Putative Activation Domain Research Articles

Cloning, Chromosomal Localization, Physical Mapping, and Genomic Characterization ofHKR3

TheKrüppel-type zinc finger proteins are members of a conserved family of transcription factors that are important in developmental regulation. Altered expression of several of these proteins has been implicated in human diseases, including cancer. We report the cloning, mapping, and characterization of the zinc finger geneHuman Krüppel-Related 3(HKR3). Genomic clones ofHKR3were isolated from a P1 library and localized to human chromosome subband 1p36.3 by human–rodent somatic cell hybrid mapping and fluorescencein situhybridization. The gene was physically mapped to within 40 kb ofD1S214by YAC content and long-range restriction mapping.HKR3spans 9.5 kb of genomic DNA and is contained in 11 exons. Sequencing defined each of the exon/intron splice site junctions and identified a CpG island in the 5′ region of the gene.HKR3is ubiquitously expressed in human tissues as at least two major transcripts, the shorter of which excludes a conserved finger-associated box and a putative acidic activation domain contained in the full-length transcript.HKR3is a novel zinc finger gene that maps to a region of the genome commonly rearranged or deleted in human cancers.

A Novel Cytoplasmic Domain of the p55 Tumor Necrosis Factor Receptor Initiates the Neutral Sphingomyelinase Pathway

The human p55 tumor necrosis factor (TNF) receptor (TR55) initiates at least two independent signaling cascades. The acidic sphingomyelinase (A-SMase) pathway involves a phosphatidylcholine-specific phospholipase C, an endosomal A-SMase, and controls expression of multiple TNF-responsive genes through induction of transcription factors such as NF-kappaB. The neutral sphingomyelinase (N-SMase) pathway comprises a membrane-bound N-SMase, proline-directed protein kinases, as well as phospholipase A2 and appears critical for the inflammatory responses induced by TNF. While the domain of TR55 that induces A-SMase is probably identical to the death domain, the exact location and extent of a putative N-SMase activation domain are still unknown. Structure-function analysis of TR55 deletion mutants revealed a novel region of 11 amino acids at position 309-319 that is both necessary and sufficient for activation of N-SMase. The N-SMase activation domain is distinct from the death domain and incapable of induction of A-SMase, NF-kappaB, and cytotoxicity. Taken together, our results suggest that a functionally independent region of TR55 is responsible for selectively initiating the N-SMase pathway that couples to an important inflammatory signaling cascade.

A new Cys2/His2 zinc finger gene, rKr1, expressed in oligodendrocytes and neurons.

The myelination of nerve fibers is essential for the function of the vertebrate nervous system as a prerequisite for fast saltatory conduction of action potentials. In the central nervous system (CNS), myelin is produced by oligodendrocytes. In order to identify gene regulatory proteins involved in the differentiation of this glial cell type or in the expression of myelin-specific genes, we have constructed a cDNA library from a highly enriched population of rat oligodendrocytes and screened this library for members of the Krüppel family of Cys2/His2 zinc finger proteins. One of the identified clones, named rKr1, encodes a novel protein of 650 amino acids which contains 12 carboxy-terminal zinc finger domains and an amino-terminal acidic domain. On Northern blots, a single rKr1 mRNA of 4.3 kb is detected. This message is present in all adult rat tissues tested, with the highest levels found in the CNS and testis. In situ hybridization on the P15 brain revealed that the transcript is expressed in differentiated oligodendrocytes and in subtypes of neurons. Particularly high message levels are found in motor neurons of the brainstem and the spinal cord. The modular structure of the rKr1 protein, in which a potential DNA binding region (the zinc fingers) is combined with a putative activation domain (the acidic region), suggests a function as sequence-specific transcriptional activator.

Structure of the membrane binding domain of CTP:phosphocholine cytidylyltransferase.

It has been proposed that the domain of the regulatory enzyme, CTP:phosphocholine cytidylyltransferase, which mediates reversible binding of the enzyme to membranes, is an amphipathic alpha-helix of approximately 60 amino acid residues and that this domain is adjacent to the putative active site domain of this enzyme. Circular dichroism indicated that the secondary structures of two overlapping peptides spanning this region were predominantly alpha-helical in the presence of PG vesicles or sodium dodecyl sulfate micelles. Interproton distances were obtained from two-dimensional NMR spectroscopic measurements to solve the structures of these two peptides. The C-terminal 22 amino acid peptide segment (corresponding to Val267-Ser288) was a well-defined alpha-helix over its length. The N-terminal 33-mer (corresponding to Asn236-Glu268) was composed of an alpha-helix from Glu243 to Lys266, a well-structured bend of about 50 degrees at Tyr240-His241-Leu242, and an N-terminal four-residue helix. It is proposed that the three residues involved in generating the bend act as the hinge between the catalytic and regulatory domains. The nonpolar faces of the 33-mer and 22-mer were interrupted by Ser260, Ser271, and Ser282. These residues may serve to limit the hydrophobicity and facilitate reversible and lipid-selective membrane binding. The hydrophobic faces of the helices were flanked by a set of basic amino acid residues on one side and basic amino acid residues interspersed with glutamates on the other. The disposition of these side chains gives clues to the basis for the specificities of these peptides for anionic surfaces.

The Activation Domain of Simian Immunodeficiency Virus SIVmac239 Rev Protein Is Structurally and Functionally Analogous to the HIV-1 Rev Activation Domain

The Rev proteins of primate immunodeficiency viruses are essential transactivators for the switch from early to late phase in the viral replication cycle. By mutational analysis, a putative activation domain (AD) has been assigned to the carboxy-terminus. This leucine-rich stretch of amino acids proved to be essential for the transactivating properties of HIV-1 Rev. Some mutants in the AD transdominantly inhibit the function of wild-type Rev protein very efficiently We identified a similar domain structure for SIVmac239 Rev by sequence comparison and in vitro mutagenesis. The leucine/isoleucine residues of the SIYmac239 Rev activation domain appeared to be of similar importance for function. The mutants of these residues in the SIV AD displayed a dominant negative phenotype on both HIV-1 and SIVmac239 rev-responsive elements (RRE). The prokaryotically expressed wild-type and mutant proteins were analyzed for RNA-binding properties in a gel-shift assay in vitro . This assay revealed a similar binding pattern of wild-type and transdominant proteins on either RRE.

GATA-3 Dominant Negative Mutant: FUNCTIONAL REDUNDANCY OF THE T CELL RECEPTOR α AND β ENHANCERS

The GATA family of transcription factors regulates a wide variety of genes, including those involved in differentiation of erythrocytes and T lymphocytes. We report here the creation of a dominant negative mutant of GATA-3, KRR, which effectively blocks wild-type GATA-1, GATA-2, and GATA-3 transactivation when co-expressed in transient assays. KRR was generated by site-directed mutagenesis while investigating a putative activation domain of GATA-3, located between its two zinc fingers. The GATA-3 KRR mutation does not affect expression, nuclear translocation, or the ability to bind to a consensus GATA sequence. KRR can suppress the activity of the minimal T cell receptor (TCR) alpha and beta enhancers by 12- and 3.4-fold, respectively. However, KRR did not have a significant effect on the activity of larger TCR-alpha and -beta enhancer fragments. Thus, functional redundancy in the TCR-alpha and -beta enhancers can compensate for the loss of GATA-3 activity.

Expression cloning of a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase).

A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.

Transcriptional activation by CTF proteins is mediated by a bipartite low-proline domain.

Members of the CCAAT-binding transcription factor (CTF) family of proteins stimulate the initiation of adenovirus DNA replication and act as transcriptional activators. To investigate the mechanisms underlying CTF-mediated transactivation patterns, we expressed several natural CTF variants in Saccharomyces cerevisiae and determined their transactivating activities in enzymatic assays. CTF7, which lacks the entire proline-rich region previously thought to mediate transcriptional activation by CTF proteins, enhances transcription to a greater degree than full-length CTF1, which contains the putative activation domain. CTF2, which contains a partially deleted proline-rich activation region, does not stimulate transcription at all. These findings indicate that the proline-rich region of CTF proteins is not essential for transcriptional activation in yeast. Our studies also suggest a bipartite two-domain structure of CTF-type transcriptional activation domains.

Distinct activation domains within cAMP response element-binding protein (CREB) mediate basal and cAMP-stimulated transcription

Activation of protein kinase A (PKA) by cAMP results in phosphorylation of cAMP response element-binding protein (CREB) and induction of specific gene expression. However, whether CREB participates directly in basal (PKA-independent) transcription is still an open question, and existing studies conflict over the identification of putative basal activation domains. In the present study, the activation domain of CREB, whether fused to the GAL4 DNA binding domain (CRG) or in native CREB, stimulated basal activity of the minimal tk promoter, but not the minimal SV40 early promoter. Cotransfection with PKI, a specific inhibitor of PKA, blocked PKA-induced expression of both promoters, but did not block CRG-mediated basal expression of the tk promoter. In addition, both CRG and a PKA phosphorylation site mutant provided comparable stimulation of basal tk promoter activity. Examination of a series of CREB deletion mutants mapped basal activity to interacting domains, located on either side of the previously identified PKA activation domain (amino acids 98-142). This PKA-independent activity mapped primarily to a bipartite COOH-terminal basal activation domain (amino acids 165-252). Its major component bears no obvious homology to previously identified activation domains, whereas a minor component is glutamine-rich. This COOH-terminal domain acts independently and provides the majority of basal activation but requires an NH2-terminal domain (amino acids 41-86) to provide full basal activity. A repressor domain (amino acids 142-165), deletion of which enhanced both basal and PKA-activated transcription, was also identified. This work establishes that CREB contains distinct basal and PKA-activated domains, that they operate independently for both loss of function and gain of function, and that they work on different promoters in different cell types.

Mouse monoclonal antibodies recognizing the activation domains of HIV-1 rev transactivator

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. So far, at least four necessary functional domains have been assigned to the HIV-1 rev protein: (1) one for RNA binding; (2) a second for nuclear/nucleolar localization that may be indistinguishable from the RNA binding motif; (3) two domains for multimerization; and (4) a putative activation domains (AD) that is suppressed in trans by dominant-negative mutant rev protein. We report three IgG 1κ mouse monoclonal antibodies (mabs) that were independently raised against rev protein expressed in Escherichia coli. Epitopes are mapped by immunoprecipitation and Western blot screening with 40 different rev mutant peptides. Surprisingly, monoclonal antibodies from all three hybridomas recognized the activation domains of HIV-1 rev.

Genetic evidence for transcriptional activation by the yeast IME1 gene product.

IME1 is required in yeast for meiosis and for expression of IME2 and other early meiotic genes. IME1 is a 360-amino acid polypeptide with central and C-terminal tyrosine-rich regions. We report here that a fusion protein composed of the lexA DNA-binding domain and IME1 activates transcription in vivo of a reporter gene containing upstream lexA binding sites. Activation by the fusion protein shares several features with natural IME1 activity: both are dependent on the RIM11 gene product; both are impaired by the same ime1 missense mutations; both are restored by intragenic suppressors. The central tyrosine-rich region is sufficient to activate transcription when fused to lexA. Deletion of this putative activation domain results in a defective IME1 derivative. Function of the deletion derivative is restored by fusion to the acidic Herpesvirus VP16 activation domain. The C-terminal tyrosine-rich region is dispensable for transcriptional activation; rather it renders activation dependent upon starvation and RIM11. Immunofluorescence studies indicate that an IME1-lacZ fusion protein is concentrated in the nucleus. These observations are consistent with a model in which IME1 normally stimulates IME2 expression by providing a transcriptional activation domain at the IME2 5' regulatory region.

Membrane topology and biogenesis of eukaryotic signal peptidase.

The signal peptidase complex (SPC) is a hetero-oligomeric membrane protein containing subunits of 12, 18, 21, 22/23, and 25 kDa. The 18- and 21-kDa subunits are mammalian homologs of SEC11 protein, which is necessary for signal peptide processing and cell viability in the yeast Saccharomyces cerevisiae. The functional and/or structural contributions of the 12-, 22/23-, and 25-kDa subunits to SPC activity have not yet been elucidated. To explore the structure of SPC subunits and their relationships to signal peptide processing and protein translocation, we have examined their endoplasmic reticulum (ER) membrane topology and biogenesis. Signal peptidase activity and SPC subunits are resistant to protease treatment in intact and detergent-solubilized membranes. Heat-denatured SPC subunits and SPC subunits translated in vitro are, however, protease sensitive, suggesting that the assembly of the oligomeric complex confers protease resistance. To define the membrane topology of SPC subunits, both wild-type subunits and subunit fusion proteins containing additional sites for N-linked glycosylation were assembled into microsomal membranes in vitro. Despite the presence of multiple hydrophobic domains, each subunit is anchored to the ER membrane by a single amino-terminal transmembrane domain in an Ncytoplasmic Cexoplasmic (type II) orientation. This topology places the bulk of the protein mass in the ER lumen and positions a putative serine-containing active site domain in SPC 18 and 21 at the same relative distance from the membrane as the analogous region in Escherichia coli leader peptidase. These studies have also revealed that, in spite of the temporal and perhaps physical association of the SPC with the process of protein translocation, SPC subunits integrate into the ER membrane by a signal recognition particle-dependent pathway and, hence, rely on the existence of a preformed translocation apparatus for their own membrane assembly.

Structural and functional analysis of the amdR regulatory gene of Aspergillus oryzae

We have isolated the Aspergillus oryzae homologue of the amdR regulatory gene of Aspergillus nidulans by cross hybridization. Sequence analysis and functional studies have shown that the amdR genes are highly conserved and functionally interchangeable between the two species. The homology between the two genes extends throughout most of the coding sequences, including sequences encoding the DNA-binding domain and putative activation domains. Two regions of nonconserved sequence were also identified. Studies using various amdS:: lacZ fusion constructs indicate that the A. oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A. nidulans protein. Inactivation of the A. oryzae gene results in the inability to grow on y-amino-butyric acid (GABA) as a carbon and/or nitrogen source indicating that GABA utilization is amdR-dependent in A. oryzae as it is in A. nidulans.

Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate-early 1 transcriptional regulatory protein.

Autographa californica multicapsid nuclear polyhedrosis virus-infected insect cells express a viral immediate-early transcriptional regulatory protein, IE1, that has been shown by transient-expression assays to stimulate the expression of certain baculovirus delayed-early (DE) promoters and to inhibit the expression of other immediate-early (IE) genes. It is believed that certain DE promoters are activated, in part, by direct interactions between IE1 and enhancer elements located in regions adjacent to these genes. We have used transient cotransfection and DNA-binding assays to examine the function of mutant forms of IE1. Our results indirectly show that IE1 has at least two separable domains that are essential for its role in the modulation of baculovirus gene expression. A domain rich in acidic residues and essential for transactivation is located within the N-terminal 145 amino acids of the polypeptide. A second domain, located in the C-terminal 437 amino acids of IE1, is required for inhibitory and DNA-binding activities. Several nontransactivating IE1 mutants trans-dominantly interfered with wild-type IE1 transactivation of enhancer-linked DE genes. trans-dominant interference was expressed only by IE1 mutants that retained the N-terminal putative acidic activation domain, suggesting that this region may be involved in associations with a factor(s) essential for activation of enhancer-linked genes.

Molecular cloning of cDNAs for human fibroblast nucleotide pyrophosphatase

Nucleotide pyrophosphatase (NPPase) activity is markedly elevated in cultured skin fibroblasts from patients of Lowe's syndrome. cDNA clones encoding the NPPase were isolated using synthetic oligonucleotide probes designed on the basis of partial amino acid sequence of the enzyme purified from human placenta. The complete sequences of these clones yielded a merged sequence of 3508 bases. The polypeptide chain of the enzyme was deduced to comprise 873 amino acids with a calculated molecular weight of 99,703 and had the characteristics of a class II transmembrane protein. Ten potential N-glycosylation sites were detected in the protein. RNA blot analysis showed that human fibroblasts contain two minor mRNAs of 7.0 and 8.2 kb, respectively, in addition to a major 3.6-kb species that coincides with the merged cDNA in size. A computer search of a nucleotide sequence database revealed that plasma cell membrane glycoprotein PC-1, whose function was unknown at the time, is identical with the NPPase. Comparison of the amino acid sequence of the NPPase with the active site sequence of bovine 5′-nucleotide phosphodiesterase allowed the assignment of a putative active site domain to the central region of the COOH-terminal extracellular domain of the NPPase. The gene for human NPPase was localized to chromosome 6 at q22–q23 by in situ hybridization with a fragment of the NPPase cDNA.

A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.

A herpesvirus proteinase activity has been identified and partially characterized by using the cloned enzyme and substrate genes in transient transfection assays. Evidence is presented that the proteinase gene of cytomegalovirus strain Colburn encodes a 590-amino acid protein whose N-terminal 249 residues contain the proteolytic activity and two domains that are highly conserved in the homologous protein of other herpesviruses. Insertion of a short amino acid sequence between these domains abolished proteinase activity, suggesting that this region constitutes part or all of the enzyme active site. Plasma desorption mass spectrometry was used to identify the C terminus of the mature assembly protein as alanine, enabling the recognition of a consensus proteinase cleavage sequence of V/L-X-A decreases S/V, near the C-terminal end of all herpesvirus assembly protein homologs. Interestingly, the proteinase and its substrate, the assembly protein precursor, are encoded by opposite halves of the same open reading frame.

Isolation and molecular characterization of PosF21, an Arabidopsis thaliana gene which shows characteristics of a b‐Zip class transcription factor.

We have isolated a putative transcription factor gene, PosF21, from Arabidopsis thaliana using an indirect cross-hybridization approach. cDNA clones were isolated which encode single repeating amino acids. Such sequences may function as activation domains in transcription factors and may be indicative for such proteins. The clone PosF21 encodes a region very rich in glutamines. Besides this putative activation domain it encodes a protein sequence which shows all the characteristics of a basic-domain/leucine zipper type of DNA-binding domain. PosF21 is expressed constitutively at a low level in young seedlings and in roots, stems and leaves of mature Arabidopsis plants. A genomic clone of PosF21 was isolated and the gene structure was analyzed. Related sequences in Arabidopsis and a wide range of other plants were detected using the putative DNA-binding domain as a probe in cross-hybridization experiments. Transient transformations in tobacco protoplasts were performed using the beta-glucuronidase (GUS) gene as reporter gene. Approximately 400 bp of the 5' genomic region of PosF21 promote expression of the GUS gene in tobacco protoplasts. Evidence for a regulatory function of PosF21 was obtained since co-expression of the full PosF21 protein or its DNA-binding region alone specifically stimulated GUS gene expression directed from the PosF21 promoter by 6-8-fold.

An X-linked zinc finger gene mapping to Xq21.1–q21.3 closely related to ZFX and ZFY: possible origins from a common ancestral gene

We describe a new zinc finger gene sequence (CMPX1 or HGM symbol ZNF6; isolated by cross-hybridization of ZFY to clones in a testis cDNA library) which possesses a zinc finger domain closely related to the transcriptional activator gene ZFX. The putative acidic activation domain is only 11.5% homologous with ZFX, whereas the putative DNA binding domain shares 75% homology and shows the same organisation composed of a basic two fingered repeat unit. ZNF6 has an unusually large 5' untranslated region (UTR) of 1.2 Kb which contains 26 potential ATG initiation codons, only one of which is associated with a long open reading frame. Southern and Northern blot analysis has shown that this 5' UTR is shared with many other sequences in the genome and transcribed associated with a large range of mRNA species. In situ hybridisation, analysis of somatic cell hybrids and male individuals carrying deleted X chromosomes have mapped the gene to Xq21.1-q21.3. The gene is highly conserved amongst the primates, in the mouse and can be detected weakly in the genome of a metatherian mammal (possum). Dosage in male and female mice indicates that it is also X-linked in this species. Possible origins of ZFX, ZFY and CMPX1 from a common ancestral gene are discussed.