huang S. Strasser A. Cell. 2000; 103: 839-842Google Scholar, 13Fesik S.W. Cell. 2000; 103: 273-282Google Scholar). The BH4 domain, a heterodimerization region, is responsible for Bcl-2-directed targeting of Raf-1 to mitochondria and the cooperation between Bcl-2 and Raf-1 in the suppression of apoptosis (50Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Google Scholar). The loop region of Bcl-2 regulates its protective function (14Chang B.S. Minn A. Muchmore S.W. Fesik S.W. Thompson C.B. EMBO J. 1997; 16: 968-977Google Scholar). Bcl-2 that lacks its C-terminal domain inefficiently associates with cellular membranes and is incapable of preventing apoptosis (15Nguyen M. Branton P. Walton P. Oltvai Z. Korsmeyer S. Shore G. J. Biol. Chem. 1994; 269: 16521-16524Google Scholar, 16Tanaka S. Saito K. Reed J. J. Biol. Chem. 1993; 268: 10920-10926Google Scholar). We previously demonstrated that overexpressed Bcl-2 prevents cells that express v-Ha-rasfrom undergoing apoptosis in response to protein kinase C (PKC) down-regulation. This protection coincides with the binding of Bcl-2 to Ras (17Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498Google Scholar, 18Chen C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar). However, the molecular mechanism by which the interaction of Bcl-2 and Ras regulates apoptosis has remained unknown. The biological effects of Ras on cell growth or apoptosis depend strongly upon the kind of stimulus, cell type, or regulatory environment (19Downward J.D. Curr. Opin. Genet. Dev. 1998; 8: 49-54Google Scholar). Human or mouse lymphocytes expressing activatedras undergo apoptosis in response to PKC down-regulation and this apoptotic process is blocked by overexpression of bcl-2(17Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498Google Scholar, 18Chen C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar, 20Chen C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Google Scholar). Ras activity is involved in Fas-regulated, multiple signaling pathways (13Fesik S.W. Cell. 2000; 103: 273-282Google Scholar, 21Gulbins E. Brenner B. Koppenhoefer U. Linderkamp O. Lang F. J. Leukoc. Biol. 1998; 78: 247-306Google Scholar). However, Bcl-2 partially protects lymphocytes from Fas-induced apoptosis, possibly through decreasing the permeability of the mitochondrial membrane (22Itoh N. Tsujimoto Y. Nagata S. J. Immunol. 1993; 151: 621-627Google Scholar, 23Jaattela M. Benedict M. Tewari M. Shayman J.A. Dixit V.M. Oncogene. 1995; 10: 2297-2305Google Scholar). It is well known that upon activation or mitogenic stimulation a series of post-translational modifications, including prenylation, -AAX proteolysis, and carboxyl methylation, occur on the C terminus of Ras. This process allows Ras to associate with membrane compartments, especially with plasma membrane (24Bartels D.J. Mitchell D.A. Dong X. Deschenes R.J. Mol. Cell. Biol. 1999; 19: 6775-6787Google Scholar, 25Clark S. Annu. Rev. Biochem. 1992; 61: 355-386Google Scholar, 26Deschenes R.J. Resh M.D. Broach J.R. Curr. Opin. Cell Biol. 1990; 2: 1108-1113Google Scholar). Ras family proteins (Ki-, Ha-, and N-Ras) have also been demonstrated to associate with mitochondria in murine lymphokine-dependent TS1αβ cells (27Rebollo A. Perey-Sala D. Martiney-A C. Oncogene. 1999; 18: 4930-4939Google Scholar). The function of mitochondrial Ras has not been fully investigated yet. Furthermore, the CAAX motif of Ras may regulate its N-terminal conformation and further its to with other proteins A. Harris A.W. Huang D.C. Krammer P.H. Cory S. EMBO J. 1995; 14: 6136-6147Google Scholar, 26Deschenes R.J. Resh M.D. Broach J.R. Curr. Opin. Cell Biol. 1990; 2: 1108-1113Google Scholar, A. J. J. Cell Biol. Scholar). It on the CAAX motif of Ras is required for its apoptotic signaling. Fas-induced apoptosis, Ras in cells is activated the signaling (22Itoh N. Tsujimoto Y. Nagata S. J. Immunol. 1993; 151: 621-627Google Scholar, 23Jaattela M. Benedict M. Tewari M. Shayman J.A. Dixit V.M. Oncogene. 1995; 10: 2297-2305Google Scholar, E. R. A. S. W. T. G. G. G. Lang F. D. 1995; 2: Scholar). cells have been to to which may to of Bcl-2 C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar, M.J. J.R. Nature. 1993; Scholar). to further the of Bcl-2 protection against Ras-mediated apoptotic we bcl-2 or as well as and the in the of or PKC We demonstrate that, apoptotic newly synthesized Bcl-2 is in mitochondria, and binds to mitochondrial Ras. in cells that express of Bcl-2 or mouse cells that activated by various we the BH4 domain of Bcl-2 as the interaction with Ras, and this domain is for protection against Ras-mediated apoptotic signaling. Furthermore, we that the of the CAAX motif of Ras and its are for apoptotic and regulate the of Ras to associate with cells from cells by and from J. The cell and the cell from S. of The cells from the of mouse by T. with a C-terminal the The of by (17Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498Google Scholar). bcl-2 and various bcl-2 by T. Parslow of at ER and a a for the of ER the with and in of J. J.D. P.H. S. D.A. Nature. 1990; Scholar). The cell to a and by through a The at for to and the of the cell and the at for The and the in the for on and at The (50Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Google Scholar). the ER an the mitochondrial cells in and to on for the in on a C. S. A. C. F. Krammer P.H. EMBO J. 1998; Scholar). with for for cells or for cell and protein in each the to and J. J.D. P.H. S. D.A. Nature. 1990; Scholar). for The with or Bcl-2 for at with protein and on a Bcl-2 or Ras with or or used as cell the cells with for and with a with the with for for cells or for cells with with and with and We previously demonstrated that apoptosis the activity of PKC is and that the overexpression of Bcl-2 blocks this possibly through its with Ras C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar). The mechanism of protection against Ras apoptotic signaling is Ras is involved in Fas-induced apoptosis and Bcl-2 partially protects cells from Fas-induced apoptosis C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Google Scholar, N. Tsujimoto Y. Nagata S. J. Immunol. 1993; 151: 621-627Google Scholar, 23Jaattela M. Benedict M. Tewari M. Shayman J.A. Dixit V.M. Oncogene. 1995; 10: 2297-2305Google Scholar, K. Y. C. P. Cell. 1998; Scholar, A. J. Immunol. 1998; we Bcl-2 interacts with Ras Fas-induced or cells or and a The cells with for or to and the of the cells with by cells that to Fas-induced apoptosis the other cell The of cells with at of and that activated Ras Fas-induced However, in cells not and at The of in cells between the in and It that overexpressed in the to protects cells from cell or the of the apoptotic also an not apoptosis not of also to the that of the of The of not or overexpressed We Bcl-2 or Ras protein or cells of The of Bcl-2 in a cell from and cells or and not further of the protein Ras a in which the express of the protein with the cell not protein the cells express a of Bcl-2 or Ras, we used and to the of Bcl-2 and Ras. The from cell with or for Bcl-2 in the mitochondrial of and the protein in response to Bcl-2 in the ER growth the of Bcl-2 in ER Bcl-2 not in the from as The of Ras in the also by of Ras protein in various membrane from cells growth and not the protein and of Ras protein in the mitochondrial and plasma membrane from and the not upon The that apoptotic stimulation by the of Bcl-2 in the compartments, not of Ras. The of the from the with or by for the of mitochondrial an endoplasmic and plasma membrane the apoptotic stimulation, of the proteins in in to the Bcl-2 in various also in and results which the of not We of Bcl-2 in occur in and cells to a PKC for and the mitochondrial and ER for response to PKC the of Bcl-2 in the ER from cells which with protein in PKC suppression is not a death to the of the protein in the from the cells not which that the of Bcl-2 in mitochondria of cells PKC suppression is to The of the from the with or by the It is known that Bcl-2 is in some cells S. M. C. D. F. R. E. A. Cell. 1991; Scholar). and cell used to Bcl-2 the We also mouse cells mouse cell and mouse cell from the of The of Bcl-2 or Ras in cells by of Bcl-2 or Ras in or The of proteins in each by the with We or cells to to apoptosis, a the cells with HMG, the of the cells with by The inhibitor not apoptosis in which is with in which cells not in response to PKC suppression (17Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498Google Scholar, 18Chen C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar). However, the of not the cells to cell that overexpression of Bcl-2 blocks the apoptotic of mouse cells with this apoptotic process in The of Bcl-2 in of the cells also The ER and mitochondrial from the cells with or and with an of Bcl-2 in the ER from and and the protein in the ER from Bcl-2 in the ER in and not in mitochondria, a of the protein in the However, the protein in cells in response to The indicate that Bcl-2 redistributes and protects the cells against The of the from the with or by We to the of Bcl-2 in the apoptosis is to the or of the of ER or mitochondrial Bcl-2 in with or protein Bcl-2 in the ER growth and a of the protein in the of Bcl-2 in ER in the protein in mitochondria in the protein by the of ER with or to in to the the mitochondrial in the of the protein to an The that the of mitochondrial Bcl-2 by in is from the newly synthesized not from Bcl-2 this cells with the ER and mitochondrial for or and the Bcl-2 in ER, with or the mitochondrial a of Bcl-2 also a and Bcl-2 in the ER and the mitochondrial Bcl-2 the results the that newly synthesized upon is in We previously demonstrated that, in Bcl-2 to Ras in response to PKC down-regulation C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar). Here, we to further this by apoptotic and to the of the The of Bcl-2 and Ras of in the cell from or cells an Bcl-2 with the from and The not in or which is to the that cells express of Bcl-2 and Ras. The from the and of the mitochondrial in which a of Bcl-2 in the from cells and a of the protein in The interaction not in the ER from any of the four cell a the with Ab, an an of Ras in the from the cell or mitochondrial of and a of Ras in the of in response to Ras not with the in ER from any of the four cell in response to The of Ras or Bcl-2 in the from also by the with the or the a used for in cell from the by with or to the of binding of proteins of Bcl-2 or Ras with The results from the mitochondrial not The and of Bcl-2 and Ras, in response to PKC also in and cells The interaction between Bcl-2 and Ras, in or of Bcl-2 by the in The not in cells in response to the PKC is not a death to the The of Ras in the by the with the the that the of Bcl-2 with Ras apoptotic and in an in mitochondrial and Bcl-2 regulates the permeability to apoptosis S. J. Biol. Chem. 1997; Scholar, Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: Scholar, G. K. Dixit V.M. J. Biol. Chem. 1998; 273: Scholar). We or mitochondrial to from and at various for in the from cells of the protein the apoptotic the protein in the of Ab, and in The of in cells to The that the anti-apoptotic effects of bcl-2 and the pro-apoptotic to the of mitochondrial from Fas-induced apoptotic signaling. The of the from the with or by not The of the death-inducing signaling of and is the in signaling R.J. U. S. A. 1997; Scholar). that the of Bcl-2 and its interaction with Ras depend upon an DISC, or cells or The in the of is to or the of Bcl-2 by Ras in the cells not and the as in the cells as The of the from the with or by for or and also with cell from the cells as well as cells a Bcl-2 not with Ras in the cells the apoptotic that the interaction the apoptotic signaling. The of Ras in the by the with the we the region of Bcl-2 that is responsible for the bcl-2 Bcl-2 proteins that at BH4 BH3 BH1 and BH2 and the of the proteins by growth Bcl-2 proteins not in mitochondria not Bcl-2 proteins in The of Bcl-2 to bind to Ras by and growth there interaction of Bcl-2 or with Ras in and proteins with Ras in this However, the protein not with Ras the from the not The of Ras protein in the by the with the the that the BH4 domain of Bcl-2 is for its interaction with Ras, and apoptotic The of Ras with or in mitochondria not in the cells However, and in the Ras the protein not with Ras in The of Ras protein in the also by the with the that the BH4 domain of Bcl-2 is the binding region for its interaction with Ras. However, Bcl-2 that lacks the BH4 domain is of to mitochondria upon of the death It is known that the CAAX domain of Ras regulates its signaling in response to mitogenic stimulation (24Bartels D.J. Mitchell D.A. Dong X. Deschenes R.J. Mol. Cell. Biol. 1999; 19: 6775-6787Google Scholar, 25Clark S. Annu. Rev. Biochem. 1992; 61: 355-386Google Scholar, 26Deschenes R.J. Resh M.D. Broach J.R. Curr. Opin. Cell Biol. 1990; 2: 1108-1113Google Scholar). C-terminal The Ras in cell from with on the The of the Ras in mitochondria also and the protein not in this membrane with or the of the C terminus of Ras its interaction with Bcl-2 apoptosis, cell from or cells for and The Ras not bind to Bcl-2 We the of the the interaction of Ras with farnesyl transferase inhibitor cells with for to in to of activated Ras. the and in the cell The of Ras with Bcl-2 in the cells blocked by The of the Ras or Ras by the with the not the that may a in Bcl-2 or in Ras and a that not by the and with the in or with to not the of the proteins to the that the of Ras is required for its interaction with Bcl-2 the apoptotic The of the bcl-2 to suppress Fas-induced apoptosis with a of the of Bcl-2 not cells from The of in the cells to that in which express the BH1 BH3 and BH2 of Bcl-2 to apoptosis as that by which is in with the that the pro-apoptotic is not involved in Fas-induced apoptosis K. Y. C. P. Cell. 1998; Scholar, R. A. R. J.M. Cell. 1998; Scholar). The protection by Bcl-2 also in and results not We also Ras apoptotic of to suppress prenylation, the of cells with in response to The of not the of or cells to Fas-induced We that Bcl-2 may Ras apoptotic signaling in cells its BH4 to its in mitogenic stimulation, Ras to its apoptotic cells undergo apoptosis in response to and overexpressed Bcl-2 partially protects the cells from this apoptotic process C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Google Scholar, N. Tsujimoto Y. Nagata S. J. Immunol. 1993; 151: 621-627Google Scholar, 23Jaattela M. Benedict M. Tewari M. Shayman J.A. Dixit V.M. Oncogene. 1995; 10: 2297-2305Google Scholar, E. R. A. S. W. T. G. G. G. Lang F. D. 1995; 2: Scholar, A. J. Immunol. 1998; Scholar). Ras activity is involved in the of Fas-induced apoptosis C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Google Scholar, E. R. A. S. W. T. G. G. G. Lang F. D. 1995; 2: Scholar). We previously demonstrated that overexpressed Bcl-2 in cells blocks apoptosis, possibly through its with Ras (17Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498Google Scholar, 18Chen C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar). Experiments further the mechanism by which Bcl-2 with apoptotic signaling elicited by activated The results that newly synthesized upon or PKC redistributes in the mitochondrial interacts there with Ras, and blocks Ras-mediated apoptotic signaling. activated Ras the apoptotic Bcl-2 and Ras are overexpressed apoptosis a bcl-2 that the protein domains demonstrate that Bcl-2 binds to mitochondrial Ras through its BH4 domain, and this region is also required for its suppression of Ras-mediated apoptotic signaling. However, the of the or heterodimerization domains of including have on its by or PKC Experiments with C-terminal-truncated Ras or that of the motif or of not the apoptotic activity of Ras, also its with The of and Bcl-2 family proteins have been in cell as well as in and A. Harris A.W. Huang D.C. Krammer P.H. Cory S. EMBO J. 1995; 14: 6136-6147Google Scholar, 9Reed C.J. Nature. 1997; 387: 773-776Google Scholar, N. Tsujimoto Y. Nagata S. J. Immunol. 1993; 151: 621-627Google Scholar, J. J. S. K. X. Korsmeyer S.J. J. Biol. Chem. 1996; 271: Scholar). that Ras activity is elicited signaling in the multiple apoptotic pathways by and that the Fas-induced death process (17Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498Google Scholar, 20Chen C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Google Scholar, E. R. A. S. W. T. G. G. G. Lang F. D. 1995; 2: Scholar). as a of apoptosis, Bcl-2 blocks cell death by a of and partially Fas-induced cell death A. Harris A.W. Huang D.C. Krammer P.H. Cory S. EMBO J. 1995; 14: 6136-6147Google Scholar, N. Tsujimoto Y. Nagata S. J. Immunol. 1993; 151: 621-627Google Scholar, 23Jaattela M. Benedict M. Tewari M. Shayman J.A. Dixit V.M. Oncogene. 1995; 10: 2297-2305Google Scholar, J. J. S. K. X. Korsmeyer S.J. J. Biol. Chem. 1996; 271: Scholar). to cells from apoptosis, Bcl-2 interacts with various factors and in in mitochondria M.D. G. C. T. E. M. T. M.J. J. Cell Biol. 1999; Scholar, Thompson C.B. Cell. 1997; Scholar). It is that, through binding to Ras, Bcl-2 the apoptotic signaling by Ras, not other the apoptotic Ras activity is involved in of the multiple Fas-induced death the interaction of proteins may the of the of the cells to It has been that apoptotic signaling to the of the cell death B. J. Immunol. 1995; C.-Y. Faller D.V. J. Biol. Chem. 1990; Scholar). of some of the as well as the activation of and B. J. Immunol. 1995; Scholar, C.-Y. Faller D.V. J. Biol. Chem. 1990; Scholar). Bcl-2 redistributes to mitochondria, there is protection against Ras-mediated Furthermore, in Bcl-2 not with Ras Fas-induced apoptosis, and further protects Bcl-2 as to function C.-Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar, T. X. B. J. Biol. Chem. 1997; is that Ras-mediated apoptotic signaling a that regulates Bcl-2 and further the anti-apoptotic activity of The C terminus of Bcl-2 a of hydrophobic amino acids that the protein in the of the membrane (4Gonzalez-Garcia M. Perez-Ballestero R. Ding L. Duan L. Biose L.H. Thompson C.B. Nunez G. Development. 1994; 120: 3033-3042Google Scholar, 5Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714Google Scholar, 6Yang T. Kozopas K. Craig R. J. Cell Biol. 1995; 128: 1173-1184Google Scholar). Bcl-2 that lacks the C-terminal domain the to associate with cellular membranes and to suppress cell death Z. J. Mol. Cell Biol. Scholar, M. W. M. Korsmeyer S.J. U. S. A. 1991; Scholar). Bcl-2 family such as or undergo in response to various apoptotic stimuli R.J. U. S. A. 1997; Scholar). Here, we demonstrate that overexpressed Bcl-2 is in membrane compartments, in the or PKC Bcl-2 or its domain express in The results that of Bcl-2 is an the apoptotic This process the C-terminal region of not the interaction the that the mitochondrial membrane has been as of the Bcl-2 and that in mitochondrial have been in apoptosis by Ras H.-G. Rapp U.R. Reed J.C. Cell. 1996; 15: 629-638Google Scholar, M.D. G. C. T. E. M. T. M.J. J. Cell Biol. 1999; Scholar, Thompson C.B. Cell. 1997; Scholar, Faller D.V. J. Biol. Chem. 2000; is to that apoptotic Bcl-2 to this Bcl-2 by signaling or PKC is mitochondria, and or blocks an in mitochondrial preventing the of or It is well that targeting of Ras protein multiple of post-translational at its C-terminal CAAX motif in response to growth or stimulation, such as (24Bartels D.J. Mitchell D.A. Dong X. Deschenes R.J. Mol. Cell. Biol. 1999; 19: 6775-6787Google Scholar, 25Clark S. Annu. Rev. Biochem. 1992; 61: 355-386Google Scholar, 26Deschenes R.J. Resh M.D. Broach J.R. Curr. Opin. Cell Biol. 1990; 2: 1108-1113Google Scholar). mitogenic or growth stimulation, Ras from to membrane compartments, to plasma However, is not Ras this for its apoptotic activity. We that of the of activated Ras with its binding to as well as its apoptotic activity. We also demonstrate that activated Ras proteins in various intracellular membrane and not undergo or upon apoptotic we that activated Ras in may have in biological upon the of the stimulation or the of the signaling of its known as that growth or from membrane to apoptotic stimulation, mitochondrial Ras may with a of to the death have the of multiple pathways in apoptosis (17Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498Google Scholar, 20Chen C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Google Scholar, E. R. A. S. W. T. G. G. G. Lang F. D. 1995; 2: Scholar). response to binds and to the which results in the activation of apoptotic signaling (50Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Google Scholar, P. C.J. J. J. Curr. Biol. 1998; 8: Scholar, C. A. M. Krammer P.H. EMBO J. 1997; 16: Scholar, M. K. A. J. C. J.D. M. R. M. Krammer P.H. Dixit V.M. Cell. 1996; 85: Scholar). Experiments from or cells or from the of in the of cell death Krammer P.H. J. Immunol. 1997; Scholar, J. D. A. A. Nature. 1998; Scholar, S. K. Cell 1998; Scholar). have also demonstrated that upon such as bind to and apoptotic signaling P. C.J. P. J. Cell 1999; 10: Scholar). The results of with and cell indicate that and are required for the interaction between Ras and and that Ras is a of and Fas-induced However, results not the of signaling pathways that may from that are not to the interaction or are of Ras activity. a has been between the of bcl-2 and the of bcl-2 in a cell and the of M. D. N. G. J. Immunol. 1994; Scholar, M. U. A. Reed J.C. A. J. 1995; Scholar). that Ras apoptosis in overexpressed Bcl-2 to the death the overexpression of Bcl-2 Ras cell of of Fas-induced apoptosis by activated Ras to the death process by Bcl-2 is to at an essential in the that is to for a of the between Bcl-2 and Ras in response to mitogenic activated Ras at the plasma membrane to and the growth death stimulation, apoptotic factors mitochondrial Ras a process which Bcl-2 We domains of Bcl-2 and Ras that this the BH4 domain of Bcl-2 its with Ras and protection against apoptotic We that BH4 domain of Bcl-2 may to in cell of the pathways or by may to or We T. Parslow of J. and J. for the of various bcl-2 mutants, cell and