Chromatin domain boundaries delimited by a histone-binding protein in yeast.

When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.

Lipid binding by the unique and SH3 domains of c-Src suggests a new regulation mechanism

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase,SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus.

Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.

LaTBP1: A Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. (C) 2007 Elsevier B.V. All rights reserved.

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242–335 of the oxygenase domain. In this study, iNOS8−9− and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8−9− in 293 cell line resulted in generation of iNOS8−9− mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8−9− did not form dimers. Similar to iNOSFL, iNOS8−9− exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two “control” iNOS deletion mutants were synthesized, lacking either residues 45–52 of the oxygenase domain or residues 1131–1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zα. Here we report the solution structure of free Zα and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zα)2/Z-DNA complex shows that most Z-DNA contacting residues in free Zα are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (α+β)helix–turn–helix/B-DNA complexes suggests that binding of Zα to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The α-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel α-subunit, which does form a CaM-modulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence.

Conformational changes in tertiary structure near the ligand binding site of an integrin I domain

For efficient ligand binding, integrins must be activated. Specifically, a conformational change has been proposed in a ligand binding domain present within some integrins, the inserted (I) domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333–1340]. This proposal remains controversial, however, despite extensive crystal structure studies on the I domain [Lee, J., Bankston, L., Arnaout, M. & Liddington, R. C. (1995) Structure (London) 3, 1333–1340; Liddington, R. & Bankston, L. (1998) Structure (London) 6, 937–938; Qu, A. & Leahy, D. J. (1996) Structure (London) 4, 931–942; and Baldwin, E. T., Sarver, R. W., Bryant, G. L., Jr., Curry, K. A., Fairbanks, M. B., Finzel, B. C., Garlick, R. L., Heinrikson, R. L., Horton, N. C. & Kelly, L. L. (1998) Structure (London) 6, 923–935]. By defining the residues present in the epitope of a mAb against the human Mac-1 integrin (αMβ2, CD11b/CD18) that binds only the active receptor, we provide biochemical evidence that the I domain itself undergoes a conformational change with activation. This mAb, CBRM1/5, binds the I domain very close to the ligand binding site in a region that is widely exposed regardless of activation as judged by reactivity with other antibodies. The conformation of the epitope differs in two crystal forms of the I domain, previously suggested to represent active and inactive receptor. Our data suggests that conformational differences in the I domain are physiologically relevant and not merely a consequence of different crystal lattice interactions. We also demonstrate that the transition between the two conformational states depends on species-specific residues at the bottom of the I domain, which are proposed to be in an interface with another integrin domain, and that this transition correlates with functional activity.

Structure and function of the C-terminal PABC domain of human poly(A)-binding protein

We have determined the solution structure of the C-terminal quarter of human poly(A)-binding protein (hPABP). The protein fragment contains a protein domain, PABC [for poly(A)-binding protein C-terminal domain], which is also found associated with the HECT family of ubiquitin ligases. By using peptides derived from PABP interacting protein (Paip) 1, Paip2, and eRF3, we show that PABC functions as a peptide binding domain. We use chemical shift perturbation analysis to identify the peptide binding site in PABC and the major elements involved in peptide recognition. From comparative sequence analysis of PABC-binding peptides, we formulate a preliminary PABC consensus sequence and identify human ataxin-2, the protein responsible for type 2 spinocerebellar ataxia (SCA2), as a potential PABC ligand.

An axoplasmic myosin with a calmodulin-like light chain.

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.

Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo.

Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.