A novel mechanism for the control of translation of specific mRNAs by tumor suppressor protein Pdcd4: inhibition of translation elongation.

The tumor suppressor gene Pdcd4 (programmed cell death gene 4) has drawn considerable attention because its downregulation is involved in the development of several types of cancer. Because Pdcd4 interacts with the translation initiation factor eIF4A and inhibits its helicase activity, Pdcd4 has been implicated in the translational suppression of cellular mRNAs containing structured 5'-untranslated regions. However, Pdcd4's role in translation regulation is still poorly understood, because only very few physiological Pdcd4 target mRNAs are known. By using a Pdcd4-deficient clone of the chicken B-cell line DT40, we have discovered that the mRNA of the A-myb proto-oncogene is a novel Pdcd4 target RNA whose translation is suppressed by Pdcd4. Interestingly, the inhibitory effect of Pdcd4 is independent of the Pdcd4-eIF4A interaction, but is dependent on an RNA-binding domain at the N terminus of Pdcd4 and on sequences located within the coding region of A-myb mRNA, indicating that Pdcd4 suppresses A-myb translation by a novel mechanism. Our data show that the Pdcd4 RNA-binding domain preferentially recognizes an RNA secondary structure element formed by the part of the A-myb coding region that mediates Pdcd4-dependent suppression. Previously, we have shown that Pdcd4 also suppresses the translation of the c-myb mRNA by a similar mechanism involving binding of Pdcd4 to RNA secondary structure formed by the c-myb coding region. Surprisingly, our data show that Pdcd4 exerts its inhibitory activity only when the target region of Pdcd4 in A-myb and c-myb mRNA is itself translated, consistent with a mechanism in which Pdcd4 suppresses translation by interfering with translation elongation. Taken together, our work reveals a novel mechanism by which Pdcd4 affects the translational of cellular RNAs. Furthermore, as c-myb and A-myb are members of the Myb proto-oncogene family whose deregulation has been implicated in tumorigenesis, inhibiting their translation might contribute to the tumor-suppressive activity of Pdcd4.

The sigma enigma: in vitro/in silico site-directed mutagenesis studies unveil σ1 receptor ligand binding.

The σ1 receptor is an integral membrane protein that shares no homology with other receptor systems, has no unequivocally identified natural ligands, but appears to play critical roles in a wide variety of cell functions. While the number of reports of the possible functions of the σ1 receptor is increasing, almost no information about the three-dimensional structure of the receptor and/or possible modes of interaction of the σ1 protein with its ligands have been described. Here we performed an in vitro/in silico investigation to analyze the molecular interactions of the σ1 receptor with its prototypical agonist (+)-pentazocine. Accordingly, 23 mutant σ1 isoforms were generated, and their interactions with (+)-pentazocine were determined experimentally. All direct and/or indirect effects exerted by the mutant residues on the receptor-agonist interactions were reproduced and rationalized in silico, thus shining new light on the three-dimensional structure of the σ1 receptor and its ligand binding site.

B-Myb switches from Cyclin/Cdk-dependent to Jnk- and p38 kinase-dependent phosphorylation and associates with SC35 bodies after UV stress.

B-Myb is a highly conserved member of the Myb transcription factor family that has essential roles in cell-cycle progression. Recent work has suggested that B-Myb is also involved in the cellular DNA-damage response. Here, we have investigated the fate of B-Myb in UV-irradiated cells. UV stress leads to the appearance of phosphorylated B-Myb in nuclear SC35 speckles during transcriptional shutdown. Furthermore, we show that UV irradiation leads to a change of the phosphorylation pattern of B-Myb, which is caused by a switch from Cyclin/Cdk-dependent to Jnk and p38 kinase-dependent phosphorylation. Taken together, we have identified Jnk and p38 kinase as novel regulators of B-Myb and established the localization of phosphorylated B-Myb in SC35 speckles as a potential novel regulatory mechanism for B-Myb in UV irradiated cells.

Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46.

The tumor suppressor protein Pdcd4 is a nuclear/cytoplasmic shuttling protein that has been implicated in the development of several types of human cancer. In the nucleus, Pdcd4 affects the transcription of specific genes by modulating the activity of several transcription factors. We have identified the Daxx protein as a novel interaction partner of Pdcd4. Daxx is a scaffold protein with roles in diverse processes, including transcriptional regulation, DNA-damage signaling, apoptosis and chromatin remodeling. We show that the interaction of both proteins is mediated by the N-terminal domain of Pdcd4 and the central part of Daxx, and that binding to Pdcd4 stimulates the degradation of Daxx, presumably by disrupting the interaction of Daxx with the de-ubiquitinylating enzyme Hausp. Daxx has previously been shown to serve as a scaffold for protein kinase Hipk2 and tumor suppressor protein p53 and to stimulate the phosphorylation of p53 at serine 46 (Ser-46) in response to genotoxic stress. We show that Pdcd4 also disrupts the Daxx-Hipk2 interaction and inhibits the phosphorylation of p53. We also show that ultraviolet irradiation decreases the expression of Pdcd4. Taken together, our results support a model in which Pdcd4 serves to suppress the phosphorylation of p53 in the absence of DNA damage, while the suppressive effect of Pdcd4 is abrogated after DNA damage owing to the decrease of Pdcd4. Overall, our data demonstrate that Pdcd4 is a novel modulator of Daxx function and provide evidence for a role of Pdcd4 in restraining p53 activity in unstressed cells.

Inhibition of Myb-dependent gene expression by the sesquiterpene lactone mexicanin-I.

The c-myb proto-oncogene encodes a transcription factor that is highly expressed in the progenitor cells of the hematopoietic system, where it regulates the expression of genes important for lineage determination, cell proliferation and differentiation. There is strong evidence that deregulation of c-myb expression is involved in the development of human tumors, particularly of certain types of leukemia, and breast and colon cancer. The c-Myb protein is therefore an interesting therapeutic target. Here, we have investigated the potential of natural sesquiterpene lactones (STLs), a class of compounds that are active constituents of a variety of medicinal plants, to suppress Myb-dependent gene expression. We have developed a test system that allows screening of compounds for their ability to interfere with the activation of Myb target genes. Using this assay system, we have identified the STL mexicanin-I as the first cell-permeable, low-molecular-weight inhibitor of Myb-induced gene expression.

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation.

Pdcd4 is a novel tumor suppressor protein that functions in the nucleus and the cytoplasm, and appears to be involved in the regulation of transcription and translation. In the cytoplasm, Pdcd4 has been implicated in the suppression of translation of mRNAs containing structured 5'-untranslated regions; however, the mechanisms that recruit Pdcd4 to specific target mRNAs and the identities of these mRNAs are mostly unknown. In this study, we have identified c-myb mRNA as the first natural translational target mRNA of Pdcd4. We have found that translational suppression of c-myb mRNA by Pdcd4 is dependent on sequences located within the c-myb-coding region. Furthermore, we have found that the N-terminal domain of Pdcd4 has an important role in targeting Pdcd4 to c-myb RNA by mediating preferential RNA binding to the Pdcd4-responsive region of c-myb mRNA. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA and provides the first evidence for a key role of the RNA-binding domain in targeting Pdcd4 to a specific mRNA.

Disruption of the Pdcd4 tumor suppressor gene in chicken DT40 cells reveals its role in the DNA-damage response.

The programmed cell death gene 4 (Pdcd4) gene has been implicated as a new tumor suppressor gene in the development of several types of human cancer. Pdcd4 interacts with the translation initiation factor, eIF4A, and is thought to act as a translation inhibitor. Here, we have used the chicken B-cell line DT40 to disrupt the Pdcd4 gene by homologous recombination. Our study shows that cells lacking a functional Pdcd4 gene are viable and have no obvious defects when cultivated under normal growth conditions. However, Pdcd4 knockout cells show an increased sensitivity to agents that cause DNA damage, such as UV light, etoposide or ethyl-methanesulfonate. In summary, our findings show that Pdcd4 has an important function in the cellular response to DNA damage. Low Pdcd4 expression, which is frequently observed in tumor cells, might therefore contribute to tumorigenesis by disturbing the cellular DNA-damage response.

v-Myc inhibits C/EBPbeta activity by preventing C/EBPbeta-induced phosphorylation of the co-activator p300.

Myc, a key regulator of cellular proliferation, differentiation and apoptosis, exerts its biological functions by activating or suppressing the transcription of specific sets of target genes. C/EBP transcription factors play important roles during differentiation of various cell types and have been identified as critical targets for v-Myc- and c-Myc-dependent suppression of myeloid and fat cell differentiation. Here, we have addressed the mechanism by which v-Myc suppresses the activity of C/EBPbeta. We show that v-Myc is recruited to the aminoterminal domain of C/EBPbeta and interferes with the cooperation of C/EBPbeta and the co-activator p300 by preventing C/EBPbeta-induced phosphorylation of p300. We have identified the protein kinase responsible for C/EBPbeta-induced phosphorylation of p300 as homeo-domain interacting protein kinase 2 (HIPK2) and show that v-Myc displaces the kinase from the C/EBPbeta-p300 complex. Overall, our findings that the modulation of the C/EBPbeta-induced phosphorylation of p300 as a new mechanism of transcriptional suppression by v-Myc.

siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Waf1/Cip1) expression.

The transformation suppressor gene, programmed cell death gene 4 (Pdcd4), inhibits tumor-promoter-mediated transformation of mouse keratinocytes and has been implicated as a tumor suppressor gene in the development of human cancer. The Pdcd4 protein interacts with translation initiation factors eIF4A and eIF4G and binds to RNA, suggesting that it might be involved in regulating protein translation or other aspects of RNA metabolism. To study the function of Pdcd4 in more detail, we have downregulated Pdcd4 expression in HeLa cells by stable expression of shRNA. We have found that diminished Pdcd4 expression leads to increased expression of p21(Waf1/Cip1) and several other p53-regulated genes. Reporter gene studies demonstrate that Pdcd4 interferes with the activation of p53-responsive promoters genes by p53. Pdcd4 knockdown cells show decreased apoptosis and increased survival after UV irradiation. Taken together, our observations suggest a model in which low Pdcd4 expression after DNA damage favors the survival of cells, which would be eliminated by apoptosis under normal levels of Pdcd4 expression. Our results provide the first evidence that Pdcd4 is important role in the DNA-damage response and suggest that low levels of Pdcd4 expression observed in certain tumor cells contribute to tumorigenesis by affecting the fate of DNA-damaged cells.

Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity.

Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.

Structure of the C-terminal MA-3 domain of the tumour suppressor protein Pdcd4 and characterization of its interaction with eIF4A.

Programmed cell death protein 4 (Pdcd4) is a novel tumour suppressor protein, which is involved in the control of eukaryotic transcription and translation. The regulation of translation involves specific interactions with eukaryotic initiation factor (eIF)4A and eIF4G, which are mediated via the two tandem MA-3 domains. We have determined the structure of the C-terminal MA-3 domain of Pdcd4 (Pdcd4 MA-3(C)), characterized its interaction with eIF4A and compared the features of nuclear magnetic resonance (NMR) spectra obtained from the single domain and tandem MA-3 region. Pdcd4 MA-3(C) is composed of three layers of helix-turn-helix hairpins capped by a single helix and shows close structural homology to the atypical HEAT repeats found in many eIFs. The sequence conservation and NMR data strongly suggest that the tandem MA-3 region is composed of two equivalent domains connected by a somewhat flexible linker. Pdcd4 MA-3(C) was found to interact with the N-terminal domain of eIF4A through a conserved surface region encompassing the loop connecting alpha5 and alpha6 and the turn linking alpha3 and alpha4. This site is strongly conserved in other MA-3 domains known to interact with eIF4A, including the preceding domain of Pdcd4, suggesting a common mode of binding.

Identification of the myb-inducible promoter of the chicken Pdcd4 gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which disrupts the myelomonocytic differentiation program and transforms myelomonocytic cells in vivo and in vitro. It is thought that v-Myb exerts its biological effects by deregulating the expression of specific target genes, most of which are still unknown. c-myb, the cellular progenitor of v-myb, is expressed in all immature hematopoietic cells and is presumed to regulate the expression of genes that are essential for the development of the hematopoietic system. Recently, we have identified the chicken Pdcd4 gene as a novel v-myb target gene. Pdcd4 has originally been identified in a screen for genes upregulated in apoptotic cells and, more recently, has been implicated in tumor progression. As a myb-regulated gene Pdcd4 is of interest because unlike most other myb target genes it is expressed in a broad spectrum of hematopoietic cells. As a first step to study the regulation of Pdcd4 expression in more detail, we here report the identification and preliminary characterization of the myb-inducible promoter of the Pdcd4 gene.

The chicken Pdcd4 gene is regulated by v-Myb.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the ability of avian myeloblastosis virus (AMV) to transform myelomonocytic cells. v-Myb is thought to disrupt the differentiation of myelomonocytic cells by affecting the expression of specific target genes. To identify such genes we have analysed the gene expression in a myelomonocytic chicken cell line that carries an estrogen inducible version of v-Myb by differential display. Here we describe the identification of the chicken homolog of the mouse Pdcd4 gene as a novel v-Myb target gene. Pdcd4 is also known as MA-3, TIS and H731 and has recently been shown to suppress the transformation of epidermal cells by tumor promoters. Our results provide the first evidence that v-Myb directly regulates the expression of a potential tumor suppressor gene.

Crosstalk between Myc and activating transcription factor 2 (ATF2): Myc prolongs the half-life and induces phosphorylation of ATF2.

Myc is a key regulator of cell growth, differentiation and apoptosis, and affects cell fate decisions by activating as well as by inhibiting the expression of cellular genes. Myc is a member of the basic region-helix-loop-helix-leucine zipper (b-HLH-Zip) class of transcription factors, which heterodimerizes with the Max protein and recognizes a consensus Myc binding motif. Stimulation of gene expression by Myc is thought to be mediated by direct binding of Myc-Max heterodimers to specific target genes. So far, only a few genes have been identified as direct binding targets of Myc, raising the possibility that Myc affects gene expression also by indirect mechanisms. In this work we present evidence that v-Myc encoded by the avian retrovirus MC29 stimulates activating transcription factor 2 (ATF2)-dependent transcription. Analysis of the effect of Myc on ATF2 shows that v-Myc prolongs the half-life of ATF2 and induces the phosphorylation of N-terminal sites of ATF2 (Thr-69 and Thr-71) which have previously been identified as the target sites of stress-activated protein kinases and implicated in the regulation of ATF2 activity. Taken together, our results suggest that v-Myc can affect gene expression indirectly by modulating the activity of ATF2.

An alternatively spliced isoform of B-Myb is a transcriptional inhibitor.

B-Myb is a highly conserved member of the Myb transcription factor family. The primary transcript of the B-myb gene is spliced alternatively in two mRNAs which either contain or lack a sequence corresponding to the so-called exon 9A of c-myb. Recent studies showed that full-length B-Myb containing the exon 9A encoded amino acids is a cell cycle regulated transcription factor whose activity is stimulated by cyclin A/Cdk 2-dependent phosphorylation at the carboxyl-terminus of B-Myb. We have now investigated in more detail the transactivation potential of the shorter isoform of B-Myb lacking exon 9A. Here, we show that B-Myb lacking exon 9A has no transactivation activity even in the presence of cyclin A. This inactivity of the shorter isoform of B-Myb is not due an improper subcelluar localization. Our work suggests that B-Myb lacking exon 9A may act as an inhibitor for full-length B-Myb mediated transactivation. Furthermore, by analysing the transactivation potential of Gal4/B-Myb fusion proteins we have identified the amino-terminal part of the exon 9A as the principal transactivation domain of full-length B-Myb. The results presented here demonstrate that B-myb encodes both an activator and an inhibitor of transcription and, thus, reveal an additional level of regulation of B-Myb activity beside the known cyclin dependent mechanisms.

Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway.

B-Myb, a highly conserved member of the Myb oncoprotein family, is a 110 kDa sequence-specific DNA binding protein expressed in virtually all proliferating cells. B-myb expression reaches its maximum at the G1/S phase boundary and during the S phase of the cell cycle. We have previously shown that B-Myb activity is cell cycle regulated and it is controlled by the antagonistic effects of cyclin D1 and A. Here we show that ectopic expression of cyclin A causes a pronounced reduction of B-Myb protein level. We provide evidence that in addition to triggering B-Myb activity an important effect of cyclin A is to facilitate multiple ubiquitination of B-Myb. The C-terminal domain of B-Myb is of key importance in mediating this effect of cyclin A. Contrary to full-length B-Myb, a C-terminal deletion mutant displays activity irrespective of cyclin A expression, does not undergo ubiquitination, and its half-life is not affected by cyclin A. Ectopic expression of either Cdc34 or the F-box protein p45Skp2, respectively the E2 and E3 components of a ubiquitination pathway that regulates the G1/S transition, accelerates degradation of B-Myb. We show that B-Myb physically and functionally interacts with components of the Cdc34-SCFp45Skp2 ubiquitin pathway and propose that B-Myb degradation may be required for controlling the correct alternation of events during progression through the cell division cycle. Oncogene (2000).

Regulation of B-Myb activity by cyclin D1.

Evidence obtained during recent years suggests that B-Myb, a highly conserved member of the Myb transcription factor family, plays a key role in cell proliferation. We have shown previously that the activity of B-Myb is stimulated by cyclin A/Cdk2-dependent phosphorylation of the carboxyl-terminus of B-Myb. We have now investigated in more detail the effect of other cyclins on B-Myb. Here, we show that cyclin D1, in contrast to cyclin A, strongly inhibits the activity of B-Myb. This inhibitory effect does not involve increased phosphorylation of B-Myb but seems to rely on the formation of a specific complex of B-Myb and cyclin D1. Our work identifies B-Myb as an interacting partner for cyclin D1 and suggest that the activity of B-Myb during the cell cycle is controlled by the antagonistic effects of cyclin D1 and A. The results presented here suggest a more general role of cyclin D1 as regulator of transcription in addition to the known effect on RB phosphorylation.

The v-Myb oncoprotein activates C/EBPbeta expression by stimulating an autoregulatory loop at the C/EBPbeta promoter.

Previous studies have implicated the CCAAT box/enhancer binding protein beta (C/EBPbeta) in the regulation of cell-type specific gene expression in myelomonocytic cells and in the activation of target genes by the transcription factor v-Myb. To better understand the role of C/EBPbeta in myelomonocytic cells we have cloned the chicken C/EBPbeta gene and studied its regulation. The chicken C/EBPbeta promoter contains a number of C/EBP binding sites and is activated by C/EBPbeta, suggesting that the C/EBPbeta gene is autoregulated by its own protein product. Interestingly, the C/EBPbeta promoter is not activated by C/EBPalpha, another C/EBP family member highly expressed in myelomonocytic cells, indicating that the autoregulation is specific for C/EBPbeta. Comparison of different C/EBP inducible promoters shows that the relative transactivation potential of C/EBPalpha and beta is extremely dependent on the promoter context. By using the promoters of the mim-1 and C/EBPbeta genes and by exchanging the DNA-binding domains between C/EBPalpha and beta we show that the observed promoter preferences of C/EBPalpha and beta are not due to differential DNA-binding but instead depend on the transactivation domains of these proteins. The C/EBPbeta promoter also contains several Myb binding motifs, suggesting that the C/EBPbeta gene is also myb-inducible. We show that the C/EBPbeta promoter is activated synergistically by v-Myb and C/EBPbeta and that transcription of the endogenous C/EBPbeta gene is increased by v-Myb. Thus, our results identify the C/EBPbeta gene as a novel v-Myb target gene. Taken together, our data suggest a model for the regulation of C/EBPbeta expression in which v-Myb stimulates the synthesis of C/EBPbeta by enhancing an autoregulatory loop acting on the C/EBPbeta promoter.

Myb and Ets transcription factors cooperate at the myb-inducible promoter of the tom-1 gene.

Transformation of myeloid cells by the retroviral oncogene v-myb is thought to be caused by deregulated expression of specific cellular genes that act as targets of v-Myb in myeloid cells. Recently, we have identified the chicken tom-1 gene as a direct target for v-Myb. tom-1 has two promoters, only one of which (the tom-1A promoter) is activated by v-Myb. Here, we show that v-Myb activates the tom-1A promoter by cooperating with Ets-2, a member of the Ets transcription factor family. Interestingly, we find that the ability of v-Myb to cooperate with Ets proteins differs from that of its non-oncogenic cellular counterpart c-Myb. c-Myb cooperates with Ets-1 and Ets-2, whereas v-Myb only cooperates with Ets-2. Truncation of the N-terminus of c-Myb, which is known to activate the oncogenic potential of c-Myb, specifically abrogates the ability of the protein to cooperate with Ets-1. Our findings, therefore, reveal a novel function for the N-terminus of c-Myb and raise the possibility that oncogenic activation of c-Myb is linked to the loss of cooperation between Myb and c-Ets-1.