Cleavage of 14-3-3 Protein by Caspase-3 Facilitates Bad Interaction with Bcl-x(L) during Apoptosis

Abstract

The 14-3-3ϵ protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3ϵ protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3ϵ protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3ϵ protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3ϵ protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3ϵ protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L). The 14-3-3ϵ protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3ϵ protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3ϵ protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3ϵ protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3ϵ protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3ϵ protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L). During the early events of apoptosis, mitochondria have been thought to act as central coordinators of cell death (1Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 2Reed J.C. Green D.R. Mol. Cell. 2002; 9: 1-3Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Some apoptotic signal cascades induce mitochondrial membrane permeabilization under the control of Bcl-2-related proteins in the mitochondria. The loss of transmembrane potential in the mitochondria then induces the release of apoptotic activators such as cytochrome c, Smac/DIABLO, HtrA2/Omi, AIF, Endo G, and caspases from the mitochondria (3Ravagnan L. Roumier T. Kroemer G. J. Cell. Physiol. 2002; 192: 131-137Crossref PubMed Scopus (435) Google Scholar). Released cytochrome c activates caspase-9, binds to Apaf-1, and induces Apaf-1 oligomerization to form apoptosome (4Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The apoptosome recruits and activates procaspase-9, which, in turn, activates inactive procaspase-3 into the active caspase-3, the executor molecule of apoptosis. Cytochrome c release was reported to be blocked by anti-apoptotic Bcl-2 or Bcl-x(L) and accelerated by pro-apoptotic Bax (5Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4266) Google Scholar, 6Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4395) Google Scholar). Bcl-2 family proteins are key regulatory proteins that play critical roles in mediating the signal transduction path that leads to apoptosis. The multi-BH domain members of the Bcl-2 protein family either suppress (Bcl-2 or Bcl-x(L)) or promote (Bax or BAK) apoptosis, whereas “BH3-only members” (Bad or Bid) exclusively promote apoptosis (7Huang D.C. Strasser A. Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 8Conradt B. Horvitz H.R. Cell. 1998; 93: 519-529Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Bad is a death-promoting BH3-only member of Bcl-2 family proteins and heterodimerizes with anti-apoptotic proteins such as Bcl-2 and Bcl-x(L). Survival kinases including Akt, protein kinase A (PKA), and ribosomal S6 kinase 1 (RSK1) phosphorylate serine residues of Bad (9Lizcano J.M. Morrice N. Cohen P. Biochem. J. 2000; 349: 547-557Crossref PubMed Scopus (256) Google Scholar, 10Tan Y. Demeter M.R. Ruan H. Comb M.J. J. Biol. Chem. 2000; 275: 25865-25869Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 11Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (533) Google Scholar, 12Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Korsmeyer S.J. Mol. Cell. 1999; 3: 413-422Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Bad phosphorylation has been shown to be necessary for the release of Bad from its association with Bcl-x(L). 14-3-3 proteins are conserved dimeric regulators in eukaryotic cells. There are seven isotypes of 14-3-3 proteins in mammals and two in yeast. 14-3-3 proteins are known for their ability to bind multiple cellular protein ligands. As many as 100 cellular proteins have been found to interact with 14-3-3 proteins to date. 14-3-3 proteins require phosphorylation of their target proteins for the interaction (13Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar, 14Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). The large number and diversity of ligands for 14-3-3 proteins implicate a suggestion that 14-3-3 proteins are involved in many different cellular events, including signal transduction, cell cycle regulation, apoptosis, stress response, cytoskeleton organization, and malignant transformation (15van Hemert M.J. Steensma H.Y. van Heusden G.P. Bioessays. 2001; 23: 936-946Crossref PubMed Scopus (472) Google Scholar). Despite the fact that the function of 14-3-3 proteins still remains unknown, there exist some general themes about the anti-apoptotic function of 14-3-3 proteins. 14-3-3 proteins have been implicated in signaling for apoptosis through interaction with pro-apoptotic molecules such as Bad (11Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (533) Google Scholar, 16Wang H.G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (960) Google Scholar, 17Kennedy S.G. Kandel E.S. Cross T.K. Hay N. Mol. Cell. Biol. 1999; 19: 5800-5810Crossref PubMed Scopus (589) Google Scholar), FKHRL1 (18Brunet 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 (5381) Google Scholar), ASK1 (19Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (313) Google Scholar), and Nur77 (20Masuyama N. Oishi K. Mori Y. Ueno T. Takahama Y. Gotoh Y. J. Biol. Chem. 2001; 276: 32799-32805Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Phosphorylated Bad by survival kinases interacts with 14-3-3 proteins resulting in the retention of Bad in the cytoplasm and the subsequent prevention of cytotoxic interaction with Bcl-x(L) at the mitochondrial membrane. Caspases play important roles in the execution of apoptosis and inflammatory responses. A large set of cellular proteins has been surveyed for their ability to be cleaved by caspases, and various signaling proteins were found to be caspase substrates (21Miller D.K. Semin. Immunol. 1997; 9: 35-49Crossref PubMed Scopus (191) Google Scholar, 22Villa P. Kaufmann S.H. Earnshaw W.C. Trends Biochem. Sci. 1997; 22: 388-393Abstract Full Text PDF PubMed Scopus (551) Google Scholar). In this study, we identified the 14-3-3ϵ protein as one of the caspase-3 substrates using a modified yeast two-hybrid genetic system. We explored the possibility that the cleavage of 14-3-3 proteins by caspase-3 during apoptosis might contribute to cell death by preventing the association of 14-3-3 proteins with Bad and facilitating Bad translocation to the mitochondria in which Bad associates with Bcl-x(L). Yeast Strains—The expression of the fusion protein was selected in the yeast EGY48 strain (Mat α, his3, trp1, ura3, LexAop-Leu2). The YM4271 strain (Mat a, ura3–52, his 3–200, lys2–801, ade2–101, ade 5, trp1–901, leu 2–3, 112, tyr1–501, gal4, gal80, ade5::hisG) was the mating partner for the EGY48 strain and was used to introduce plasmids expressing human caspases. Plasmids and cDNA Library—The B42 transcriptional activation domain of pJG4-5 (Clontech) was cloned into the XhoI and XbaI sites of pEG202 (Clontech) to generate pSub. Human PARP 1The abbreviations used are: PARP, poly(ADP)ribose polymerase; HA, hemagglutinin; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde. and its mutant, PARPD214A, were cloned into the XhoI and PvuII sites of pSub to generate pSub-PARP and pSub-PARPD214A. The BALB/c mouse brain cDNA library was constructed using Universal RiboClone cDNA synthesis system (Promega) and was inserted into pSub to generate pSub-cDNAs (23Aguan K. Kusano T. Suzuki N. Kitagawa Y. Nucleic Acids Res. 1990; 18: 1071Crossref PubMed Scopus (7) Google Scholar). The B42 transcriptional activation domain of pJG4-5 was deleted to generate the caspase expression vector pExp. Human caspase-1 or 3 was cloned into the KpnI and XhoI sites of pExp for the expression in yeast YM4271 cells. Human Bad was cloned into a mammalian GST fusion vector, pEBG, to produce GST-fused Bad. Human 14-3-3ϵ and its mutants, D238 and D238A, were cloned into the EcoRI and XhoI sites of cloning vector pcDNA3 (Invitrogen). Hemagglutinin was inserted into the BamHI and HindIII sites of pcDNA3 to generate the N-terminal HA-tagged 14-3-3ϵ and its mutants. Yeast Transformation and Mating—Yeast EGY48 cells were transformed with p80lacZ (Clontech) bearing the LexA-responsive lacZ gene. EGY48 cells transformed with pSub-cDNA and YM4271 cells transformed with a human caspase expression vector pExp-C3 were mated on a single plate containing YPD-rich media (24Bendixen C. Gangloff S. Rothstein R. Nucleic Acids Res. 1994; 22: 1778-1779Crossref PubMed Scopus (100) Google Scholar). After incubation for 6 h at 30 °C, the plate was replica-plated to the plates containing the selective media consisting of 40 mg/liter X-gal either with 2% dextrose or 2% galactose plus 1% raffinose. Caspase Activity Assay—The activities of caspase-1 or 3 were measured using a fluorogenic peptide substrate, zVAD-AMC (Bachem) as described (25Vanags D.M. Porn-Ares M.I. Coppola S. Burgess D.H. Orrenius S. J. Biol. Chem. 1996; 271: 31075-31085Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Yeast YM4271 cells transformed with human caspase expression vector encoding caspase-1 or 3 were cultured in the media containing 1% raffinose for 12 h. The expression of caspases was induced by the addition of 2% galactose to the media. After incubation for 6 h, cell pellets were placed in liquid nitrogen and lysed using glass beads (Sigma) in a disruption buffer containing 20 mm Tris, pH 7.9, 10 mm MgCl2, 1 mm EDTA, 5% glycerol, and protease inhibitor mix (1 μg/ml leupeptin, 1 μg/ml pepstatin, and 50 μg/ml phenylmethylsulfonyl fluoride). Cell lysate proteins (25 μg) were incubated with 50 μm zVAD-AMC in a standard interleukin-1β-converting enzyme (ICE) reaction buffer (25Vanags D.M. Porn-Ares M.I. Coppola S. Burgess D.H. Orrenius S. J. Biol. Chem. 1996; 271: 31075-31085Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Cleavage of zVAD-AMC was monitored by a fluorescence spectrophotometer at an excitation/emission wavelength pair of 355 nm/460 nm. In Vitro Cleavage of 14-3-3ϵ Protein by Caspase-3—Human 14-3-3ϵ and its mutant, D238A, were cloned into pcDNA3 (Invitrogen). Site-directed mutagenesis was performed using the procedures of QuikChange site-directed mutagenesis kit (Stratagene). 35S-labeled 14-3-3ϵ protein and D238A were produced using the tnt T7 coupled reticulocyte lysate system (Promega). In vitro translated 35S-14-3-3ϵ protein or 35S-D238A was incubated with Escherichia coli BL21(DE3) cell lysate containing the recombinant human caspase-3 at 30 °C for 8 h. Proteolytic cleavage of 35S-labeled 14-3-3ϵ protein and D238A was analyzed by 12% SDS-PAGE and autoradiography. Immunoblotting and Co-immunoprecipitation—COS cells were harvested 10 h after staurosporine treatment or 24 h after UVC irradiation. Cells were lysed in lysis buffer A containing 50 mm Tris, pH 7.5, 120 mm NaCl, 0.5% Nonidet P-40, 100 mm NaF, 200 μm sodium orthovanadate, and 1 mm phenylmethylsulfonyl fluoride. Cellular 14-3-3ϵ protein was resolved by 12% SDS-PAGE, and cleavage of the 14-3-3ϵ protein and PARP was analyzed by immunoblotting using antibodies directed against the 14-3-3ϵ protein (Santa Cruz Biotechnology) and PARP (Santa Cruz Biotechnology). Effects of 14-3-3ϵ protein cleavage on Bad association with cellular Bcl-x(L) were examined in human 293T cells. Cells were co-transfected with GST-Bad and constructs of HA-14-3-3ϵ by the calcium phosphate method as described (26Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001: 16.19-16.20Google Scholar). After incubation for 24 h, cells were lysed in lysis buffer A. Bad and its associated proteins were then “pulled down” using glutathione-Sepharose 4B (Amersham Biosciences). HA-14-3-3ϵ protein and Bcl-x(L), which were associated with Bad, were analyzed by immunoblotting using antibodies directed against HA and Bcl-x(L) (Transduction Laboratories), respectively. Apoptosis—Apoptosis was measured by fluorescence-activated cell sorter (FACS) analysis. Cells were harvested 24 h after transfection and fixed with cold ethanol. DNA were determined by after DNA of cells at were used as the for the of apoptosis. of 14-3-3ϵ Protein as Caspase by the Yeast the for caspase substrates in the yeast genetic system. The encoding caspase substrates were the domain and the B42 activation Yeast EGY48 cells expressing the fusion produced the under the on plates containing the The to lacZ and as plasmids the cDNA that caspases were by and the fusion were cleaved by caspases. In a known caspase-3 substrate, was examined for its ability to be cleaved by caspase-3 in the modified yeast two-hybrid system. Yeast cells expressing the fusion containing PARP lacZ under the and as after the of caspase-3 by However, cells expressing the fusion containing PARP in which at the was with J. S. J. Cell Sci. 2000; Google caspase-3 was by the of the modified yeast two-hybrid system as a genetic system for caspase caspase-1 and 3 were active in yeast YM4271 and their activities were by the caspase the that yeast be used as a system for caspase the mouse brain cDNA fusion library in the of 14-3-3ϵ residues was as one of the caspase-3 substrates by the modified yeast genetic system. 35S-labeled 14-3-3ϵ protein was by in vitro procedures and cleaved by the recombinant human caspase-3, PARP was cleaved under the The cleavage was by a caspase-3 The cleavage of the 14-3-3ϵ protein in cells in response to apoptosis inducers was examined in that apoptosis inducers such as UVC or staurosporine induced the cleavage of the 14-3-3ϵ protein in cells. The cleavage of PARP the activation of caspase-3 in cells after UVC or staurosporine the activities of caspase-3 were in with the cleavage of cellular 14-3-3ϵ in cells after UVC of the cleavage that caspase-3 the 14-3-3ϵ protein at Asp238 of A mutant 14-3-3ϵ protein in which Asp238 was with was by caspase-3 of 14-3-3ϵ protein at Asp238 by the 35S-labeled 14-3-3ϵ protein and PARP were produced by in vitro 35S-labeled 14-3-3ϵ protein or 35S-labeled PARP was cleaved by the recombinant human caspase-3, and the were analyzed by 12% SDS-PAGE and subsequent autoradiography. was used as a caspase-3 cells were with staurosporine or UVC and incubated for 10 h or 24 h respectively. Cell lysate proteins μg) were on 12% of the cellular 14-3-3ϵ protein and PARP were examined by using antibodies against the 14-3-3ϵ protein and PARP, respectively. the 35S-labeled 14-3-3ϵ protein and the uncleavable D238A were produced by in vitro The cleavage of the 35S-labeled proteins by the recombinant human caspase-3 was analyzed by 12% SDS-PAGE and subsequent autoradiography. was used as a caspase-3 Cleavage of 14-3-3ϵ Protein with Bad and and 14-3-3ϵ constructs were in cultured 293T cells. The cleaved 14-3-3ϵ protein with Bad with lower affinity than its wild type or the uncleavable Bad with the Bcl-x(L) in cells co-expressing Bad either with the wild type 14-3-3ϵ protein or the uncleavable of the truncated 14-3-3ϵ protein D238 with Bad Bad association with cellular Bcl-x(L) and the Bad association with Bcl-x(L) to contribute to apoptosis. apoptosis was by the expression of the wild type 14-3-3ϵ protein of the uncleavable D238A The expression of the truncated D238 also apoptosis, whereas the expression of the uncleavable D238A apoptosis in 293T cells after staurosporine treatment Cleavage of the 14-3-3ϵ protein by caspase-3 to the of 14-3-3ϵ protein wild type 14-3-3ϵ protein was with HA-tagged wild type 14-3-3ϵ protein the truncated D238 or uncleavable D238A in 293T cells. HA-14-3-3ϵ to proteins were examined by analysis. with the protein as HA-14-3-3ϵ or with of the cleaved 14-3-3ϵ protein Bad and the 14-3-3ϵ protein were in 293T cells. After transfection for 24 h, cells were harvested and fixed with cold ethanol. Cells were with for h after The of apoptosis was by DNA of cells at using the from the was with HA-14-3-3ϵ in 293T cells. and its associated proteins were analyzed by as described in wild The of modified yeast two-hybrid system as a genetic system to proteins was shown in The 14-3-3ϵ protein was cleaved at Asp238 of by caspase-3 The cleavage site was in the the of which is 14-3-3 isotypes K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). In the 14-3-3ϵ protein has been reported to be one of the signaling proteins that were cleaved in response to the apoptotic induced by and in cultured mammalian cells C. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). There are that 14-3-3 proteins anti-apoptotic through interaction with Bad (11Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (533) Google Scholar, 16Wang H.G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (960) Google Scholar). that the cleavage of the 14-3-3ϵ protein by the to apoptosis by releasing Bad from its association with the 14-3-3 protein and facilitates Bad translocation from the to the mitochondria and its interaction with Bcl-x(L). Cleavage of the 14-3-3ϵ protein to the ability of its with Bad 14-3-3 proteins signal transduction through their with the target proteins containing different and and is have been identified in known 14-3-3 proteins. Cell death Bad two 14-3-3 sites with of and and associates with the 14-3-3 protein at (13Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar, H. Cell 2000; PubMed Scopus Google Scholar). in 3 that Bad from 14-3-3 to the mitochondria and heterodimerizes with Bcl-x(L). was by the of J. H. E. J. Korsmeyer S.J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google that the 14-3-3 protein Bad and Bad from with Bcl-x(L). The conserved 14-3-3 proteins have been known to be present as multiple isotypes in many eukaryotic A. van Heusden T. G. J. Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar, Science. 1994; PubMed Scopus Google Scholar). The function of mammalian 14-3-3 protein isotypes is to apoptosis. 14-3-3 proteins have been thought to the pro-apoptotic function of their of which Bad is a S.G. Kandel E.S. Cross T.K. Hay N. Mol. Cell. Biol. 1999; 19: 5800-5810Crossref PubMed Scopus (589) Google Scholar). H. S. C. A. J. 2000; 19: PubMed Scopus Google 14-3-3 proteins as general anti-apoptotic reported that the mutant of the 14-3-3 protein the anti-apoptotic function of the wild type 14-3-3 protein and apoptosis in response to irradiation. In the present study, we that the 14-3-3ϵ protein is cleaved by the caspase-3 to promote cell death during to apoptotic The cleaved 14-3-3ϵ protein its affinity to Bad and Bad to to the mitochondrial membrane Bad heterodimerizes with Bcl-x(L). 14-3-3 proteins have been shown to apoptosis through interaction at the phosphorylation sites of Bad (11Yano S. Tokumitsu H. Soderling T.R. Nature. 1998; 396: 584-587Crossref PubMed Scopus (533) Google Scholar, 16Wang H.G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (960) Google Scholar). A number of have described the dimeric of 14-3-3 proteins that interacts with 14-3-3 proteins at two 14-3-3 The of the truncated 14-3-3ϵ protein (D238) on apoptosis in 293T cells be by the of the association of D238 with Bad the cellular 14-3-3ϵ protein in 293T cells expressing D238 also have with Bad. The in 4B an that the truncated D238 with the 14-3-3ϵ protein and the of wild type 14-3-3ϵ that Bad in co-transfected cells. 14-3-3 proteins form as as the truncated D238 also with of 14-3-3 proteins in 293T cells. is that cleavage of the 14-3-3ϵ protein by caspase-3 the of during apoptosis, the residues of 14-3-3 involved in the are conserved mammalian D.H. S. Aitken A. PubMed Scopus Google Scholar). The fact that D238 with of 14-3-3 proteins the interaction of D238 with Bad, the different of the 14-3-3 protein were reported to interact with Bad K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). The ability of with Bad was with that of HA-14-3-3ϵ or the uncleavable in an in which HA-14-3-3ϵ or were with or wild type 14-3-3ϵ protein in 293T cells. of the 14-3-3ϵ protein with the affinity of with Bad, that cleavage of the 14-3-3ϵ protein by its affinity with Bad of the of the association of the cleaved 14-3-3ϵ protein The death Bad then heterodimerizes with Bcl-x(L) in the membrane of the mitochondria. The of Bad to Bcl-x(L) might Bcl-x(L) to interact with pro-apoptotic Bax to release cytochrome c or Bcl-x(L) activities (3Ravagnan L. Roumier T. Kroemer G. J. Cell. Physiol. 2002; 192: 131-137Crossref PubMed Scopus (435) Google Scholar), to the of cell