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Query: UNIPROT:P10415 (Bcl-2)
 33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.
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PMID:R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism. 774 59

R-ras is a member of the ras family of small GTPases that associates with the apoptosis-suppressing proto-oncogene product Bcl-2. Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase. This interaction requires only the N-terminal regulatory domain (amino acids 1-256) of Raf-1, and is observed with both the wild type and a constitutively active R-ras mutant, but not with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding. Moreover, using an in vitro binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81-amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131). Taken together, these data indicate that R-ras may exert its biological effect by means of modulating the activity of the Raf-1 kinase as its direct downstream effector.
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PMID:The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner. 800 32

The Bcl-2 protein is over-produced in many types of human tumors and suppresses apoptosis induced by a wide-variety of stimuli, including chemotherapeutic drugs and gamma-irradiation. The biochemical mechanism of action of the Bcl-2 protein however remains enigmatic. Here we show that Bcl-2 can be co-immunoprecipitated with the serine/threonine-specific Raf-1 kinase both in a mammalian hemopoietic cell 32D.3 and when the two proteins are produced in Sf9 insect cells using recombinant baculoviruses. Though analysis of Raf-1 deletion mutants suggested that the C-terminal half of the protein which contains the catalytic domain is sufficient for co-immunoprecipitation with Bcl-2, Raf-1 does not appear to induce phosphorylation of Bcl-2 protein in 32D.3 and Sf9 cells. Furthermore, a mutant form of Raf-1 that lacks kinase activity could still be co-immunoprecipitated with Bcl-2 in Sf9 cells, suggesting that the interaction of these proteins does not reflect a kinase-substrate relation. Gene transfer experiments using 32D.3 hemopoietic cells demonstrated functional synergy between Bcl-2 and Raf-1 with regards to suppression of apoptosis induced by growth factor withdrawal. Taken together, these observations for the first time functionally link Bcl-2 to a signal transducing protein and suggest that the interaction of the Bcl-2 and Raf-1 proteins may be responsible for their ability to cooperate in the suppression of apoptosis.
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PMID:Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase. 805 42

c-Raf-1 (Raf-1) is a central component of signal transduction pathways stimulated by various growth factors, protein kinase C, and other protein kinases. Raf-1 activation is thought to be initiated at the plasma membrane after its recruitment by Ras. Raf-1 activation is associated primarily with proliferation and cell survival, but it has also been implicated in apoptosis. Raf-1 has also been shown to form complexes with both R-Ras and Bcl-2, raising the possibility that this component of cellular Raf-1 plays a role in apoptosis. Recently, taxol was reported to induce Bcl-2 phosphorylation and inactivation. We have previously demonstrated Raf-1 activation following taxol in MCF7 cells. We now present evidence that taxol fails to stimulate either apoptosis or phosphorylation of Bel-2 in the absence of Raf-1. Moreover, Raf-1 activation by taxol coincided with Bel-2 phosphorylation, showing similar dose and time dependence. Thus, our data support a role for a distinct subcellular component of Raf-1, which is taxol but not phorbol myristate acetate sensitive, in mediating an apoptotic pathway involving Bc1-2.
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PMID:Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. 862 May 3

The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1.
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PMID:Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. 869 45

The Bcl-2 protein blocks a distal step in an evolutionarily conserved pathway for programmed cell death and apoptosis. To gain better understanding of how this protein functions, we have undertaken a structure-function analysis of this protein, focusing on domains within Bcl-2 that are required for function and for interactions with other proteins. Four conserved domains are present in Bcl-2 and several of its homologs: BH1 (residues 136-155), BH2 (187-202), BH3 (93-107) and BH4 (10-30). Deletion of the BH1, BH2, or BH4 domains of Bcl-2 abolishes its ability to suppress cell death in mammalian cells and prevents homodimerization of these mutant proteins, though these mutants can still bind to the wild-type Bcl-2 protein. These mutants also fail to bind to BAG-1 and Raf-1, two proteins that we have shown can associate with protein complexes containing Bcl-2 and which cooperate with Bcl-2 to suppress cell death. Deletion of either BH1 or BH2 nullifies the ability of Bcl-2 to: (a) suppress death in mammalian cells: (b) block Bax-induced lethality in yeast; and (c) heterodimerize with Bax. In contrast, deletion of the BH4 domain of Bcl-2 nullifies anti-apoptotic function and homodimerization, but does not impair binding to the pro-apoptotic protein Bax. Taken together, the data suggest the possibility that both Bcl-2/Bcl-2 homodimerization and Bcl-2/Bax heterodimerization are necessary but insufficient for the anti-apoptotic function of the Bcl-2 protein. Homodimerization of Bcl-2 with itself involves a head-to-tail interaction, in which an N-terminal domain where BH4 resides interacts with the more distal region of Bcl-2 where BH1, BH2, and BH3 are located. In contrast, Bcl-2/Bax heterodimerization involves a tail-to-tail interaction, that requires the portion of Bcl-2 where BH1, BH2, and BH3 reside and a central region in Bax where the BH3 domain is located. The BH3 domain of Bax is also required for Bax/Bax homodimerization and pro-apoptotic function in both yeast and mammalian cells. Thus, Bcl-2 may suppress cell death at least in part by binding to Bax via the BH3 domain and thereby preventing formation of Bax/Bax homodimers. Further studies however are required to delineate the full significance of Bcl-2/Bcl-2, Bcl-2/Bax, and Bax/Bax dimers and the biochemical mechanisms by which Bcl-2 family proteins ultimately control cell life and death.
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PMID:Structure-function analysis of Bcl-2 family proteins. Regulators of programmed cell death. 891 Jun 75

A green fluorescent protein (GFP)-Raf-1 fusion protein was used to show that Bcl-2 can target this kinase to mitochondria. Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD, a proapoptotic Bcl-2 homolog. Plasma membrane-targeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-1 and ERK-2. Untargeted active Raf-1 improved Bcl-2-mediated resistance to apoptosis, whereas a kinase-inactive Raf-1 mutant abrogated apoptosis suppression by Bcl-2. Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD or possibly other protein substrates involved in apoptosis regulation.
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PMID:Bcl-2 targets the protein kinase Raf-1 to mitochondria. 892 27

Paclitaxel has been shown to activate Raf-1 and cause phosphorylation of Bcl-2, which has been correlated with paclitaxel-induced apoptosis of cancer cells. In the present studies, we demonstrate that in human AML HL-60 cells that express Bcl-2 but little Bcl-xL (HL-60/neo cells), paclitaxel-induced phosphorylation of Bcl-2 is followed by increased intracellular free Bax levels. This, in turn, is followed by the cleavage and activation of the key cysteine protease, CPP32beta/Yama, and cleavage of poly(ADP-ribose) polymerase, resulting in the DNA fragmentation of apoptosis. Cotreatment with the benzoquinone ansamycin Geldanamycin depleted Raf-1 but did not decrease Bcl-2 levels or impair paclitaxel-induced Bcl-2 phosphorylation in HL-60/neo cells. Also, Geldanamycin did not affect paclitaxel-induced apoptosis of HL-60/neo cells. As compared to the control HL-60/neo, HL-60/Bcl-xL cells contain Bcl-2 as well as an enforced overexpression of Bcl-xL. Immunoprecipitation studies with anti-Bcl-2 and/or anti-Bcl-x antibodies demonstrated that HL-60/Bcl-xL cells possess lower free Bax but higher levels of Bax heterodimerized to Bcl-2 and Bcl-xL. Following treatment of HL-60/Bcl-xL cells with paclitaxel, although Bcl-2 phosphorylation was observed, it was not followed by increased free Bax levels, cleavage of CPP32beta/Yama and poly(ADP-ribose) polymerase, or induction of the DNA fragmentation of apoptosis. These findings indicate the order of molecular events leading to paclitaxel-induced apoptosis and show that Raf-1 may not be involved in paclitaxel-induced phosphorylation of Bcl-2 or apoptosis of HL-60 cells.
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PMID:Bcl-xL overexpression inhibits progression of molecular events leading to paclitaxel-induced apoptosis of human acute myeloid leukemia HL-60 cells. 906 80

The expression and function of the newly identified Bcl-2- and Raf-1- binding protein, Bag-1, during the cytokine-regulated growth of B and T cell lines was examined. Immunoblot analysis of lysates from the interleukin-3 (IL-3)-dependent B cell line Ba/F3, and the PRL-dependent T cell line Nb2, revealed that variations in Bag-1 levels paralleled alterations in cellular proliferation, viability, and apoptosis induced by the presence or absence of growth factor. To test whether up-regulation of Bag-1 levels altered cellular survival and proliferation, Ba/F3 cells were transfected with a Bag-1 expression construct. The overexpression of Bag-1 in transfected Ba/F3 cells induced an IL-3-independent state. Such transfectants demonstrated sustained viability and proliferation, with minimal apoptosis, in the complete absence of exogenous IL-3. Bag-1 expression was also compared in glucocorticoid-sensitive Nb2 cells and a PRL-independent, glucocorticoid-resistant subline, SFJCD1, during culture of these lines in dexamethasone (Dex). Bag-1 levels were profoundly decreased by the addition of Dex to Nb2 cells, precedent to the onset of apoptotic cell death. In contrast, Dex treatment or PRL withdrawal had no effect on levels of Bag-1 within the SFJCD1 line. These findings establish that the overexpression of Bag-1 in the appropriate cellular context promotes cellular survival and growth, events that may result from the juxtaposition of this protein with mitogenic and antiapoptotic signaling pathways.
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PMID:Role of Bag-1 in the survival and proliferation of the cytokine-dependent lymphocyte lines, Ba/F3 and Nb2. 913 4

In a previous study, we demonstrated that bufalin, which is an active principle of Chinese medicine, chan'su, caused apoptosis in human leukemia U937 cells by anomalous activation of mitogen-activated protein kinase (MAPK) via the signaling pathway of Ras, Raf-1, and MAPK kinase-1. Here, we report the effect of overexpression of bcl-2 in U937 cells on the signaling pathway of apoptosis that is induced by bufalin. The results indicated that the apoptosis induced by bufalin in U937 cells was significantly inhibited by overexpression of the Bcl-2 protein. No significant difference was detected in the activation of MAPK kinase-1 that is induced by bufalin in wild-type or Bcl-2-overexpressed U937 cells; however, the activation of MAPK by bufalin was significantly attenuated in the cells overexpressing Bcl-2. Bufalin treatment activated activator protein-1 transcriptional activity; however, this activation was decreased to 40% in bcl-2-overexpressed U937 cells. These results indicate that Bcl-2 acts downstream of MAPK kinase-1 but upstream of MAPK and suggest that, in the signaling pathway of the apoptotic process induced by bufalin, the transcriptional activity of activator protein-1 may be down-regulated through the inhibition of MAPK activity by Bcl-2.
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PMID:Bcl-2 protein inhibits bufalin-induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells. 924 31


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