ARF-GEP100, a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP100 activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP100, a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP100 in those areas. No similar coincidence of ARF-GEP100 with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

Mechanisms of Transforming Growth Factor-β Receptor Endocytosis and Intracellular Sorting Differ between Fibroblasts and Epithelial Cells

Transforming growth factor-βs (TGF-β) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-β type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-β receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2α

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.

Target gene search for the metal-responsive transcription factor MTF-1

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding α-fetoprotein, the liver-enriched transcription factor C/EBPα and tear lipocalin/von Ebner’s gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns γ-glutamylcysteine synthetasehc (γ-GCShc), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. γ-GCShc mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

The role of distinct p185neu extracellular subdomains for dimerization with the epidermal growth factor (EGF) receptor and EGF-mediated signaling

The extracellular domain of p185c-neu can be viewed as a complex structure of four subdomains, two of which are cysteine-rich subdomains. We have investigated the contribution of these distinct p185c-neu extracellular subdomains to p185/epidermal growth factor receptor (EGFR) heteromer formation and EGF-induced heteromeric signaling. Our studies indicate that at least two separate p185 subdomains, a region spanning subdomains I and II and subdomain IV are involved in association of p185 with the EGFR. We also demonstrated that subdomain IV reduced the heteromeric signaling and transforming activities induced by EGF after associating with EGFR. When 126 aa were deleted from subdomain IV, this small subdomain IV-derived fragment could still lead to heterodimers with EGFR and suppress EGF-induced mitogen-activated protein kinase activation and subsequent transformation abilities. These data provide information about trans-inhibitory mechanisms of mutant p185 species and also indicate that both the entire and a part of subdomain IV may represent a therapeutic target for erbB-overexpressing tumors. Finally, these studies define a basic feature of receptor-receptor associations that are determined by cystine-knot containing subdomains.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor α (TGFα), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Cleavage and polyadenylation of mRNA 3′ ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual protein–protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.

RNA polymerase II transcription initiation: A structural view

In eukaryotes, RNA polymerase II transcribes messenger RNAs and several small nuclear RNAs. Like RNA polymerases I and III, polymerase II cannot act alone. Instead, general initiation factors [transcription factor (TF) IIB, TFIID, TFIIE, TFIIF, and TFIIH] assemble on promoter DNA with polymerase II, creating a large multiprotein–DNA complex that supports accurate initiation. Another group of accessory factors, transcriptional activators and coactivators, regulate the rate of RNA synthesis from each gene in response to various developmental and environmental signals. Our current knowledge of this complex macromolecular machinery is reviewed in detail, with particular emphasis on insights gained from structural studies of transcription factors.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants

The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants. RF2a, a b-Zip protein from rice, is known to bind to the Box II cis element near the TATA box of the promoter. Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides −164 to +45, result in phloem-specific expression of β-glucuronidase (GUS) reporter genes in transgenic tobacco plants. When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2–20-fold. The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive. Constitutive expression of RF2a did not induce morphological changes in the transgenic plants. In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants. These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants.

An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.

Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis.

Human complement factor H controls spontaneous activation of complement in plasma and appears to play a role in distinguishing host cells from activators of the alternative pathway of complement. In both mice and humans, the protein is composed of 20 homologous short consensus repeat (SCR) domains. The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a significant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant protein to be fully active. Mutants of this full-length protein have now been prepared, purified, and examined for cofactor activity and binding to C3b and heparin. The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is located in SCR domains 1-4, as has been shown by others, (iii) that a second site exists in the domain 6-10 region, (iv) that a third site resides in the SCR 16-20 region, and (v) that two heparin binding sites exist in factor H, one near SCR 13 and another in the SCR 6-10 region. Functional assays demonstrated that only the first C3b site located in SCR 1-4 expresses factor I cofactor activity. Mutant proteins lacking any one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocytes. The identification of multiple functionally distinct sites on factor H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bound C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alternative pathway of complement.

Organization and chromosomal localization of the gene encoding the mouse acid labile subunit of the insulin-like growth factor binding complex.

After birth, most of insulin-like growth factor I and II (IGFs) circulate as a ternary complex formed by the association of IGF binding protein 3-IGF complexes with a serum protein called acid-labile subunit (ALS). ALS retains the IGF binding protein-3-IGF complexes in the vascular compartment and extends the t1/2 of IGFs in the circulation. Synthesis of ALS occurs mainly in liver after birth and is stimulated by growth hormone. To study the basis for this regulation, we cloned and characterized the mouse ALS gene. Comparison of genomic and cDNA sequences indicated that the gene is composed of two exons separated by a 1126-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide and contributes the first nucleotide of codon 6. Exon 2 contributes the last 2 nt of codon 6 and encodes the remaining 17 amino acids of the signal peptide as well as the 580 amino acids of the mature protein. The polyadenylylation signal, ATTAAA, is located 241 bp from the termination codon. The cDNA and genomic DNA diverge 16 bp downstream from this signal. Transcription initiation was mapped to 11 sites over a 140-bp TATA-less region. The DNA fragment extending from nt -805 to -11 (ATG, +1) directed basal and growth hormone-regulated expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. Finally, the ALS gene was mapped to mouse chromosome 17 by fluorescence in situ hybridization.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.