PubMed Health⌕ Search

Biomedical subjects

C Kedinger

Publications and source records attributed to C Kedinger.

At least 37 records · Page 2Linked to original sources
PubMed1996

In vivo association of ATFa with JNK/SAP kinase activities.

The human ATFa proteins belong to the CREB/ATF family of transcription factors. We have previously shown that the ATFa proteins may contribute to the modulation of the transcriptional activity of the Jun/Fos complexes (Chatton et al. (1994). Oncogene, 9, 375-385). We now show that a protein kinase activity is strongly associated with ATFa in vivo, as revealed by coimmunoprecipitation of ATFa/kinase complexes from whole cell extracts, with antibodies against ATFa. Two independent regions were found to be implicated in kinase binding: a major interaction site is located within the N-terminal 82 residues comprising an important metal-chelating element; a weaker binding site corresponds to the basic sequence element preceding the C-terminal leucine-zipper of ATFa. Induction experiments suggest that each of these ATFa domains may interact with different kinases. The major activity is associated with the ATFa N-terminal domain. Based on its response to various inducers, on both in vitro and in vivo binding assays, and on its immunological properties, this activity most likely corresponds to the 54/55 kDa JNK2 protein. Taken together, these observations suggest that the ATFa proteins, among other CREB/ATF proteins, may be important effectors of cell signalling pathways.

Activating Transcription Factors↗
PubMed1996

The gene (POLR2L) encoding the hRPB7.6 subunit of human RNA polymerase.

The gene (POLR2L) encoding a 7.6-kDa subunit (hRPB7.6) of human RNA polymerase has been cloned. It compromises two exons, 116 and 227 bp, respectively, interspaced with an intron of about 2.1 kb. This gene, whose localization has been assigned to the short arm of chromosome 11 (position 11p15), is transcribed in HeLa cells as one major messenger RNA, which encodes a 67-residue polypeptide (7645 Da) that shares strong homologies with the corresponding subunits of other eukaryotic and archaeal RNA polymerase subunits. Like its yeast counterpart (ABC10 beta, encoded by the RPB10 gene), the hRPB7.6 subunit may be shared by all three classes of human nuclear RNA polymerase. Cysteine residues characteristic of an atypical zinc-binding domain are conserved in the homologous sequences of all six species analyzed. A small, related RNA polymerase subunit from vaccinia virus exhibits an identical set of cysteines, suggesting that these residues may be contribute to a crucial function in the multimeric RNA polymerases.

Amino Acid Sequence↗
PubMed1996

Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter.

The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the binding of both DEF-A and DEF-B complexes to the downstream elements. Finally, as revealed by immunomicroscopy, pIVa2 is targeted to the nucleus, where it distributes to restricted locations in the nucleoplasm, as well as to the nucleoli. Altogether, these results demonstrate that pIVa2 plays a critical role in the transition from the early to the late phase of the lytic cycle. Furthermore, pIVa2 may serve additional functions yet to be uncovered, as suggested by its presence within the cell nucleolus.

Adenoviruses, Human↗
PubMed1996

Nucleoplasmic and nucleolar distribution of the adenovirus IVa2 gene product.

Sequence elements (DE) located downstream of the adenovirus major late promoter start site have previously been shown to be essential for the activation of this promoter after the onset of viral DNA replication. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner and contribute to this activation. DEF-B corresponds to a dimer of the adenovirus IVa2 gene product (pIVa2, 449 residues), while DEF-A is a heteromeric protein also comprising pIVa2. As revealed by specific immunofluorescence staining of infected cells, pIVa2 is targeted to the nucleus, where it distributes to both nucleoplasmic and nucleolar structures. We have identified the pIVa2 nuclear localization signal (NLS) as a basic peptide element at the C terminus of the protein (residues 432 to 449). An element essential for nucleolar localization (NuLS) has been mapped in the N-terminal part of pIVa2 (between residues 50 and 136). While NuLS activity is dependent upon an intact NLS, we show that both NLS and NuLS functions are independent of specific DNA-binding activity. As visualized by immunoelectron microscopy, pIVa2 is detected in the nucleoplasm at the level of the fibrillogranular network which is active in viral transcription. More surprisingly, pIVa2 accumulates within electron-dense amorphous inclusions found both in the nucleoplasm and in the nucleolus. Altogether, these results suggest that, besides controlling major late promoter transcription, pIVa2 serves additional, as yet unknown functions.

Adenoviridae↗
PubMed1995

Eukaryotic GST fusion vector for the study of protein-protein associations in vivo: application to interaction of ATFa with Jun and Fos.

We describe a multipurpose eukaryotic expression vector that incorporates the following features: restriction sites for in-frame insertion of cDNAs of interest between sequences encoding the glutathione-S-transferase (GST) and an oligohistidine element, allowing expression of the corresponding fusion proteins; a phosphorylation site for protein kinase A for in vitro labeling of the fusion protein; a T7 promoter for in vitro transcription and subsequent translation; and signals for single-stranded DNA production in bacteria. We have used this vector to demonstrate the formation in vivo of complexes between the transcription factor ATFa, a member of the family of ATF/CRE binding proteins, and the c-Jun or c-Fos proteins. Such interactions could be detected in crude extracts from cells transfected with vectors expressing the GST-ATFa fusion protein, as well as the c-Jun or c-Fos proteins. Complexes containing both ATFa and either c-Jun or c-Fos were specifically retained on glutathione (GSH)-agarose beads as revealed by immunoblot analyses. We also show that the leucine zipper domain of ATFa is essential for this interaction.

Activating Transcription Factors↗
PubMed1994

Chromosomal localization of human RNA polymerase II subunit genes.

The eukaryotic DNA-dependent RNA polymerase II (or B) is composed of 10 to 14 polypeptides ranging from 220 to 10 kDa. To gain further insight into the molecular structure and function of these subunits, we have undertaken the molecular cloning of nucleotide sequences corresponding to the human enzyme. The cDNAs of five subunits (hRPB220, hRPB140, hRPB33, hRPB25, and hRPB14.5) have been isolated. Using in situ hybridization, we show that the genes of these subunits have distinct chromosomal locations (17p13, 4q12, 16q13-q21, 19p13.3, and 19q12, respectively). Thus, if assembly of active polymerase molecules requires coordinated expression from these independent genes, mechanisms that ensure tight coregulation of the corresponding promoters must exist.

Chromosome Banding↗
PubMed1994

The product of the adenovirus intermediate gene IVa2 is a transcriptional activator of the major late promoter.

During the course of lytic infection, the adenovirus major late promoter (MLP) is induced to high levels after replication of viral DNA has started. We had previously shown that sequence elements located downstream of the MLP start site were implicated in this late-specific transcriptional activation (DE1, between +85 and +98; DE2, between +100 and +120). Two positive transcription factors involved in this activation have been detected. DEF-A, which specifically binds to DE1 and also to the 3' portion of DE2 (DE2a), and DEF-B, which interacts with the 5' part of DE2 (DE2b). When present together, these two proteins cooperatively assemble onto the DE2 element. We now report the purification of DEF-B and show that it is identical to the product of the adenovirus IVa2 gene product. This conclusion is based on microsequence analysis of DEF-B as well as on the inhibitory effect of antibodies against IVa2 on the DNA-binding activity of DEF-B and also on DE-dependent in vitro transcription. In addition, we show that bacterially synthesized IVa2 protein binds to the DE sequences with the same specificity as DEF-B. Finally, in transfected cells, a recombinant IVa2 protein stimulates MLP activity in a DE-dependent fashion. The physiological implications of these findings are discussed.

Adenoviridae↗
PubMed1994

A 14.4 KDa acidic subunit of human RNA polymerase II with a putative leucine-zipper.

The cDNA of a small subunit (hRPB14.4) of RNA polymerase II (or B) from HeLa cells has been cloned. A 127 residue peptide sequence (calculated molecular weight of 14,478; isoelectric point of 3.7) was deduced and compared to that of the homologous subunit of Saccharomyces cerevisiae polymerase (ABC23, encoded by the RPB6/RPO26 gene). About 50% of the total residues were found to be conserved between yeast and man, with the C-terminal two-third being the most conserved (72% identity). A putative leucine-zipper comprising four properly spaced leucine residues, but not preceded by a basic domain, was identified near the C-terminal end of both proteins.

Amino Acid Sequence↗
PubMed1994

Jun and Fos heterodimerize with ATFa, a member of the ATF/CREB family and modulate its transcriptional activity.

Three related clones encoding proteins (ATFa1, 2 and 3) with specific ATF/CRE DNA-binding activities have been isolated from HeLa cell cDNA libraries. All three isoforms have weak effects on the basal activity of the adenovirus E2a promoter. We present evidence suggesting that a C-terminal element of the ATFa molecules negatively interferes with the intrinsic activation function of these proteins. We also show that coexpression of ATFa with c-Jun, Jun-B or Jun-D stimulates ATFa-dependent reporter activity, while coexpression of c-Fos has no effect. Deletion analyses indicate that the metal-binding region of ATFa is dispensible for this effect, but that the domain comprising the leucine-zipper region of ATFa is required. Reciprocal co-immunoprecipitation experiments and electrophoretic band-shift assays with in vitro synthesized proteins reveal direct interactions between ATFa and Jun or Fos. The ATFa/c-Jun heterodimers, but not the ATFa/c-Fos complexes, bind efficiently to ATF, CRE or AP1 sites. The detection of ATFa-Jun complexes in crude extracts from HeLa cells transfected with ATFa and c-Jun expression vectors suggests that such ATFa/c-Jun heterodimers also form in vivo. Altogether these results indicate that the ATFa proteins may contribute to the modulation of the activity of the Jun/Fos complexes by altering their DNA-binding and transcriptional properties.

Activating Transcription Factors↗
PubMed1993

Structure of the gene encoding the 14.5 kDa subunit of human RNA polymerase II.

The structure of the gene encoding the 14.5 kDa subunit of the human RNA polymerase II (or B) has been elucidated. The gene consists of six exons, ranging from 52 to over 101 bp, interspaced with five introns ranging from 84 to 246 bp. It is transcribed into three major RNA species, present at low abundance in exponentially growing HeLa cells. The corresponding messenger RNAs contain the same open reading frame encoding a 125 amino acid residue protein, with a calculated molecular weight of 14,523 Da. This protein (named hRPB14.5) shares strong homologies with the homologous polymerase subunits encoded by the Drosophila (RpII15) and yeast (RPB9) genes. Cysteines characteristic of two zinc fingers are conserved in all three corresponding sequences and, like the yeast protein, the hRPB14.5 subunit exhibits zinc-binding activity.

Amino Acid Sequence↗
PubMed1993

Transcriptional activation by the adenovirus larger E1a product is mediated by members of the cellular transcription factor ATF family which can directly associate with E1a.

We recently isolated three cDNA clones encoding closely related proteins (ATFa1, ATFa2, and ATFa3) that belong to the activating transcription factor-cyclic AMP-responsive element family of cellular transcription factors. Using cotransfection experiments, we showed that these proteins mediate the transcriptional activation induced by the adenovirus E1a 13S mRNA gene product and that the zinc-binding domains present in both E1a conserved region 3 and the most N-terminal portion of the ATFa proteins play crucial roles in this activity. Reciprocal coimmunoprecipitation experiments demonstrated direct interactions between these proteins. Neither the conserved region 3 domain of E1a nor the N-terminal metal-binding element of ATFa is essential for these interactions. The simultaneous alteration of both the N-terminal and the C-terminal domains of ATFa abolished E1a binding, while either mutation alone failed to impair these interactions.

Activating Transcription Factors↗
PubMed1993

Rb may act as a transcriptional co-activator in undifferentiated F9 cells.

The reversible interaction of the retinoblastoma susceptibility gene product (Rb) with the cellular transcription factor E2F has recently been demonstrated. Activation of the adenovirus E2a promoter by the products of the viral E1a gene correlates with the ability of both early E1a proteins to sequester Rb, thereby releasing E2F from inactive complexes with this protein. The E2a promoter is also efficiently stimulated by a product (17.5 kDa) of the viral E4 gene. The specific interaction of this E4 protein with E2F results in the formation of complexes that bind cooperatively to the two neighboring E2F binding sites in the E2a promoter. We have previously shown that in undifferentiated F9 cells (F9EC) the E2a promoter is refractory to E2F-mediated activation by E1a, but not by E4. Using both band-shift and transfection experiments, we now demonstrate (i) that in F9EC cells the E4 product, in combination with E2F, recruits Rb into a stable multiprotein complex and (ii) that in these undifferentiated cells, as opposed to their differentiated counterpart, Rb is actively involved in the transcriptional stimulation of the E2a promoter by E4. Our results suggest that, depending on the cell state, Rb may behave either as a transcriptional activator (F9EC cells) or as a transcriptional inhibitor (differentiated F9 cells).

Adenovirus E2 Proteins↗
PubMed1992

Identification of a novel downstream binding protein implicated in late-phase-specific activation of the adenovirus major late promotor.

The adenovirus major late promotor (MLP) is induced to very high levels after the onset of the viral DNA replication. Previous studies have identified sequence elements located downstream of the MLP startsite (DE1, between +85 and +98; DE2, between +100 and +120) implicated, together with the upstream promoter element, in this late-phase-specific transcriptional activation. One protein (DEF, now renamed DEF-A), induced during the late phase of viral infection, has been identified and shown to bind to the DE1 element (Jansen-Durr et al., 1989, J. Virol. 63, 5124-5132). Here we report about a distinct late-phase-specific protein (DEF-B) and its interactions with DEF-A. DNA-binding studies reveal that DEF-B interacts with the 5' part of DE2 (DE2b), whereas DEF-A, besides its interaction with DE1, also binds to the 3' portion of DE2 (DE2a), but with a lower affinity than for DE1. Furthermore, when added together, DEF-A and DEF-B cooperatively assemble onto the DE2 element as a heteromeric complex which is substantially more stable than the complexes formed by each protein alone. Using an in vivo transcriptional assay of the MLP, we show that DEF-A and DEF-B both have intrinsic transactivating properties.

Adenoviridae↗
PubMed1990

Class II ribonuclease H comigrates with, but is distinct from, the third largest subunit of calf thymus RNA polymerase I.

It has been reported (Iborra et al. (1979) J. Biol. Chem. 254, 10920-10924) that the third and the fifth largest subunit of yeast RNA polymerase I exhibit ribonuclease H activity. The authors suggested that the third largest subunit is identical with the chromatin-associated ribonuclease H49, the putative yeast equivalent of bovine ribonuclease H IIb. Although the third largest subunit of calf thymus RNA polymerase I and ribonuclease H IIb display nearly identical molecular masses under denaturing conditions, serological analysis reveals that, in contrast to their counterparts in yeast, these mammalian proteins are distinct entities. Interestingly, sera from some patients with mixed connective tissue disease which contain antibodies directed against RNA polymerase I, also react with ribonuclease H IIb epitopes. This observation suggests that a protein displaying ribonuclease H IIb antigenicity could be associated with RNA polymerase I. Additional indications supporting this conclusion are discussed.

Animals↗
PubMed1990

Isolation and characterization of two novel, closely related ATF cDNA clones from HeLa cells.

ATF or CRE binding proteins are cellular transcription factors involved in the regulation of adenovirus Ela-responsive and cellular cAMP-inducible promoters. We report the isolation from a HeLa cell cDNA library of two clones that encode proteins with specific ATF/CRE DNA binding activity. The two clones differ by a 63 bp element which is retained in one (ATF-a) and deleted from the other (ATF-a delta) and which may correspond to an alternative exon. The peptide sequences (483 and 462 amino acids, respectively) derived from each of these cDNAs are identical, except for the additional 21 amino acids in ATF-a, but clearly differ from the other ATF/CREB proteins reported. All of them, however, share a conserved leucine zipper domain also found in other transcription factors. ATF-a and ATF-a delta therefore represent two closely related members of a larger multigene family of proteins that interact with conserved promoter elements.

Amino Acid Sequence↗
PubMed1988

EIa-mediated stimulation of the adenovirus EIII promoter involves an enhancer element within the nearby EIIa promoter.

The transcriptional induction of adenovirus early genes by the viral immediate early gene EIa constitutes an attractive model system for the study of control mechanisms involved in eucaryotic promoter function. The EIa-mediated activation of the divergently transcribed EIIa early (EIIaE) and EIII promoters was investigated in experiments in which recombinant plasmids containing the entire EIIa-EIII control region were cotransfected with a plasmid expressing the EIa 13S mRNA. First, both promoters were activated by low levels of EIa, but the extent of EIII induction decreased with increasing EIa concentrations, whereas EIIaE stimulation remained unchanged. Second, transcriptional analysis of deletion mutants revealed that an element of the EIIaE promoter contributed to maximal EIa responsiveness of the nearby EIII promoter. This element, located between positions -82 and -71 with respect to the EIIaE major cap site, corresponded to the central portion of an EIa-dependent enhancer, originally mapped between about -110 and -50 (P. Jalinot and C. Kédinger, Nucleic Acids Res. 14:2651-2669, 1986). The implication of these observations in the coordinate expression from the EIIaE and EIII promoters during lytic infection is discussed.

Adenovirus Early Proteins↗
PubMed1988

Genomic footprinting detects factors bound to major late and IVa2 promoters in adenovirus-infected HeLa cells.

We used DNase I footprinting assays on nuclei isolated from adenovirus-infected cells to examine the nucleoprotein configuration of a 250-base-pair segment which encompasses the adenovirus type 5 major late (ML) and IVa2 promoters. At 12 and 20 h postinfection (p.i.), fine DNase I digestion mapping of wild-type adenovirus-infected cells revealed specific sequences protected from digestion which corresponded to promoter elements required for expression of the ML gene in vivo. At 12 h p.i., a G+C-rich region which lies upstream of the IVa2 cap site and is important for maximal IVa2 activity was also found masked to nuclease activity. At 20 h p.i., however, this element became more sensitive to nuclease attack, while the ML promoter elements stayed protected. No major changes in DNA-protein interactions were detected in the region spanning the ML and IVa2 cap sites upon promoter activation, suggesting that the binding properties of the cognate factors for this region are not modified during the process.

Adenoviruses, Human↗