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.

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.

Phosphorylation and activation of B-Myb by cyclin A-Cdk2.

BACKGROUND: Cyclins and their catalytic partners, the cyclin-dependent kinases (Cdks), function as key regulators of the eukaryotic cell cycle. Specific cyclin-Cdk complexes are active at successive stages during the cell cycle and control cell-cycle progression by phosphorylating specific target proteins, most of which have not yet been identified. B-Myb, a conserved member of the Myb oncoprotein family, is a sequence-specific DNA-binding protein expressed in virtually all proliferating mammalian cells. Increasing evidence suggests that B-Myb plays an important role during the late G1 and early S phases of the cell cycle. In this study, we have examined the regulation of B-Myb activity by cyclin-Cdks. RESULTS: We found that the transcriptional transactivation potential of B-Myb was repressed by a regulatory domain located at the carboxyl terminus of the protein. Coexpression of B-Myb and cyclin A relieved this repression by phosphorylation of B-Myb in its carboxy-terminal region. Tryptic phosphopeptide mapping revealed that endogenous B-Myb was phosphorylated in cells undergoing S phase. CONCLUSIONS: This work provides evidence for a link between the Myb oncoprotein family and the cell-cycle machinery. We have shown that the carboxyl terminus of B-Myb acts as a cell-cycle sensor that regulates the transactivation function of B-Myb. Moreover, our studies have identified B-Myb as a target of cyclin A-Cdk2 and have indicated that B-Myb activity is regulated by phosphorylation mediated by cyclin A-Cdk2.

Interaction of C/EBPbeta and v-Myb is required for synergistic activation of the mim-1 gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which activates the myelomonocyte-specific mim-1 gene, a natural myb target gene, by cooperating with members of the C/EBP transcription factor family. The finding that v-Myb, together with C/EBP, is sufficient to activate the mim-1 gene in heterologous cell types has implicated Myb and C/EBP as a bipartite molecular switch, which regulates the expression of myelomonocyte-specific genes. To understand the relationship between v-Myb and C/EBP in more detail, we have examined the molecular basis of the activation of the mim-1 promoter by v-Myb and C/EBPbeta, a member of the C/EBP transcription factor family highly expressed in myelomonocytic cells. We have identified a composite Myb and C/EBP response element which mediates synergistic activation of the mim-1 promoter by both factors and consists of closely spaced Myb- and C/EBP-binding sites. In vitro and in vivo protein-binding studies indicate that v-Myb and C/EBPbeta interact with each other via their DNA-binding domains. We show that this interaction is essential for the synergistic activation of the mim-1 promoter by v-Myb and C/EBPbeta. Our work therefore identifies C/EBPbeta as an interaction partner of v-Myb involved in myelomonocyte gene expression.

Subnuclear localization of proteins encoded by the oncogene v-myb and its cellular homolog c-myb.

The retroviral transforming gene v-myb encodes a 45,000-Mr nuclear transforming protein (p45v-myb). p45v-myb is a truncated and mutated version of a 75,000-Mr protein encoded by the chicken c-myb gene (p75c-myb). Like its viral counterpart, p75c-myb is located in the cell nucleus. As a first step in identifying nuclear targets involved in cellular transformation by v-myb and in c-myb function, we determined the subnuclear locations of p45v-myb and p75c-myb. Approximately 80 to 90% of the total p45v-myb and p75c-myb present in nuclei was released from nuclei at low salt concentrations, exhibited DNA-binding activity, and was attached to nucleoprotein particles when released from the nuclei after digestion with nuclease. A minor portion of approximately 10 to 20% of the total p45v-myb and p75c-myb remained tightly associated with the nuclei even in the presence of 2 M NaCl. These observations suggest that both proteins are associated with two nuclear substructures tentatively identified as the chromatin and the nuclear matrix. The function of myb proteins may therefore depend on interactions with several nuclear targets.

Interaction and functional collaboration of p300 and C/EBPbeta.

Transcriptional coactivators such as p300 and CREB-binding protein (CBP) function as important elements in the transcription factor network, linking individual transactivators via protein-protein interactions to the basal transcriptional machinery. We have investigated whether p300 plays a role in transactivation mediated by C/EBPbeta, a conserved member of the C/EBP family. We show that C/EBPbeta-dependent transactivation is strongly inhibited by adenovirus E1A but not by E1A mutants defective in p300 binding. Ectopic expression of p300 reverses the E1A-dependent inhibition and increases the transactivation potential of C/EBPbeta. Furthermore, we show that C/EBPbeta and p300 interact with each other and demonstrate that the sequences responsible for interaction map to the E1A binding region of p300 and the amino terminus of C/EBPbeta. Finally, we show that the minimal C/EBPbeta binding site of p300 acts as a dominant-negative inhibitor of C/EBPbeta. These observations identify p300 as a bona fide coactivator for C/EBPbeta. C/EBPbeta is highly expressed in the myelomonocytic lineage of the hematopoietic system and cooperates with Myb to activate mim-1, a gene specifically expressed during myelomonocytic differentiation. Recent evidence has shown that Myb recruits CBP (and presumably p300) as a coactivator and, in contrast to C/EBPbeta, interacts with the CREB binding site of p300-CBP. We show that p300 not only stimulates the activity of Myb and C/EBPbeta individually but also increases the synergy between them. Thus, our results reveal a novel function of p300: in addition to linking specific transcription factors to the basal transcriptional machinery, p300 also mediates the cooperation between transactivators interacting with different domains of p300.

Rearrangement at the 5' end of amplified c-myc in human COLO 320 cells is associated with abnormal transcription.

The proto-oncogene c-myc is amplified in sublines of human COLO 320 cells carrying either homogeneously staining chromosomal regions or double minutes. COLO 320 cells carrying homogeneously staining chromosomal regions have 15 to 20 copies of an apparently normal c-myc allele and 1 to 2 copies of an abnormal c-myc allele lacking exon 1 and express high levels of a normal c-myc mRNA 2.5 kilobases in size. COLO 320 cells carrying double minutes have about 25 copies each of the normal allele and the abnormal allele but express preferentially an abnormal c-myc mRNA 2.2 kilobases in size. Nucleotide sequence analyses revealed that the break point of rearrangement resulting in the loss of exon 1 in the abnormal allele lies within a region frequently rearranged in human and murine B-cell tumors.

Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb.

We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA.

Induced differentiation of avian myeloblastosis virus-transformed myeloblasts: phenotypic alteration without altered expression of the viral oncogene.

Cells of a clone of avian myeloblastosis virus-transformed myeloblasts were induced to differentiate to adherent myelomonocytic cells by treatment with lipopolysaccharide. These adherent cells were subcultured and maintained as a line for more than 6 months with lipopolysaccharide present. Cells of this line were induced to differentiate to nondividing macrophage-like cells by the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. In this way, the following homogeneous cell populations representing three distinct stages of myeloid differentiation were obtained: I, actively dividing myeloblasts that grew in suspension: II, actively dividing adherent cells; and III, fully differentiated nondividing cells resembling macrophages. When the expression of v-myb (the oncogene of avian myeloblastosis virus) was examined in cells of these three differentiation stages, it was found that the protein encoded by v-myb (p45v-myb) continued to be synthesized in similar quantities and showed no obvious alteration (assessed by partial proteolytic digestion and two-dimensional gel electrophoresis) during differentiation. These results show that cells transformed by v-myb can be induced to differentiate without affecting the expression of v-myb and imply that, during differentiation, the effect of v-myb is suppressed by a mechanism other than altered expression of the oncogene.

Coordinate regulation of myelomonocytic phenotype by v-myb and v-myc.

Both avian myeloblastosis virus (by the action of v-myb) and avian myelocytomatosis virus MC29 (by the action of v-myc) transform cells of the myelomonocytic lineage. Whereas avian myeloblastosis virus elicits a relatively immature phenotype, cells transformed by MC29 resemble mature macrophages. When cells previously transformed by v-myb were superinfected with MC29, their phenotype was rapidly altered to that of a more mature cell. These superinfected cells expressed both v-myb (at a level similar to that found before superinfection) and v-myc. It therefore appears that the expression of v-myc can elicit certain properties of a more differentiated phenotype. In addition, unlike cells transformed by v-myb alone, the cells expressing both v-myb and v-myc could not be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to differentiate to fully mature macrophages. Cells with a morphology similar to that of the superinfected cells were elicited by simultaneously infecting yolk sac macrophages with avian myeloblastosis virus and MC29. Such cells expressed both v-myb and v-myc. These results indicate that expression of v-myb and v-myc in infected cells coordinately regulates myelomonocytic phenotype and that the two viral oncogenes vary in their ability to interfere with tumor promoter-induced differentiation. Our findings also sustain previous suggestions that the oncogenes v-myb and v-myc may not transform target cells by simply blocking differentiation.

Interaction of myb proteins with nuclear matrix in vitro.

Fractionation studies of isolated nuclei have shown that the proteins encoded by the retroviral oncogene v-myb and its cellular homologue c-myb are associated to a variable extent with the nuclear matrix, suggesting that the nuclear matrix might contain a cellular target for myb proteins. I have explored the possible existence of such a target by incubating soluble v-myb and c-myb protein with nuclear matrix prepared from a separate source. The results presented here suggest that nuclear matrices from various cells contain binding sites for myb proteins. Matrix-binding appears to be an intrinsic property of myb proteins. In addition to myb proteins I have demonstrated the existence of a small group of proteins possessing nuclear matrix binding activity. These findings suggest that the nuclear matrix serves as a target for a specific set of proteins, including the products of myb genes.

Association of v-myc protein with chromatin.

The subnuclear distribution of proteins encoded by v-myb and v-myc was analysed in a cell-line of AMV-transformed chicken myeloblasts superinfected by the myc-containing retrovirus MC29. p45v-myb and p110gag-myc, co-expressed in these cells, were released in similar fashion when nuclei were treated with salt or DNAase. Analysis of nucleoprotein complexes extracted from nuclease-treated nuclei shows that p45v-myb and p110gag-myc are associated with a chromatin fraction of enhanced nuclease sensitivity. v-myb and v-myc proteins thus share the same subnuclear location and apparently interact directly with the cellular DNA.

Methylation-sensitive DNA binding by v-myb and c-myb proteins.

The retroviral oncogene v-myb and its cellular homolog c-myb encode nuclear DNA-binding phosphoproteins (v-MYB and c-MYB) that function as transcriptional regulators. v-MYB and c-MYB recognize a nucleotide sequence motif, PyAACG/TG, that is present in the promoter region of the myb-inducible mim-1 gene and is required for regulation of mim-1 expression by v-MYB and c-MYB. Since the myb-binding motif contains a CpG dinucleotide that constitutes a potential target for methylation, we have investigated whether recognition of the binding site by myb proteins is sensitive to CpG methylation of the binding motif. The results presented here demonstrate that bacterially expressed v-MYB as well as authentic v-MYB and c-MYB bind to the myb binding site in a methylation-sensitive manner. Our observations raise the interesting possibility that myb function can be regulated by methylation of myb binding sites.

B-Myb and cyclin D1 mediate heat shock element dependent activation of the human HSP70 promoter.

Previous studies have shown that B-Myb, a conserved member of the Myb transcription factor family, is a potent activator of the promoter of the human HSP70 gene but does not activate promoters containing Myb binding sites. We have now investigated the transactivation properties of B-Myb in more detail. We here report that B-Myb activates the HSP70 promoter by a novel mechanism which involves the heat shock element (HSE). Deletion analysis of B-Myb shows that a specific domain in the center of B-Myb, but not the DNA-binding domain is required for HSE-dependent transactivation. We also show that deletion of the C-terminal domain of B-Myb does not affect HSE-dependent transactivation but allows the protein to activate a promoter containing Myb binding sites. This suggests that the ability to activate Myb binding site containing promoters is repressed in the context of full length B-Myb and that HSE dependent and Myb binding site dependent transactivation are distinct functions of B-Myb. Finally, we report that cyclin D1 like B-Myb strongly activates the HSP70 promoter via the HSE. HSE-dependent transactivation is a novel activity of cyclin D1 and appears to be independent of the phosphorylation of the Rb protein. Our results reveal an interesting and unexpected connection between HSE-dependent gene activation and proteins expressed during the G1/S-transition of the cell cycle.

Linking Myb to the cell cycle: cyclin-dependent phosphorylation and regulation of A-Myb activity.

A-myb, a conserved member of the Myb proto-oncogene family, encodes a sequence-specific DNA binding protein (A-Myb) that binds to and transactivates promoters containing myb-binding sites. Previous work has suggested that the C-terminus of A-Myb functions as a regulatory domain, however, the physiological signals that control the activity of A-Myb have not yet been identified. The presence of potential phosphorylation sites for cyclin-dependent kinases in the C-terminus of A-Myb has prompted us to examine the possibility that the function of A-Myb is controlled by the cell cycle. We here show that the transactivation potential of A-Myb is repressed by the C-terminal domain and that phosphorylation of A-Myb, induced by cyclins A and E, relieves this inhibitory effect. Our work provides the first evidence that the function of A-Myb is regulated by the cell cycle machinery and that the carboxy-terminal domain of A-Myb acts as a cell cycle sensor. In addition, we show that A-myb mRNA expression is also cell cycle regulated and attains maximal levels during the late G1- and early S-phase. Thus, A-Myb appears to be controlled by two different mechanisms resulting in maximal A-Myb activity during the G1/S-transition and the S-phase of the cell cycle.

The chicken A-myb protein is a transcriptional activator.

c-myb encodes a trans-activator that is essential for the proliferation of most hematopoietic precursor cells but lacks a major role in non-hematopoietic cells. The recent identification of two myb-related genes (A-myb and B-myb) in several vertebrate species has raised the possibility that these genes perform c-myb like functions in non-hematopoietic cells. Here, we report the isolation and preliminary characterization of the chicken A-myb gene. Like other members of the myb family, the A-myb protein contains an N-terminally located repeat domain that is highly homologous to the DNA-binding domain of the c-myb protein. Unlike c-myb, A-myb is expressed in fibroblasts, suggesting that it functions in non-hematopoietic cells. We demonstrate that A-myb transactivates myb-responsive reporter genes. Furthermore, when stably expressed in a chicken macrophage cell line. A-myb causes activation of several myb-inducible endogenous genes. These findings show that A-myb encodes a transactivating member of the myb family whose functional properties resemble those of the c-myb and v-myb proteins. Thus, our results support the idea that A-myb is a functional equivalent of c-myb.

The chicken adenosine receptor 2B gene is regulated by v-myb.

The retroviral oncogene v-myb is a mutated and truncated version of the c-myb proto-oncogene and encodes a transcription factor (v-Myb) that specifically transforms myelomonocytic cells. v-Myb is thought to transform myelomonocytic cells by affecting the expression of specific target genes, most of which as yet remain unknown. To identify novel v-Myb regulated genes we have employed 'differential display', using a myelomonocytic chicken cell line that expresses a conditional version of v-Myb. Here we describe the identification of the gene encoding the A2b adenosine receptor, a member of the seven transmembrane receptor superfamily, as a v-Myb target gene. Our results provide the first evidence that v-Myb directly regulates a gene encoding a membrane receptor and establish a link between Myb function and adenosine receptor signaling.

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.

Expression of mouse c-myb during embryonic development.

Three members of the myb gene family, designated as A-myb, B-myb, and c-myb, have been described in many vertebrates. A large body of evidence indicates that the c-myb gene is essential for the development of most hematopoietic lineages. By contrast, the functions of A-myb and B-myb are less well understood. To further explore the relationship between the different myb family members we have compared the expression of A-myb and c-myb during mouse embryogenesis by Northern blotting and by in situ hybridization. In accordance with the important role of c-myb in the hematopoietic system, we detect high levels of c-myb expression in hematopoietic organs such as the fetal liver and the thymus. Surprisingly, we find that high levels of c-myb expression are not restricted to hematopoietic cells. We show that c-myb is strongly expressed in the neural retina and in epithelia of the respiratory tract. The side-by side analysis of c-myb and A-myb expression clearly shows that both genes are expressed in different, but overlapping sets of tissues. Our results suggest that the function of c-myb may not be restricted to hematopoietic cells.