Two binding modes reveal flexibility in kinase/response regulator interactions in the bacterial chemotaxis pathway

The crystal structure at 2.0-Å resolution of the complex of the Escherichia coli chemotaxis response regulator CheY and the phosphoacceptor-binding domain (P2) of the kinase CheA is presented. The binding interface involves the fourth and fifth helices and fifth β-strand of CheY and both helices of P2. Surprisingly, the two heterodimers in the asymmetric unit have two different binding modes involving the same interface, suggesting some flexibility in the binding regions. Significant conformational changes have occurred in CheY compared with previously determined unbound structures. The active site of CheY is exposed by the binding of the kinase domain, possibly to enhance phosphotransfer from CheA to CheY. The conformational changes upon complex formation as well as the observation that there are two different binding modes suggest that the plasticity of CheY is an essential feature of response regulator function.

A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, ΔesaR, and ΔesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

Apoptosis regulator Bcl-w is essential for spermatogenesis but appears otherwise redundant

Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated the bcl-w gene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. The bcl-w−/− mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Cα is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Cα and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Cα inactivated CFTR Cl− channels; reactivation required readdition of kinase. Finally, coexpression of PP2Cα with CFTR in epithelia reduced the Cl− current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

The helical domain of a G protein α subunit is a regulator of its effector

The α subunit (Gα) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Gα structure essentially comprises a GTPase “Ras-like” domain (RasD) and a unique α-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Gα (Gαt) and the closely related gustducin (Gαg), but not Gαi1, Gαs, or Gαq synergistically enhance guanosine 5′-γ[-thio]triphosphate bound Gαt (GαtGTPγS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GαtGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Gαt with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Gαt within the PDE catalytic core in addition to the sites for the inhibitory Pγ subunits. The HD moiety of GαtGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GαtGTPγS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Gαt activation enhances the PDE activation produced by subsaturating levels of Gαt, suggesting a HD-moiety synergism from a transient conformation of Gαt. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.

Xenopus Zic3, a primary regulator both in neural and neural crest development

Xenopus Zic3 is a Xenopus homologue of mouse Zic and Drosophila pair-rule gene, odd-paired. We show here that Zic3 has significant roles both in neural and neural crest development in Xenopus embryo. Expression of Zic3 is first detected in prospective neural plate region at gastrulation. Onset of the expression was earlier than most proneural genes and followed chordin expression. The expression was induced by blockade of BMP4 signal. Overexpression of Zic3 resulted in hyperplastic neural and neural crest derived tissue. In animal cap explant, the overexpression of Zic3 induced expression of all the proneural genes and neural crest marker genes. These findings suggest that Zic3 can determine the ectodermal cell fate and promote the earliest step of neural and neural crest development.

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30–100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant ΔF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein

The bacterial iron response regulator (Irr) protein mediates iron-dependent regulation of heme biosynthesis. Pulse–chase and immunoprecipitation experiments showed that Irr degraded in response to 6 μM iron with a half-life of ≈30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron. Addition of heme to low iron (0.3 μM) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum of heme to a shorter wavelength. A Cys29 → Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.

Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3

YY1 is a mammalian zinc-finger transcription factor with unusual structural and functional features. It has been implicated as a positive and a negative regulatory factor that binds to the CCATNTT consensus DNA element located in promoters of many cellular and viral genes. A mammalian cDNA that encodes a YY1-binding protein and possesses sequence homology with the yeast transcriptional factor RPD3 has been identified. A Gal4 DNA binding domain–mammalian RPD3 fusion protein strongly represses transcription from a promoter containing Gal4 binding sites. Association between YY1 and mammalian RPD3 requires a glycine-rich region on YY1. Mutations in this region abolish the interaction with mammalian RPD3 and eliminate transcriptional repression by YY1. These data suggest that YY1 negatively regulates transcription by tethering RPD3 to DNA as a cofactor and that this transcriptional mechanism is highly conserved from yeast to human.

Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with α-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

Inhibition of regulator of G protein signaling function by two mutant RGS4 proteins

Regulators of G protein signaling (RGS) proteins limit the lifetime of activated (GTP-bound) heterotrimeric G protein α subunits by acting as GTPase-activating proteins (GAPs). Mutation of two residues in RGS4, which, based on the crystal structure of RGS4 complexed with Giα1-GDP-AlF4−, directly contact Giα1 (N88 and L159), essentially abolished RGS4 binding and GAP activity. Mutation of another contact residue (S164) partially inhibited both binding and GAP activity. Two other mutations, one of a contact residue (R167M/A) and the other an adjacent residue (F168A), also significantly reduced RGS4 binding to Giα1-GDP-AlF4−, but in addition redirected RGS4 binding toward the GTPγS-bound form. These two mutant proteins had severely impaired GAP activity, but in contrast to the others behaved as RGS antagonists in GAP and in vivo signaling assays. Overall, these results are consistent with the hypothesis that the predominant role of RGS proteins is to stabilize the transition state for GTP hydrolysis. In addition, mutant RGS proteins can be created with an altered binding preference for the Giα-GTP conformation, suggesting that efficient RGS antagonists can be developed.

Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization

The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.

PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis

Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli

The global regulator FNR (for fumarate nitrate reduction) controls the transcription of >100 genes whose products facilitate adaptation of Escherichia coli to growth under O2-limiting conditions. Previous Mössbauer studies have shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster that, on exposure to oxygen, is converted into a [2Fe-2S]2+ cluster, a process that decreases DNA binding by FNR. Using 57Fe Mössbauer spectroscopy of E. coli cells containing overexpressed FNR, we show here that the same cluster conversion also occurs in vivo on exposure to O2. Furthermore, the data show that a significant amount of the [4Fe-4S]2+ cluster is regenerated when the cells are shifted back to an anaerobic environment. The present study also demonstrates that 57Fe Mössbauer spectroscopy can be employed to study the in vivo behavior of (overexpressed) proteins. The use of this technique to study other iron-containing cell components is discussed.