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At least 613 records · Page 34Linked to original sources
PubMed1991

c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site.

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

Animals↗
PubMed1991

An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

Base Sequence↗
PubMed1991

Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

Animals↗
PubMed1993

Three downstream sites repress transcription of a Ty2 retrotransposon in Saccharomyces cerevisiae.

Transcription of Ty1 and Ty2 retrotransposons of the yeast Saccharomyces cerevisiae is modulated by multiple downstream regulatory sites. Both transposon families include a positively acting site within the transcribed region which resembles a higher eukaryotic enhancer. We have demonstrated the existence of a repression site distal to the enhancer of the Ty2-917 element. Here we describe experiments investigating the internal structure of this site. We show that this 200-bp region includes three distinct repression sites which we term DRSI (downstream repression site I), DRSII, and DRSIII. Individually each site causes almost twofold repression, and together the sites repress eightfold. Unexpectedly, when the entire region encompassing the DRS sites is moved outside the transcription unit, it acts as a qualitatively positively acting element. In this context the DRS sites still repress transcription, since eliminating them increases transcription further. That the region can activate transcription implies that it includes activation sites in addition to the three repression sites. The change from qualitatively negatively acting to positively acting must reflect a change in the relative effects of the multiple positive and negative sites; when moved outside the transcription unit, the activators predominate. Importantly, DRSII and DRSIII repress transcription autonomously when inserted upstream of a heterologous promoter activated by the transcriptional activator GCN4, showing that they are indeed transcriptional repression sites.

Base Sequence↗
PubMed1993

Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites.

Little is known about the transcriptional events which occur downstream of polyadenylation sites. Although the polyadenylation site of a gene can be easily identified, it has been difficult to determine the site of transcription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach, we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 and GAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of the ADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of the poly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sized nonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. A GAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site. However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site. Mutations of GAL7 or ADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis of the extended poly(A)- transcript. Since transcription of the mutant template continues through this region in vivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data support the hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcription termination.

Cell-Free System↗
PubMed1996

TFIIIC1 acts through a downstream region to stabilize TFIIIC2 binding to RNA polymerase III promoters.

An in vitro system reconstituted with highly purified RNA polymerase III, TFIIIC2, and TFIIIB has been used to identify two chromatographically distinct human RNA polymerase III transcription factors, TFIIIC1 and TFIIIC1', which are functionally equivalent to the previously defined TFIIIC1 (S. T. Yoshinaga, P. A. Boulanger, and A. J. Berk, Proc. Natl. Acad. Sci. USA 84:3585-3589, 1987). Interactions between TFIIIC2, TFIIIC1 (or TFIIIC1'), and the VA1 and tRNA1(Met) templates have been investigated by DNase I footprint analysis. Homogeneous TFIIIC2 alone shows only a weak footprint over the B-box region of the VA1 and tRNA1(Met) templates, whereas TFIIIC1 (or TFIIIC1') alone shows both a strong interaction over the downstream termination region and a very weak interaction near the A-box region. Importantly, when both factors are present simultaneously, TFIIIC1 (or TFIIIC1') dramatically enhances the level of TFIIIC2 binding and extends the footprint to a region that includes the A box. The downstream termination region is essential for this cooperative interaction between TFIIIC2 and TFIIIC1 (or TFIIIC1') on the VA1 and tRNA1(Met) templates and plays a role in the overall accuracy and efficiency of RNA polymerase III transcription.

Binding Sites↗
PubMed1996

cis-acting sequences located downstream of the human immunodeficiency virus type 1 promoter affect its chromatin structure and transcriptional activity.

We have examined the roles of AP-1, AP-3-like, DBF1, and Sp1 binding sites, which are located downstream of the human immunodeficiency virus type 1 (HIV-1) promoter, in regulating basal transcriptional activity directed by the integrated viral long terminal repeat (LTR). Point mutations affecting all four of these elements functionally inactivated the HIV-1 LTR when it was constrained in a chromatin configuration. Analyses of the chromatin structures of the transcriptionally active wild-type and inactive mutated HIV-1 promoters revealed several differences. In the active promoter, the 3' half of the U3 region, including the basal promoter, the enhancer, and the putative upstream regulatory sequences are situated within a nuclease-hypersensitive region. However, the far upstream U3 region appears to be packaged into a nuclease-resistant nucleosomal structure, whereas the R, U5, and gag leader sequences are associated with a region of altered chromatin that is sensitive to restriction endonucleases. In the inactive template, only the basal promoter and enhancer element remain sensitive to nucleases, and the adjacent upstream and downstream regions are incorporated into nuclease-resistant nucleosomal structures. Taken together, these results indicate that the chromatin structure of the integrated HIV-1 LTR plays a critical role in modulating basal transcriptional activity.

Base Sequence↗
PubMed2000

Sli2 (Ypk1), a homologue of mammalian protein kinase SGK, is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast.

ISP-1 is a new type of immunosuppressant, the structure of which is homologous to that of sphingosine. In a previous study, ISP-1 was found to inhibit mammalian serine palmitoyltransferase, the primary enzyme involved in sphingolipid biosynthesis, and to reduce the intracellular pool of sphingolipids. ISP-1 induces the apoptosis of cytotoxic T cells, which is triggered by decreases in the intracellular levels of sphingolipids. In this study, the inhibition of yeast (Saccharomyces cerevisiae) proliferation by ISP-1 was observed. This ISP-1-induced growth inhibition was also triggered by decreases in the intracellular levels of sphingolipids. In addition, DNA duplication without cytokinesis was detected in ISP-1-treated yeast cells on flow cytometry analysis. We have cloned multicopy suppressor genes of yeast which overcome the lethal sphingolipid depletion induced by ISP-1. One of these genes, SLI2, is synonymous with YPK1, which encodes a serine/threonine kinase. Kinase-dead mutants of YPK1 did not show any resistance to ISP-1, leading us to predict that the kinase activity of the Ypk1 protein should be essential for this resistance to ISP-1. Ypk1 protein overexpression had no effect on sphingolipid biosynthesis by the yeast. Furthermore, both the phosphorylation and intracellular localization of the Ypk1 protein were regulated by the intracellular sphingolipid levels. These data suggest that the Ypk1 protein is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. The Ypk1 protein was reported to be a functional homologue of the mammalian protein kinase SGK, which is a downstream kinase of 3-phosphoinositide-dependent kinase 1 (PDK1). PDK1 phosphotidylinositol (PI) is regulated by PI-3,4,5-triphosphate and PI-3,4-bisphosphate through the pleckstrin homology (PH) domain. Overexpression of mammalian SGK also overcomes the sphingolipid depletion in yeast. Taking both the inability to produce PI-3,4, 5-triphosphate and PI-3,4-bisphosphate and the lack of a PH domain in the yeast homologue of PDK1, the Pkh1 protein, into account, these findings further suggest that yeast may use sphingolipids instead of inositol phospholipids as lipid mediators.

Fatty Acids, Monounsaturated↗
PubMed2000

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders.

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

5' Untranslated Regions↗
PubMed2000

Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins.

The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.

3T3 Cells↗
PubMed2000

Feedback phosphorylation of the yeast a-factor receptor requires activation of the downstream signaling pathway from G protein through mitogen-activated protein kinase.

The two yeast pheromone receptors, the a and alpha-factor receptors, share many functional similarities: both G protein-coupled receptors couple to the same downstream signal transduction pathway, and both receptors undergo feedback regulation involving increased phosphorylation on their C-terminal domains in response to ligand challenge. The present work, which focuses on the signaling mechanism controlling this feedback phosphorylation, indicates one striking difference. While the alpha-factor-induced phosphorylation of the alpha-factor receptor does not require activation of the downstream G protein-directed signaling pathway (B. Zanolari, S. Raths, B. Singer-Kruger, and H. Riezman, Cell 71:755-763, 1992), the a-factor-induced phosphorylation of the a-factor receptor (Ste3p) clearly does. Induced Ste3p phosphorylation was blocked in cells with disruptions of various components of the pheromone response pathway, indicating a requirement of pathway components extending from the G protein down through the mitogen-activated protein kinase (MAPK). Furthermore, Ste3p phosphorylation can be induced in the absence of the a-factor ligand when the signaling pathway is artificially activated, indicating that the liganded receptor is not required as a substrate for induced phosphorylation. While the activation of signaling is critical for the feedback phosphorylation of Ste3p, pheromone-induced gene transcription, one of the major outcomes of pheromone signaling, appears not to be required. This conclusion is indicated by three results. First, ste12Delta cells differ from cells with disruptions of the upstream signaling elements (e.g., ste4Delta, ste20Delta, ste5Delta, ste11Delta, ste7Delta, or fus3Delta kss1Delta cells) in that they clearly retain some capacity for inducing Ste3p phosphorylation. Second, while activated alleles of STE11 and STE12 induce a strong transcriptional response, they fail to induce a-factor receptor phosphorylation. Third, blocking of new pheromone-induced protein synthesis with cycloheximide fails to block phosphorylation. These findings are discussed within the context of a recently proposed model for pheromone signaling (P. M. Pryciak and F. A. Huntress, Genes Dev. 12:2684-2697, 1998): a key step of this model is the activation of the MAPK Fus3p through the G(betagamma)-dependent relocalization of the Ste5p-MAPK cascade to the plasma membrane. Ste3p phosphorylation may involve activated MAPK Fus3p feeding back upon plasma membrane targets.

Alleles↗
PubMed2000

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase.

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.

Animals↗
PubMed2000

Multiple ras downstream pathways mediate functional repression of the homeobox gene product TTF-1.

Expression of oncogenic Ras in thyroid cells results in loss of expression of several thyroid-specific genes and inactivation of TTF-1, a homeodomain-containing transcription factor required for normal development of the thyroid gland. In an effort to understand how signal transduction pathways downstream of Ras may be involved in suppression of the differentiated phenotype, we have tested mutants of the Ras effector region for their ability to affect TTF-1 transcriptional activity in a transient-transfection assay. We find that V12S35 Ras, a mutant known to interact specifically with Raf but not with RalGDS or phosphatidylinositol 3-kinase (PI3 kinase) inhibits TTF-1 activity. Expression of an activated form of Raf (Raf-BXB) also inhibits TTF-1 function to a similar extent, while the MEK inhibitors U0126 and PD98059 partially relieve Ras-mediated inactivation of TTF-1, suggesting that the extracellular signal-regulated kinase (ERK) pathway is involved in this process. Indeed, ERK directly phosphorylates TTF-1 at three serine residues, and concomitant mutation of these serines to alanines completely abolishes ERK-mediated phosphorylation both in vitro and in vivo. Since activation of the Raf/MEK/ERK pathway accounts for only part of the activity elicited by oncogenic Ras on TTF-1, other downstream pathways are likely to be involved in this process. We find that activation of PI3 kinase, Rho, Rac, and RalGDS has no effect on TTF-1 transcriptional activity. However, a poorly characterized Ras mutant, V12N38 Ras, can partially repress TTF-1 transcriptional activity through an ERK-independent pathway. Importantly, concomitant expression of constitutive activated Raf and V12N38 Ras results in almost complete loss of TTF-1 activity. Our data indicate that the Raf/MEK/ERK cascade may act in concert with an as-yet-uncharacterized signaling pathway activated by V12N38 Ras to repress TTF-1 function and ultimately to inhibit thyroid cell differentiation.

Animals↗
PubMed2001

An upstream AG determines whether a downstream AG is selected during catalytic step II of splicing.

Specific mechanisms must exist to ensure fidelity in selecting the AG dinucleotide that functions as the 3' splice site during the second transesterification step of splicing. Here we show that the optimal location for this AG is within a narrow distance (19 to 23 nucleotides [nt]) downstream from the branch point sequence (BPS). Contrary to previous expectations, AGs located less than 23 nt from the BPS are always recognized, even when a second AG located more optimally downstream is used in the transesterification reaction. Indeed, the AG closest to the BPS actually dictates the precise location of the AG that engages in the reaction. This mechanism, in which the AG is identified by a general localization step followed by a precise localization step, may be used to achieve fidelity while allowing flexibility in the location of 3' splice sites.

Cell Nucleus↗
PubMed2003

GATA-4 and GATA-5 transcription factor genes and potential downstream antitumor target genes are epigenetically silenced in colorectal and gastric cancer.

The GATA family of transcription factors participates in gastrointestinal (GI) development. Increases in GATA-4 and -5 expression occur in differentiation and GATA-6 expression in proliferation in embryonic and adult settings. We now show that in colorectal cancer (CRC) and gastric cancer promoter hypermethylation and transcriptional silencing are frequent for GATA-4 and -5 but are never seen for GATA-6. Potential antitumor target genes upregulated by GATA-4 and -5, the trefoil factors, inhibinalpha, and disabled-2 (Dab2) are also silenced, in GI cancers, with associated methylation of the promoters. Drug or genetically induced demethylation simultaneously leads to expression, in CRC cells, of all of the GATA-4, -5, and downstream genes. Expression of exogenous GATA-5 overrides methylation at the downstream promoters to activate the target genes. Selection for silencing of both upstream transcription factors and their target genes in GI cancers could indicate that epigenetic silencing of the involved genes provides a summated contribution to tumor progression.

Cell Line, Tumor↗
PubMed2003

Translocation of CrkL to focal adhesions mediates integrin-induced migration downstream of Src family kinases.

The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.

Adaptor Proteins, Signal Transducing↗
PubMed2004

U1A inhibits cleavage at the immunoglobulin M heavy-chain secretory poly(A) site by binding between the two downstream GU-rich regions.

The immunoglobulin M heavy-chain locus contains two poly(A) sites which are alternatively expressed during B-cell differentiation. Despite its promoter proximal location, the secretory poly(A) site is not expressed in undifferentiated cells. Crucial to the activation of the secretory poly(A) site during B-cell differentiation are changes in the binding of cleavage stimulatory factor 64K to GU-rich elements downstream of the poly(A) site. What regulates this change is not understood. The secretory poly(A) site contains two downstream GU-rich regions separated by a 29-nucleotide sequence. Both GU-rich regions are necessary for binding of the specific cleavage-polyadenylation complex. We demonstrate here that U1A binds two (AUGCN(1-3)C) motifs within the 29-nucleotide sequence and inhibits the binding of cleavage stimulatory factor 64K and cleavage at the secretory poly(A) site.

Animals↗
PubMed2004

Akt inhibits apoptosis downstream of BID cleavage via a glucose-dependent mechanism involving mitochondrial hexokinases.

The serine/threonine kinase Akt/protein kinase B inhibits apoptosis induced by a variety of stimuli, including overexpression or activation of proapoptotic Bcl-2 family members. The precise mechanisms by which Akt prevents apoptosis are not completely understood, but Akt may function to maintain mitochondrial integrity, thereby preventing cytochrome c release following an apoptotic insult. This effect may be mediated, in part, via promotion of physical and functional interactions between mitochondria and hexokinases. Here we show that growth factor deprivation induced proteolytic cleavage of the proapoptotic Bcl-2 family member BID to yield its active truncated form, tBID. Activated Akt inhibited mitochondrial cytochrome c release and apoptosis following BID cleavage. Akt also antagonized tBID-mediated BAX activation and mitochondrial BAK oligomerization, two downstream events thought to be critical for tBID-induced apoptosis. Glucose deprivation, which impaired the ability of Akt to maintain mitochondrion-hexokinase association, prevented Akt from inhibiting BID-mediated apoptosis. Interestingly, tBID independently elicited dissociation of hexokinases from mitochondria, an effect that was antagonized by activated Akt. Ectopic expression of the amino-terminal half of hexokinase II, which is catalytically active and contains the mitochondrion-binding domain, consistently antagonized tBID-induced apoptosis. These results suggest that Akt inhibits BID-mediated apoptosis downstream of BID cleavage via promotion of mitochondrial hexokinase association and antagonism of tBID-mediated BAX and BAK activation at the mitochondria.

Animals↗