Amino-terminal Deletion Mutant Research Articles

Mutant and Wild-Type Androgen Receptors Exhibit Cross-Talk on Androgen-, Glucocorticoid-, and Progesterone-Mediated Transcription

Androgen, glucocorticoid, and progesterone receptors (ARs, GRs, and PRs) often can regulate transcription via composite hormone response elements in target genes. We have used artificial and natural mutant ARs from patients with androgen resistance to study their effects on dominant negative activity on wild type AR, GR, and PR function on mouse mammary tumor virus (MMTV) and tyrosine aminotransferase (TAT) promoters. Artificial ARs that contained internal deletions within the amino-terminal region had minimal transcriptional activity but blocked ligand-mediated transcription by wild type AR. Mutants containing deletions of the DNA-binding and ligand-binding domains had minimal or weak dominant negative activity. We then tested the ability of wild type and mutant ARs to modulate GR- and PR-mediated transcriptional activity. The amino-terminal deletion mutants exerted dominant negative effects on GR- and PR-mediated activity, both in the absence and presence of testosterone. Surprisingly, wild type AR, which had approximately 20% of the maximal transcriptional activity of GR on the MMTV promoter, also had dominant negative activity on dexamethasone-regulated transcription mediated by GR. This dominant negative activity likely involves DNA binding because a point mutation in the DNA-binding domain abrogated such activity of an amino-terminal deletion mutant. Additionally, natural human AR mutants from patients with androgen resistance, which do not bind either DNA or ligand, did not block dexamethasone-mediated transcription. In summary, these studies suggest that mutant and wild type ARs can display dominant negative activity on other steroid hormone receptors that bind to a composite hormone response element This cross-regulation may be important in regulating maximal transcriptional activity in tissues where these receptors are coexpressed and may contribute to the phenotype of patients with steroid hormone resistance.

Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme.

Amino-terminal and carboxy-terminal deletion mutagenesis have been used to identify structurally and functionally critical regions of recombinant wild-type human phenylalanine hydroxylase (wt-hPAH; Ser2-Lys452). The wild-type form consisted of dimeric and tetrameric forms in equilibrium, and only the isolated tetrameric form showed positive cooperativity of substrate (L-Phe) binding (Hill coefficient h = 2.2, S0.5 = 154 microM). The deletion mutants lacking the carboxy-terminal 24 amino acids hPAH (Ser2-Gln428) and hPAH(Gly103-Gln428) formed catalytically active dimers, and incubation with L-Phe did not promote the formation of tetramers, a characteristic property of dimeric wt-hPAH. The carboxyterminus thus seems to contain a motif required for dimer-dimer interaction in wt-hPAH. The deletion mutants hPAH(Asp112-Lys452), hPAH(Ser2-Gln428) and hPAH(Gly103-Gln428) were all activated by prior incubation with L-Phe, but did not reveal any positive cooperativity of substrate binding (h = 1.0). The activation by L-Phe was accompanied by a measurable conformational change (as probed by intrinsic fluorescence spectroscopy) only in the enzyme forms containing the amino-terminal sequence. i.e. wt-hPAH and the Ser2-Gln428 mutant. The amino-terminal deletion mutants hPAH(Asp112-Lys452) and hPAH(Gly103-Gln428) revealed high specific activity, increased apparent affinity for L-Phe (S0.5 = 60 microM) and a tryptophan fluorescence emission spectrum similar to that of the L-Phe-activated wt-hPAH. Moreover, prior incubation of the enzyme forms with lysophosphatidylcholine, a commonly used activator of the PAH, only increased the activity of those forms containing the wt-hPAH amino-terminal sequence. Our results are compatible with a model in which incubation of wt-hPAH with L-Phe induces both a conformational change (with cooperativity in the tetrameric enzyme) which relieves the inhibition imposed by the amino-terminal domain to the high-affinity binding of L-Phe, and an additional activation, as observed for the truncated forms lacking the amino-terminal.

Interaction of wild-type and mutant adeno-associated virus (AAV) Rep proteins on AAV hairpin DNA

Both the Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) bind to AAV terminal repeat hairpin DNA and can mediate site-specific nicking in vitro at the terminal resolution site (trs) within the terminal repeats. To define the regions of the Rep proteins required for these functions, a series of truncated Rep78 derivatives was created. Wild-type and mutant proteins were synthesized by in vitro translation and analyzed for AAV hairpin DNA binding, trs endonuclease activity, and interaction on hairpin DNA. Amino-terminal deletion mutants which lacked the first 29 or 79 amino acid residues of Rep78 did not bind hairpin DNA, which is consistent with our previous identification of a DNA-binding domain in this region. Progressive truncation of the carboxyl-terminal region of Rep78 did not eliminate hairpin DNA binding until the deletion reached amino acid 443. The electrophoretic mobility of the Rep-specific protein-DNA complexes was inversely related to the molecular weight of the Rep derivative. Analysis of the C-terminal deletion mutants by the trs endonuclease assay identified a region (amino acids 467 to 476) that is essential for nicking but is not necessary for DNA binding. When endonuclease-positive, truncated Rep proteins that bound hairpin DNA were mixed with full-length Rep78 or Rep68 protein in electrophoretic mobility shift assays, a smear of protein-DNA complexes was observed. This smear migrated at an intermediate position with respect to the bands generated by the proteins individually. An antibody recognizing only the full-length protein produced a novel supershift band when included in a mixed binding assay containing Rep68 and a truncated Rep mutant. These experiments suggest that the Rep proteins can form hetero-oligomers on the AAV hairpin DNA.

Transcription factor repression and activation of the human acetylcholinesterase gene.

Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5' deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.

Characterization of Active and Inactive Forms of the JAK2 Protein-tyrosine Kinase Produced via the Baculovirus Expression Vector System

Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C delta 795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N delta 661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phosphotyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C delta 795) was not observed; further, coproduction of rJAK2(C delta 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previously published results (Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 21411-21414). Mutant (N delta 661)rJAK2 exhibited a robust tyrosine phosphorylation signal. A second 62-kDa tyrosine phosphoprotein co-immunoprecipitated with (N delta 661)rJAK2 but not with rJAK2 or rJAK2(C delta 795). Both rJAK2 and (N delta 661)rJAK2 incorporated phosphate under in vitro kinase assay conditions, but rJAK2(C delta 795) did not. A JAK2 oligomer with interacting catalytic sites and/or inhibitory sites would provide a simple model to describe these results.

Accumulation of inactivation in a cloned transient K+ channel (AKv1.1a) of Aplysia.

1. Inactivation of a cloned Aplysia K+ channel, AKv1.1a, expressed in Xenopus oocytes was examined by a cell-attached macropatch recording. A fast macroscopic inactivation (the time constant for decay was in the range of 20-40 ms) in response to a depolarizing command pulse was insensitive to the concentration of external K+ (2-100 mM KCl). 2. By contrast, recovery from inactivation was extremely slow and dependent on external K+. When the concentration of external KCl was 2-3 mM, a patched membrane had to be held at hyperpolarized potential for > 40 s for a full recovery. The recovery was greatly accelerated if external K+ concentration was increased. A tail current following a command pulse long enough to inactivate most of the channels showed a marked rising phase. 3. A consequence of the slow recovery from inactivation was that AKv1.1a showed a marked accumulation of the inactivation following repetitive pulses, even at low frequency (< 0.1 Hz). When two depolarizing pulses were applied at a short interval, the current during a second pulse was smaller than the current at the end of the preceding pulse. This is a phenomenon called "cumulative inactivation." The onset and the extent of cumulative inactivation of AKv1.1a were voltage dependent but relatively insensitive to external K+ concentration. An amino terminal deletion mutant of AKv1.1a that lacks the fast N-type inactivation did not show cumulative inactivation. 4. These results suggest that the inactivation gating by the amino terminal region of AKv1.1a has a similarity to open-channel blockade, and that the cumulative inactivation can also be dependent on the amino terminal cytoplasmic domain of K+ channels.

Human parainfluenza virus type 3: Analysis of the cytoplasmic tail and transmembrane anchor of the hemagglutinin-neuraminidase protein in promoting cell fusion

The role of the cytoplasmic tail and transmembrane anchor of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) glycoprotein in promoting cell fusion was investigated. A series of amino terminal deletion mutants (d10, d20, d27, d31, d40, d44, and d73) were compared for processing, cell surface expression, and maintenance of their biological attributes by recombinant expression of mutant genes using a plasmid vector (pcDL-SR alpha-296) in CV-1 and HeLa cells. To determine the fusion promoting activity (FPA) of the various mutant proteins, a simple assay was developed which quantified the fusion of two different HeLa cell types. One of the cell types, HeLa-tat, constitutively expressed the human immunodeficiency virus type I (HIV-1) tat protein from a Moloney murine leukemia virus long terminal repeat (LTR), while the second cell type, HeLa β-gal, contained a reporter gene, beta-galactosidase, under the control of an HIV1-LTR. Fusion of mixed HeLa cell monolayers (50:50, HeLa-tat: HeLa β-gal), following transfection with appropriate plasmids, resulted in transactivation of the reporter gene which was then measured by direct staining of cells or using cell lysates with appropriate substrates. Cell fusion was observed only when both the HPIV3 F and functional HN proteins were both co-transfected into cells. Of the seven deletion mutants examined, only d10, d20, d27 and d31 were expressed to significant levels on the cell surface and only these four mutant proteins maintained FPA. Compared with the wt HN at 48 h post transfection, d10 and d20 had enhanced FPA (119% and 158%, respectively), while d27 and d31 were diminished (74% and > 4%, respectively). Analysis of protein expression suggested that the reason for the increase in FPA of the mutant proteins was that the levels of protein expressed at the cell surface was twofold or threefold higher for d10 and d20, respectively, compared to the wt HN.

Up- and Down-Modulation of a Cloned Aplysia K+Channel (AKv1.1a) by the Activators of Protein Kinase C

Modulation of a cloned Aplysia K+ channel, AKv1.1a, by protein kinase C (PKC) activators was examined in Xenopus oocytes expression system. Following the application of phorbol esters (phorbol 12-myristate 13-acetate, PMA; phorbol 12,13-dibutyrate, PDBu), or a diacylgrycerol analogue (1-oleoyl-2-acetyl-sn-glycerol, OAG), the fast inactivation of the AKv1.1a became slower and the peak current increased (up-modulation). However, the effect was transient. The expressed current was decreased even below control level about 15 to 20 min after the treatment (down-modulation). Both effects by PMA was blocked by the kinase inhibitor, H7, suggesting that phosphorylation by PKC is involved. The amino acid sequence of AKv1.1a contains three putative phosphorylation sites by PKC (Ser24, Thr345, Ser349). We tested their contributions to the PMA-induced modulation by site-directed mutagenesis. The results suggest that the up-modulation by PKC activators is due to the inhibition of the fast inactivation by the amino-terminal domain (N-type inactivation), thereby increase the time the channels are conductive. Phosphorylation of Ser24 may enhance the PKC-induced down-modulation, while phosphorylation of Thr345 may inhibit the down-modulation. By contrast, mutation of Ser349 did not affect the modulation. The N-type inactivation were not indispensable for the down-modulation because the amino-terminal deletion mutant also showed some down-modulation although its onset was quite slow. Thus, the down-modulation of AKv1.1a may be heterogeneous.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the Human Spuma Retrovirus Integrase by Site-directed Mutagenesis, by Complementation Analysis, and by Swapping the Zinc Finger Domain of HIV-1

The human spuma retrovirus or foamy virus integrase (HFV IN) is an enzymatically active protein consisting of domains similar to other retroviral integrases: an amino-terminal HH-CC finger, a centrally located region with the conserved D, D-35-E protein motif required for catalytic activity and oligomerization, and at least one DNA binding domain implicated in the 3' DNA processing activity and integrase. Recombinant, purified HFV IN protein carrying 10 histidine residues displays a site-specific endonuclease, an integrase, and a disintegrase activity with oligonucleotide substrates that mimic the viral long terminal repeat (LTR) ends. Site-directed mutagenesis of conserved HFV IN residues of the catalytic domain had increased endonuclease and disintegrase activities. Deletion mutants at both ends of the HFV IN protein were generated, purified, and characterized. Unexpectedly, it was found that the HFV integrase and disintegrase activities require an intact NH2-terminal sequence and that COOH-terminal deletions led to an increase in disintegrase activity. The HH-CC finger of HFV IN was exchanged with that of the human immunodeficiency virus-1 (HIV-1) IN protein. The resulting chimeric IN had a 3' processing activity that utilized the HFV LTR instead of the HIV LTR, indicating that the central domain is crucial for substrate recognition. Functional complementation of the amino-terminal deletion mutant of HFV IN was achieved by a carboxyl-terminal deletion mutant of the chimeric IN, resulting in high levels of integrase activity.

Isolation of human NuMA protein

NuMA is a protein involved in maintenance of nuclear structure and in the assembly of the mitotic spindle. Expression of amino-terminal deletion mutants results in a phenotype identical to that caused by a temperature-sensitive defect of RCC1 (regulator of chromosome condensation). Here we describe the isolation of NuMA protein from HeLa cells under mild conditions as a prerequisite to study its interactions with elements of the RCC1-Ran regulatory pathway. In an overlay assay, NuMA did not bind Ran·[γ- 32P]GTP. Thus it is clearly different from Ran·GTP binding proteins of similar M r.

Functional Map of the Alpha Subunit of Escherichia coli RNA Polymerase : Deletion Analysis of the Amino-terminal Assembly Domain

The α subunit of Escherichia coli RNA polymerase plays a major role in the assembly of the core enzyme. The amino-terminal two-thirds of the α subunit, as far as position 235, are involved in this assembly. To define the site(s) within this region required for core enzyme assembly we constructed a set of amino-terminal and internal deletion mutants of the rpoA gene. The overexpressed α derivatives were purified to apparent homogeneity and examined for their abilities to assemble β and β′ subunits into active core enzymes in vitro. Among a total of 22 α derivatives tested, only four mutants retained the activity to form active enzyme. These mutants had deletions of the extreme amino-terminal residues as far as amino acid residue 30. The minimum fragment with full activity of the core assembly was α(21 to 235), with deletions of 20 amino-terminal and 94 carboxy-terminal amino acid residues. Most of the other mutants appeared to be defective in the formation of stable α dimers as analyzed by high-pressure liquid chromatography gel filtration, although some formed self-aggregates. These results, taken together, suggest that the amino-terminal region of the α subunit with the core assembly activity is highly structured, and any deletion within this domain disrupts its ordered conformation. Deletions of the extreme amino-terminal region did not affect transcription activation by CRP at the lacPl promoter or by OmpR at the ompC promoter.

Type II regulatory subunit (RII) of the cAMP-dependent protein kinase interaction with A-kinase anchor proteins requires isoleucines 3 and 5.

Compartmentalization of the type II cAMP-dependent protein kinase is maintained by association of the regulatory subunit (RII) with A-Kinase Anchor Proteins (AKAPs). In previous studies (Scott, J. D., Stofko, R. E., McDonald, J. R., Comer, J. D., Vitalis, E. A., and Mangili J. (1990) J. Biol. Chem. 265, 21561-21566) we have shown that dimerization of RII alpha was required for interaction with the cytoskeletal component microtubule-associated protein 2. In this report we show that the localization and dimerization domains of RII alpha are contained within the first thirty residues of each RII protomer. RII des-5 (an amino-terminal deletion mutant lacking residues 1-5) was unable to bind AKAPs but retained the ability to dimerize. RII alpha I3A,I5A (a mutant where isoleucines 3 and 5 were replaced with alanine) was unable to bind a variety of AKAPs. Mutation of both isoleucines decreased AKAP binding without affecting dimerization, cAMP binding, or the overall secondary structure of the protein. Measurement of RII alpha I3A,I5A interaction with the human thyroid AKAP, Ht 31, by two independent methods suggests that mutation of isoleucines 3 and 5 decreases affinity by at least 6-fold. Therefore, we propose that two isoleucine side chains on each RII protomer are principle sites of contact with the conserved amphipathic helix binding domain on AKAPs.

The endless story of the glial fibrillary acidic protein

All intermediate filament proteins consist of an alpha-helical rod domain flanked by non-helical N-terminal head and C-terminal tail domains. The roles of the non-helical domains of various intermediate filament proteins in the assembly and co-assembly of higher-order filamentous structures have been studied by many groups but with quite contradictory results. Type III intermediate filaments are unique in that they can form homopolymers both in vitro and in vivo. The expression and assembly characteristics of carboxy- and amino-terminal deletion mutants of glial fibrillary acidic protein (GFAP), an astrocyte-specific type III intermediate filament protein, were examined by transient transfections of either vimentin-positive or vimentin-negative variants of human adrenocarcinoma-derived SW13 cell lines. Whereas complete deletion of the C-terminal tail domain of GFAP results in the formation of polymorphic aggregates, both intranuclear and cytoplasmic in self-assembly experiments, efficient co-assembly of these tail-less GFAP mutants with vimentin can be achieved as long as the KLLEGEE sequence at the C-terminal end of the rod domain is preserved. Up to one-fifth of the C-terminal end of the tail domain can be deleted without affecting the capability of GFAP to self-assemble. The highly conserved RDG-containing motif in the tail domain may be important for self-assembly but is not sufficient. The entire head domain seems to be required for self-assembly. All N-terminal deletion mutants of GFAP share the same phenotype of diffuse cytoplasmic staining when expressed in vimentin-negative SW13 cells. Although co-assembly with vimentin can still be achieved with completely head-less GFAP, preservation of some of the head domain greatly enhanced the efficiency. Our results form the basis for further, more detailed mapping of the essential regions in filament assembly of GFAP and other type III IFs.

Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine biosynthesis. This enzyme is hypothesized to consist of an amino-terminal regulatory domain and a carboxy-terminal catalytic domain. In the present studies, we have utilized recombinant DNA techniques to map the boundaries of the regulatory and catalytic domains of TH. We have isolated a full-length cDNA clone for rat pheochromocytoma TH and have expressed the enzyme in bacteria. Utilizing this bacterial expression system and polymerase chain reaction technology, we have constructed and subcloned genes for five amino-terminal deletion mutants (NΔ40, NΔ155, NΔ165, NΔ175 and NΔ200; NΔ denotes amino-terminal deletion and the numerical value denotes the number of amino acids deleted) and two carboxy-terminal deletion mutants (CΔ19 and CΔ50). The catalytic core of rat tyrosine hydroxylase has been identified to include the region from amino acid #165 to amino acid #479. The amino-terminal deletion mutants, NΔ40, NΔ155 and NΔ165 are from 1.85 to 2.5-fold more active than unmodified recombinant TH, while the removal of 19 amino acids from the C-terminus (CΔ19) results in a 70% reduction in enzyme activity. Removal of additional sequences (ten more residues from the N-terminus [NΔ175]; or an additional 31 amino acids from the C-terminus [CΔ50]) results in protein that is totally without enzyme activity. As expected, removal of 40 (or more) N-terminal amino acids abolishes the ability of the catalytic subunit of the cAMP-dependent protein kinase to phosphorylate the recombinant enzyme; serine-40 is the phosphorylation site on TH for PKA. We conclude that the N-terminal boundary for the TH catalytic domain resides between residues 165 and 175 and that removal of this N-terminal domain (totally or partially) increases the activity of the enzyme. These findings confirm previous reports that proteolytic cleavage at amino acid #158 produces an active (and activated) catalytic fragment.

Structural determinant for assembly of mammalian K+ channels

K+ channel function is regulated through the assembly of channel subunit isoforms into either homo- or heterotetrameric structures each characterized by distinct pharmacologic and kinetic properties. In studying the molecular basis of subunit association in mammalian Shaker-like K+ channels, we constructed deletion mutants of the inactivating K+ channel hKv1.4 alone and in tandem with hKv1.5 and examined the functional properties electrophysiologically in Xenopus oocytes. Deletion of 255 amino acids in the amino-terminal domain of hKv1.4 prevented the formation of hybrid channels within the subfamily but had no effect on homomultimerization or voltage-dependent gating. The amino-terminal deletion mutant of Kv2.1, a noninactivating K+ channel from a distantly related subfamily also forms functional homomultimeric channels. Although members of different K+ channel subfamilies do not coassemble, coexpression of the amino-terminal deletion mutants of hKv1.4 and Kv2.1 resulted in the formation of functional hybrid channels. These results demonstrate that the amino-terminal region of mammalian K+ channels subserves two functions. It provides a recognition site necessary for hetero- but not homomultimeric channel assembly within a subfamily and prevents coassembly between subfamilies.

E1A-mediated inhibition of myogenesis correlates with a direct physical interaction of E1A12S and basic helix-loop-helix proteins.

The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.

E1A-Mediated Inhibition of Myogenesis Correlates with a Direct Physical Interaction of E1A12S and Basic Helix-Loop-Helix Proteins

The observation that adenovirus E1A gene products can inhibit differentiation of skeletal myocytes suggested that E1A may interfere with the activity of myogenic basic helix-loop-helix (bHLH) transcription factors. We have examined the ability of E1A to mediate repression of the muscle-specific creatine kinase (MCK) gene. Both the E1A12S and E1A13S products repressed MCK transcription in a concentration-dependent fashion. In contrast, amino-terminal deletion mutants (d2-36 and d15-35) of E1A12S were defective for repression. E1A12S also repressed expression of a promoter containing a multimer of the MCK high-affinity E box (the consensus site for myogenic bHLH protein binding) that was dependent, in C3H10T1/2 cells, on coexpression of a myogenin bHLH-VP16 fusion protein. A series of coprecipitation experiments with glutathione S-transferase fusion and in vitro-translated proteins demonstrated that E1A12S, but not amino-terminal E1A deletion mutants, could bind to full-length myogenin and E12 and to deletion mutants of myogenin and E12 that spare the bHLH domains. Thus, the bHLH domains of myogenin and E12, and the high-affinity E box, are targets for E1A-mediated repression of the MCK enhancer, and domains of E1A required for repression of muscle-specific gene transcription also mediate binding to bHLH proteins. We conclude that E1A mediates repression of muscle-specific gene transcription through its amino-terminal domain and propose that this may involve a direct physical interaction between E1A and the bHLH region of myogenic determination proteins.

Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins

The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSM beta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSM beta) and a mutant (rCSM beta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSM beta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 x 10(6) and 2.78 x 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSM beta) appeared to have a comparable actin-binding affinity to that of gel-purified CSM beta homodimer (1.25 x 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 x 10(7) M-1). The amino-terminal deletion mutant rCSM beta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSM beta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSM beta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSM beta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSM beta in the actin binding and the caldesmon enhancement.

Expression of large hepatitis B envelope protein mutants using a new expression vector

Aminoterminal deletion mutants of the gene encoding the large hepatitis B surface protein were expressed in COS cells using a new expression vector. The truncated protein showed the same intracellular retention like the wild type protein. The findings show that the secretion block of the protein is not due to its aminoterminal myristylation.

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.