ABSTRACT
The Maf oncoprotein is a basic leucine zipper (bZip)-bearing transcriptional activator that recognizes the Maf recognition element (MARE) DNA sequence. In this study, we investigated the role of Maf's transactivation function in cell transformation. Replacement of the conserved amino terminus transactivator domain of Maf by a heterologous and stronger transactivator domain (the acidic transactivator domain of VP16) resulted in enhanced transformation of chicken embryo fibroblast cells. In contrast, the fusing of a transcriptional repressor domain (Sin3 interaction domain of Mxi1) with the whole Maf protein masked the transactivator function of Maf, which in turn inhibited its transforming activity. Furthermore, the leucine zipper domain of Maf, which defines its dimer-forming specificity, was exchangeable with that of GCN4 yeast protein in terms of its transactivating and cell transforming activities. Thus, heterodimer formation with other bZip factors is not required for Maf's ability to transform. These results together suggest that transactivation through MARE is necessary for Maf-induced transformation and that there exist downstream target gene(s) for transformation. Since the MARE sequence overlaps with the recognition element of another bZip oncoprotein Jun, we assessed whether Jun and Maf induce cell transformation through activating the same genes. We thus constructed a mutated version of Jun that has a GCN4 leucine zipper and lacks the transactivator domain. This mutant repressed the cell transformation not only by Jun but also by Maf. Thus, Maf and Jun share downstream target gene(s) that are involved in cell transformation.
Subject(s)
Cell Nucleus/enzymology , Cell Nucleus/metabolism , Cell Transformation, Neoplastic , DNA-Binding Proteins/metabolism , Oncogene Protein p65(gag-jun)/metabolism , Oncogene Proteins, Viral/metabolism , Viral Proteins , Animals , Base Sequence , Blotting, Western , Chick Embryo , DNA/metabolism , Dimerization , Fibroblasts , Gene Deletion , Genes, Dominant , Humans , Leucine Zippers , Luciferases/metabolism , Molecular Sequence Data , Oncogene Protein v-maf , Phenotype , Plasmids/metabolism , Point Mutation , Protein Binding , Protein Structure, Tertiary , Rabbits , Recombinant Fusion Proteins/metabolism , Time Factors , Transcriptional ActivationABSTRACT
Maf oncoprotein is a basic-leucine zipper (bZip) type of transcriptional activator. Since many transcription factors are known to form functional complexes, we searched for proteins that interact with the DNA-binding domain of Maf using the phage display method and identified two homeodomain-containing proteins, Hoxd12 and MHox/Prx1/Phox1/Pmx1. Studies with mutants of Hox and Maf proteins showed that they associate through their DNA-binding domains; the homeodomain of Hox and the bZip domain of Maf, respectively. Reflecting the high similarity of the bZip domain, all other Maf family members tested (c-/v-Maf, MafB, MafK, MafF, and MafG) also associated with the Hox proteins. Pax6, whose homeodomain is relatively similar to MHox, also could interact with Maf. However, two other bZip oncoproteins, Fos and Jun, failed to associate with the Hox proteins, while a distantly related Hox family member, Meis1, could not interact with Maf. Through interactions with the bZip domain, the Hox proteins inhibited the DNA binding activity of Maf, whereas the binding of Hox proteins to their recognition sequences was not abrogated by Maf. We further showed that coexpression of the Hox proteins repressed transcriptional activation and transforming activity of Maf. These results suggested that the interaction of a set of Hox proteins with Maf family members may interfere not only with their oncogenicity but also with their physiological roles.
Subject(s)
Cell Transformation, Neoplastic , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Amino Acid Sequence , Animals , Binding Sites , Chickens , Cloning, Molecular , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Eye Proteins , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Leucine Zippers , Molecular Sequence Data , Mutation , PAX6 Transcription Factor , Paired Box Transcription Factors , Peptide Library , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-maf , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Repressor Proteins , Trans-Activators/antagonists & inhibitors , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
We have developed a method that enables us to isolate cDNAs of putative membrane proteins. The system is designed to isolate a cDNA which can provide the transmembrane domain to the extracellular part of the IL-2 receptor alpha chain. We constructed a p18Mac vector by putting part of the IL-2 receptor alpha chain cDNA that encoded its signal sequence and extracellular domain, a cDNA cloning site and a poly(A) additional signal after a strong promoter SRalpha. If a cloned cDNA provides a transmembrane domain in-frame, the extracellular domain of the IL-2 receptor alpha chain will be expressed on the surface of the transfected cells. Otherwise, the chimeric protein will be either secreted or retained inside the transfected cells. We made a cDNA library using p18Mac and screened for cDNA clones which allowed the expression of the extracellular domain of the IL-2 receptor alpha chain on the cell surface. Of the 2000 clones screened, 5 clones were scored as positive. Partial sequence analysis revealed that one clone encoded the amyloid precursor protein, two others encoded mitochondrial proteins and the rest were new. These results suggest the system is effective in isolating cDNAs encoding putative membrane proteins.
Subject(s)
DNA, Complementary/isolation & purification , Membrane Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , CD4 Antigens/genetics , CD4 Antigens/metabolism , COS Cells , Cloning, Molecular , Epitopes , Fluorescent Antibody Technique , Gene Library , Genetic Vectors , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Sorting Signals , Receptors, Interleukin-2/genetics , Receptors, Interleukin-2/metabolism , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
We have identified a novel member of the cadherin superfamily. Among the members of the superfamily, this protein exhibited the highest overall homology with protocadherin-1 (46-49% identity). Its mRNA was predominantly expressed in the brain and heart. Hence, we named the gene BH-protocadherin (BH-Pcdh) (HGMW-approved symbol PCDH7). BH-Pcdh has an extracellular domain consisting of seven repeats of the cadherin motif (EC 1 to 7). EC2 of BH-Pcdh is unique in having a 55-amino-acid insertion in the middle of the motif. There are three isoforms of BH-Pcdh, denoted -a, -b, and -c, which have different cytoplasmic tails and a 47-amino-acid deletion in the EC2-3 region of BH-Pcdh-c. While only a 9.0-kb message was detected in normal tissues, 4.5- and 9.0-kb mRNA species were seen in the human lung carcinoma cell line A549. Furthermore, only the 4.5-kb mRNA was detected in HeLa cell S3 and human gastric cancer cell lines MKN28 and KATO-III. Southern blot analysis indicated that the BH-Pcdh gene is likely to be conserved among various vertebrates. The BH-Pcdh gene was localized to human chromosome 4p15. Interestingly, 4p15 is a region of loss of heterozygosity in some head and neck squamous cell carcinomas.
Subject(s)
Cadherins/genetics , Chromosomes, Human, Pair 4 , Amino Acid Sequence , Brain/metabolism , Cadherins/biosynthesis , Cadherins/chemistry , Chromosome Mapping , Cloning, Molecular , DNA Transposable Elements , Humans , Molecular Sequence Data , Multigene Family , Myocardium/metabolism , Organ Specificity , Protocadherins , Sequence Alignment , Sequence Homology, Amino Acid , Tumor Cells, CulturedABSTRACT
Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.
Subject(s)
DNA, Complementary , Enzymes/genetics , Gene Library , Proteins/genetics , RNA, Messenger/metabolism , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Dyneins/chemistry , Dyneins/genetics , Humans , Molecular Sequence Data , Neuroblastoma , Peptide Elongation Factor 1 , Peptide Elongation Factors/biosynthesis , Peptide Elongation Factors/genetics , Polymerase Chain Reaction , RNA Caps/metabolism , RNA, Messenger/isolation & purification , Rats , Sequence Alignment , Sequence Homology, Nucleic Acid , Tumor Cells, CulturedABSTRACT
In order to isolate promoters of mouse TGF-beta receptor genes, we used inverse PCR with highly overlapped primers corresponding to the 5' sequence of the receptor cDNAs. Nested primer sets only covered a 30- to 40-base region of the sequences. HinfI-digested and self-ligated mouse genomic DNA was used as a PCR template. Only one band for each receptor was seen after PCR. The amplified DNA fragments could direct luciferase production when the luciferase coding sequence was ligated after the fragments. The sequence of the fragment which correspond to the type II receptor showed partial homology with the promoter region of the human TGF-beta type II receptor. Thus, the inverse PCR with highly overlapped primers could be an easy way to isolate the promoter regions of many genes.