Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.

CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+ T cells

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4+ T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4+ T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.

Axotomized neonatal motoneurons overexpressing the bcl2 proto-oncogene retain functional electrophysiological properties.

Bcl2 overexpression prevents axotomy-induced neuronal death of neonatal facial motoneurons, as defined by morphological criteria. However, the functional properties of these surviving lesioned transgenic neurons are unknown. Using transgenic mice overexpressing the protein Bcl2, we have investigated the bioelectrical properties of transgenic facial motoneurons from 7 to 20 days after neonatal unilateral axotomy using brain-stem slices and whole cell patch-clamp recording. Nonaxotomized facial motoneurons from wild-type and transgenic mice had similar properties; they had an input resistance of 38 +/- 6 M omega and fired repetitively after injection of positive current pulses. When cells were voltage-clamped at or near their resting membrane potential, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), or vasopressin generated sustained inward currents. In transgenic axotomized mice, facial motoneurons could be found located ipsilaterally to the lesion; they had an input resistance of 150 +/- 30 M omega, indicating that they were smaller in size, fired repetitively, and were also responsive to AMPA, NMDA, and vasopressin. Morphological measurements achieved 1 week after the lesion have shown that application of brain-derived neurotrophic factor prevented the reduction in size of axotomized transgenic motoneurons. These data indicate that Bcl2 not only prevents morphological apoptotic death of axotomized neonatal transgenic motoneurons but also permits motoneurons to conserve functional electrophysiological properties.

Cloning and characterization of Lnk, a signal transduction protein that links T-cell receptor activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase.

A cDNA encoding a signal transduction protein with a Src homology 2 (SH2) domain and a tyrosine phosphorylation site was cloned from a rat lymph node cDNA library. This protein, which we designate Lnk, has a calculated molecular weight of 33,988. When T lymphocytes were activated by antibody-mediated crosslinking of the T-cell receptor and CD4, Lnk became tyrosine phosphorylated. In activated T lymphocytes, phospholipase C gamma 1, phosphatidylinositol 3-kinase, and Grb-2 coimmunoprecipitated with Lnk. Our results suggest that Lnk becomes tyrosine phosphorylated and links the immediate tyrosine phosphorylation signals of the TCR to the distal phosphatidylinositol 3-kinase, phospholipase C gamma 1 and Ras signaling pathways through its multifunctional tyrosine phosphorylation site.

Myc and Max: molecular evolution of a family of proto-oncogene products and their dimerization partner.

The myc gene family encodes a group of transcription factors that regulate cell proliferation and differentiation. These genes are widely studied because of their importance as proto-oncogenes. Phylogenetic analyses are described here for 45 Myc protein sequences representing c-, N-, L-, S-, and B-myc genes. A gene duplication early in vertebrate evolution produced the c-myc lineage and another lineage that later gave rise to the N- and L-myc lineages by another gene duplication. Evolutionary divergence in the myc gene family corresponds closely to the known branching order of the major vertebrate groups. The patterns of sequence evolution are described for five separate highly conserved regions, and these analyses show that differential rates of sequence divergence (= mosaic evolution) have occurred among conserved motifs. Further, the closely related dimerization partner protein Max exhibits significantly less sequence variability than Myc. It is suggested that the reduced variability in max stems from natural selection acting to preserve dimerization capability with products of myc and related genes.

Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat prolactin promoter

Ets factors play a critical role in oncogenic Ras- and growth factor-mediated regulation of the proximal rat prolactin (rPRL) promoter in pituitary cells. The rPRL promoter contains two key functional Ets binding sites (EBS): a composite EBS/Pit-1 element located at –212 and an EBS that co-localizes with the basal transcription element (BTE, or A-site) located at –96. Oncogenic Ras exclusively signals to the –212 site, which we have named the Ras response element (RRE); whereas the response of multiple growth factors (FGFs, EGF, IGF, insulin and TRH) maps to both EBSs. Although Ets-1 and GA binding protein (GABP) have been implicated in the Ras and insulin responses, respectively, the precise identity of the pituitary Ets factors that specifically bind to the RRE and BTE sites remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE) that bind to the EBSs contained in the RRE and BTE, we used EBS-RRE and BTE oligonucleotides in electrophoretic mobility shift assays (EMSAs), antibody supershift assays, western blot analysis of partially purified fractions and UV-crosslinking studies. EMSAs, using either the BTE or EBS-RRE probes, identified a specific protein–DNA complex, designated complex A, which contains an Ets factor as determined by oligonucleotide competition studies. Using western blot analysis of GH3 nuclear proteins that bind to heparin–Sepharose, we have shown that Ets-1 and GABP, which are MAP kinase substrates, co-purify with complex A, and supershift analysis with specific antisera revealed that complex A contains Ets-1, GABPα and GABPβ1. In addition, we show that recombinant full-length Ets-1 binds equivalently to BTE and EBS-RRE probes, while recombinant GABPα/β preferentially binds to the BTE probe. Furthermore, comparing the DNA binding of GH4NE containing both Ets-1 and GABP and HeLa nuclear extracts devoid of Ets-1 but containing GABP, we were able to show that the EBS-RRE preferentially binds Ets-1, while the BTE binds both GABP and Ets-1. Finally, UV-crosslinking experiments with radiolabeled EBS-RRE and BTE oligonucleotides showed that these probes specifically bind to a protein of ∼64 kDa, which is consistent with binding to Ets-1 (54 kDa) and/or the DNA binding subunit of GABP, GABPα (57 kDa). These studies show that endogenous, pituitary-derived GABP and Ets-1 bind to the BTE, whereas Ets-1 preferentially binds to the EBS-RRE. Taken together, these data provide important insights into the mechanisms by which the combination of distinct Ets members and EBSs transduce differential growth factor responses.

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.

Newly identified stress-responsive protein kinases, Krs-1 and Krs-2.

The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C2-Man-)Trp.

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

The Nef protein of HIV-1 associates with rafts and primes T cells for activation

The Nef protein is an important virulence factor of primate lentiviruses, yet the mechanisms by which it exerts this influence are imperfectly understood. Here, using an inducible system, we demonstrate that Nef increases IL-2 secretion from T cells stimulated via CD3 or CD28. This effect requires the conservation of the Nef myristoylation signal and SH3-binding proline-based motif. Together with several proteins involved in the initiation and propagation of T cell signaling, Nef associates with membrane microdomains known as rafts. The Nef-mediated superinduction of IL-2 reflects the activation of both NFAT and NFκB. Accordingly, Nef also enhances HIV-1 transcription in response to CD3 or CD28 stimulation. Nef-induced IL-2 hyperresponsiveness is also observed in primary CD4 lymphocytes. Overall, these data suggest that Nef acts at the level of rafts to prime T cells for activation. Likely consequences of this effect are the promotion of HIV-1 replication and the facilitation of virus spread.

Triplex forming oligonucleotide targeted to 3′UTR downregulates the expression of the bcl-2 proto-oncogene in HeLa cells

The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3′-untranslated region (3′UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3′UTR of the bcl-2 proto-oncogene in the regulation of bcl-2 gene expression. To do this, we have developed a novel cellular system that involves transfection of a Doxycyclin inducible expression plasmid containing the bcl-2 ORF and the 3′UTR together with a TFO targeted to the 3′UTR of the bcl-2 proto-oncogene. Phosphorothioate-modified TFO targeted to the 3′UTR of the bcl-2 gene significantly downregulated the expression of the bcl-2 gene in HeLa cells as demonstrated by western blotting. Our results indicate that blocking the functions of the 3′UTR using the TFO can downregulate the expression of the targeted gene, and suggest that triplex strategy is a promising approach for oligonucleotide-based gene therapy. In addition, triplex-based sequence targeting may provide a useful tool for studying the regulation of gene expression.

E2F-dependent transcription of the raf proto-oncogene during Drosophila development

D-raf, a Drosophila homolog of the raf proto-oncogene, has diverse functions throughout development and is transcribed in a wide range of tissues, with high levels of expression in the ovary and in association with rapid proliferation. The expression pattern resembles those of S phase genes, which are regulated by E2F transcription factors. In the 5′-flanking region of D-raf, four sequences (E2F sites 1–4) similar to the E2F recognition sequence were found, one of them (E2F site 3) being recognized efficiently by Drosophila E2F (dE2F) in vitro. Transient luciferase expression assays confirmed activation of the D-raf gene promoter by dE2F/dDP. Expression of Draf–lacZ was greatly reduced in embryos homozygous for the dE2F mutation. These results suggest that dE2F is likely to be an important regulator of D-raf transcription.