The serine and threonine residues in the Ig-α cytoplasmic tail negatively regulate immunoreceptor tyrosine-based activation motif-mediated signal transduction

The B cell antigen receptor (BCR) is a multiprotein complex consisting of the membrane-bound Ig molecule and the Ig-α/Ig-β heterodimer. On BCR engagement, Ig-α and Ig-β become phosphorylated not only on tyrosine residues of the immunoreceptor tyrosine-based activation motif but also on serine and threonine residues. We have mutated all serine and threonine residues in the Ig-α tail to alanine and valine, respectively. The mutated Ig-α sequence was expressed either as a single-chain Fv/Ig-α molecule or in the context of the complete BCR. In both cases, the mutated Ig-α showed a stronger tyrosine phosphorylation than the wild-type Ig-α and initiated increased signaling on stimulation. These findings suggest that serine/threonine kinases can negatively regulate signal transduction from the BCR.

Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells

(RS)-2-cis, 4-trans-abscisic acid (ABA), a naturally occurring plant stress hormone, elicited rapid agonist-specific changes in myo-inositol hexakisphosphate (InsP6) measured in intact guard cells of Solanum tuberosum (n = 5); these changes were not reproduced by (RS)-2-trans, 4-trans-abscisic acid, an inactive stereoisomer of ABA (n = 4). The electrophysiological effects of InsP6 were assessed on both S. tuberosum (n = 14) and Vicia faba (n = 6) guard cell protoplasts. In both species, submicromolar concentrations of InsP6, delivered through the patch electrode, mimicked the inhibitory effects of ABA and internal calcium (Cai2+) on the inward rectifying K+ current, IK,in, in a dose-dependent manner. Steady state block of IK,in by InsP6 was reached much more quickly in Vicia (3 min at ≈1 μM) than Solanum (20–30 min). The effects of InsP6 on IK,in were specific to the myo-inositol isomer and were not elicited by other conformers of InsP6 (e.g., scyllo- or neo-). Chelation of Ca2+ by inclusion of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or EGTA in the patch pipette together with InsP6 prevented the inhibition of IK,in, suggesting that the effect is Ca2+ dependent. InsP6 was ≈100-fold more potent than Ins(1,4,5)P3 in modulating IK,in. Thus ABA increases InsP6 in guard cells, and InsP6 is a potent Ca2+-dependent inhibitor of IK,in. Taken together, these results suggest that InsP6 may play a major role in the physiological response of guard cells to ABA.

Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain

Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNγ-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNγ. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.

A Stat3-interacting protein (StIP1) regulates cytokine signal transduction

Genetic and biochemical studies have led to the identification of the Stat3-Interacting Protein StIP1. The preferential association of StIP1 with inactive (i.e., unphosphorylated) Stat3 suggests that it may contribute to the regulation of Stat3 activation. Consistent with this possibility, StIP1 also exhibits an affinity for members of the Janus kinase family. Overexpression of the Stat3-binding domain of StIP1 blocks Stat3 activation, nuclear translocation, and Stat3-dependent induction of a reporter gene. These studies indicate that StIP1 regulates the ligand-dependent activation of Stat3, potentially by serving as a scaffold protein that promotes the interaction between Janus kinases and their Stat3 substrate. The ability of StIP1 to associate with several additional members of the signal transducer and activator of transcription family suggests that StIP1 may serve a broader role in cytokine-signaling events.

Regulation of the pAD1 sex pheromone response of Enterococcus faecalis by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein

The Enterococcus faecalis conjugative plasmid pAD1 (60 kb) encodes a mating response to the recipient-produced peptide sex pheromone cAD1. The response involves two key plasmid-encoded regulatory proteins: TraE1, which positively regulates all or most structural genes relating to conjugation, and TraA, which binds DNA and negatively regulates expression of traE1. In vitro studies that included development of a DNA-associated protein-tag affinity chromatography technique showed that TraA (37.9 kDa) binds directly to cAD1 near its carboxyl-terminal end and, as a consequence, loses its affinity for DNA. Analyses of genetically modified TraA proteins indicated that truncations within the carboxyl-terminal 9 residues significantly affected the specificity of peptide-directed association/dissociation of DNA. The data support earlier observations that transposon insertions near the 3′ end of traA eliminated the ability of cells to respond to cAD1.

Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation

Dexamethasone and progesterone have been found to accelerate the time of initiation and enhance the rate of myelin synthesis in Schwann cell/neuronal cocultures. The expression of mRNA for cytochrome P450scc (converts cholesterol to pregnenolone), 3β-hydroxysteroid dehydrogenase (converts pregnenolone to progesterone), and the progesterone receptor were detected and markedly induced during peak myelin formation in the cocultures. The mRNA for the glucocorticoid receptor was detected, but was found to be constituitively expressed. In addition, the specific activity of 3β-hydroxysteroid dehydrogenase was measured and found to increase by 10-fold. The mRNA for cytochrome P450scc and 3β-hydroxysteroid dehydrogenase also were found to be induced during the differentiation of O-2A precursor cells to oligodendrocytes. Fibroblast growth factor and platelet-derived growth factor were found to have proliferative effects on Schwann cells, but they had no effect on the initiation or the rate of myelin formation. These results demonstrate that myelin-forming cells have inducible enzymes responsible for steroid biosynthesis and suggest a critical role for endogenous steroid hormones in signaling the initiation and enhancing the rate of myelin formation.

Implications of macromolecular crowding for signal transduction and metabolite channeling

The effect of different total enzyme concentrations on the flux through the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in vitro was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of Escherichia coli. The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux–response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of ≈1.8 with increasing protein concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux–response coefficient, in one case to values of ≈1. PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration. This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes. High concentrations of the microsolute glycerol did not affect the combined flux–response coefficient. The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway. Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed.

Structure of a biological oxygen sensor: A new mechanism for heme-driven signal transduction

The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL’s oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the “on” state without bound ligand and one in the “off” state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.

Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation

Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86). In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining [V(D)J] recombination. Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination. It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints. The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation. An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation. To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice. We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation. Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant. These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation.

Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine2A (5HT2A) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT7 receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an ≈65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT7 receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., γ-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway.

SRPDB (Signal Recognition Particle Database)

Signal recognition particle (SRP) is a stable cytoplasmic ribonucleoprotein complex that serves to translocate secretory proteins across membranes during translation. The SRP Database (SRPDB) provides compilations of SRP components, ordered alphabetically and phylogenetically. Alignments emphasize phylogenetically-supported base pairs in SRP RNA and conserved residues in the proteins. Data are provided in various formats including a column arrangement for improved access and simplified computational usability. Included are motifs for identification of new sequences, SRP RNA secondary structure diagrams, 3-D models and links to high-resolution structures. This release includes 11 new SRP RNA sequences (total of 129), two protein SRP9 sequences (total of seven), two protein SRP14 sequences (total of 10), two protein SRP19 sequences (total of 16), 10 new SRP54 (ffh) sequences (total of 66), two protein SRP68 sequences (total of seven) and two protein SRP72 sequences (total of nine). Seven sequences of the SRP receptor α-subunit and its FtsY homolog (total of 51) are new. Also considered are β-subunit of SRP receptor, Flhf, Hbsu, CaM kinase II and cpSRP43. Access to SRPDB is at http://psyche.uthct.edu/dbs/SRPDB/SRPDB.html and the European mirror http://www.medkem.gu.se/dbs/SRPDB/SRPDB.html

Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of β-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered β-arrestin-2 binding to the receptor and internalization of AT1aR-β-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-β-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, β-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged β-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with β-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with β-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to β-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in β-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to β-arrestin-2, and the association of β-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that β-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR–LasI and RhlR–RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.

Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-Å resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 Å from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora. This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.