Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica

Bacteria employ two-component signaling to detect and respond to environmental stimuli. In essence, two-component signaling relies on a protein called a response regulator that can elicit a change in gene expression or protein function in response to phosphoryl transfer from a histidine kinase. Phosphorylation of the associated histidine kinase is regulated by detection of an environmental signal, thus linking sensing to cellular response. Recently, it has been suggested that H-NOX (Heme-nitric oxide/oxygen binding) proteins may act as nitric oxide (NO) sensors in two-component signaling systems. NO/H-NOX regulated histidine kinases have been reported, but their cognate response regulators have yet to be identified. In this work we provide biochemical characterization of a complete NO/H-NOX-regulated two-component signaling pathway in the biofilm-dwelling marine bacterium, Pseudoalteromonas atlantica. In P. atlantica, as is typical for bacteria that code for H-NOX, an hnoX gene is found in the same operon as a gene coding for a two-component signaling histidine kinase (H-NOX-associated histidine kinase; HahK). We find that HahK is capable of autophosphorylation in vitro and that NO-bound H-NOX inhibits HahK activity, implicating H-NOX as a selective NO sensor. The cognate response regulator, a protein annotated as a cyclic-di-GMP processing enzyme that we have named HarR (H-NOX-associated response regulator), was identified using bioinformatics tools. Phosphoryl transfer from HahK to HarR has been established. This report reveals the first biochemical characterization of an H-NOX-associated response regulator and contributes to a deeper understanding of NO/H-NOX signaling in bacteria.     

The molecular puzzle of two-component signaling cascades

Two-component systems constitute prevalent signaling pathways in bacteria and mediate a large variety of adaptative cellular responses. Signaling proceeds through His-Asp phosphorelay cascades that involve two central partners, the histidine protein kinase and the response regulator protein. Structural studies have provided insights into some design principles and activation mechanisms of these multi-domain proteins implicated in the control of virulence gene expression in several pathogens.     

Temporal and evolutionary dynamics of two-component signaling pathways

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Histidine kinases and response regulator proteins in two-component signaling systems.

Phosphotransfer-mediated signaling pathways allow cells to sense and respond to environmental stimuli. Autophosphorylating histidine protein kinases provide phosphoryl groups for response regulator proteins which, in turn, function as molecular switches that control diverse effector activities. Structural studies of proteins involved in two-component signaling systems have revealed a modular architecture with versatile conserved domains that are readily adapted to the specific needs of individual systems.     

Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system

We have so far cloned a cDNA encoding a hybrid-type histidine kinase (ATHK1), three cDNAs encoding phosphorelay intermediates (ATHP1-3), and four cDNAs encoding response regulators (ATRR1-4) from Arabidopsis thaliana. To determine which molecules constitute a His to Asp phosphorelay pathway, we examined protein-protein interactions between them using a pairwise yeast two-hybrid analysis, as an initial step. We detected a specific interaction between ATHK1 and ATHP1. We further examined protein-protein interactions between ATHP1-3 and other histidine kinases. We detected interactions between ETR1 and all ATHPs, and between CKI1 and ATHP1 or ATHP2. Interestingly, ERS1 could not interact with any ATHPs. We also examined protein-protein interactions between ATHP1-3 and ATRR1-4. The results indicated that ATHP2 could interact with ATRR4, and that ATHP3 could interact with ATRR1 or ATRR4. However, ATHP1 could not interact with any ATRRs. On the basis of these results, we discuss the possible phosphorelay networks in an Arabidopsis two-component system.     

Evolution of prokaryotic two-component system signaling pathways: gene fusions and fissions

Two-component systems (TCSs) are common signal transduction systems, typically comprising paired histidine protein kinase (HK) and response regulator (RR) proteins. In many examples, it appears RR and HK genes have fused, producing a "hybrid kinase " We have characterized a set of prokaryotic genes encoding RRs, HKs, and hybrid kinases, enabling characterization of gene fusion and fission. Primary factors correlating with fusion rates are the presence of transmembrane helices in HKs and the presence of DNA-binding domains in RRs, features that require correct (and separate) spatial location. In the absence of such features, there is a relative abundance of fused genes. The order of paired HK and RR genes and the nucleotide distance between encoded domains also correlate with apparent gene fusion rates. We propose that localization requirements and relative positioning of encoded domains within TCS genes affect the function (and therefore retention) of hybrid kinases resulting from gene fusion.     

Toward an interaction map of the two-component signaling pathway of Arabidopsis thaliana

Among the signal transduction pathways in higher eukaryotes, the two-component system (TCS) is unique to plants. In the model plant Arabidopsis thaliana, it consists of more than 30 proteins, including eight receptors, five phosphotransmitters and 23 response regulators. One of its important functions is to perceive and transduce the signal of the plant hormone cytokinin. The basic signal flow within the TCS is well-understood, but it is unclear how this pathway is integrated with the remainder of the proteome. Thus, knowledge about the interactions of TCS proteins should contribute to the understanding of their mode of action. Therefore, we conducted medium-scale yeast two-hybrid screens focusing on those members of the TCS, which are thought to be involved in cytokinin signaling. In total, more than 6.3 x 10 (7) transformants were screened resulting in the identification of 160 different interactions, of which 136 were novel. Most of the interacting proteins belong to the functional categories of signal transduction and protein metabolism. TCS proteins and their interactors localized to the same subcellular compartment in many cases, a prerequisite to being of biological relevance. The resulting interaction network map revealed large differences in the connectivity. Cytokinin receptors (AHK2, CRE1/AHK4) showed the highest numbers of different interaction partners. This study is the first systematic protein-protein interaction experiment for a plant signal system and provides numerous starting points for further analysis of the molecular mechanisms used to convert the signal carried by the TCS into biological processes.     

Dissecting the specificity of protein-protein interaction in bacterial two-component signaling: orphans and crosstalks

Predictive understanding of the myriads of signal transduction pathways in a cell is an outstanding challenge of systems biology. Such pathways are primarily mediated by specific but transient protein-protein interactions, which are difficult to study experimentally. In this study, we dissect the specificity of protein-protein interactions governing two-component signaling (TCS) systems ubiquitously used in bacteria. Exploiting the large number of sequenced bacterial genomes and an operon structure which packages many pairs of interacting TCS proteins together, we developed a computational approach to extract a molecular interaction code capturing the preferences of a small but critical number of directly interacting residue pairs. This code is found to reflect physical interaction mechanisms, with the strongest signal coming from charged amino acids. It is used to predict the specificity of TCS interaction: Our results compare favorably to most available experimental results, including the prediction of 7 (out of 8 known) interaction partners of orphan signaling proteins in Caulobacter crescentus. Surveying among the available bacterial genomes, our results suggest 15～25% of the TCS proteins could participate in out-of-operon "crosstalks". Additionally, we predict clusters of crosstalking candidates, expanding from the anecdotally known examples in model organisms. The tools and results presented here can be used to guide experimental studies towards a system-level understanding of two-component signaling.     

Interaction with podocin facilitates nephrin signaling

Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking CD2-associated protein (CD2AP) develop a nephrotic syndrome that resembles NPHS mutations suggesting that all three proteins are essential for the integrity of glomerular podocytes. Although the precise glomerular function of either protein remains unknown, it has been suggested that nephrin forms zipper-like interactions to maintain the structure of podocyte foot processes. We demonstrate now that nephrin is a signaling molecule, which stimulates mitogen-activated protein kinases. Nephrin-induced signaling is greatly enhanced by podocin, which binds to the cytoplasmic tail of nephrin. Mutational analysis suggests that abnormal or inefficient signaling through the nephrin-podocin complex contributes to the development of podocyte dysfunction and proteinuria.     

Pneumococcal HtrA protease mediates inhibition of competence by the CiaRH two-component signaling system

Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation in Streptococcus pneumoniae through a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression of htrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of a ciaRH+ strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.     

Co-evolving motions at protein-protein interfaces of two-component signaling systems identified by covariance analysis

Short-lived protein interactions determine signal transduction specificity among genetically amplified, structurally identical two-component signaling systems. Interacting protein pairs evolve recognition precision by varying residues at specific positions in the interaction surface consistent with constraints of charge, size, and chemical properties. Such positions can be detected by covariance analyses of two-component protein databases. Here, covariance is shown to identify a cluster of co-evolving dynamic residues in two-component proteins. NMR dynamics and structural studies of both wild-type and mutant proteins in this cluster suggest that motions serve to precisely arrange the site of phosphoryl transfer within the complex.     

Interaction between RNA molecules of a two-component trans analog of antigenomic HDV ribozyme

A study was made of the association of the RNA components forming a B:LS two-component rans analog of the antigenomic HDV ribozyme. The B:LS ribozyme differed from known trans ribozymes in the sizes and nucleotide sequences of its components (33 and 34 nt, respectively), the topology of its functional parts, and the lack of a very short cleavage product. Compared to the cis ribozyme, B:LS showed similar dependences on the reaction conditions (Mg²⁺ concentration, pH, temperature) and a similar biphasic kinetic curve of self-cleavage. The kinetic model of B:LS self-cleavage (available at www.cardio.ru/labgen/RZ_e.html) describes a possible cause of the biphasic kinetic curve as a change in the rate-limiting step of consecutive conformational transitions accompanying self-cleavage. Another possible cause is an interaction between the molecules involved in cleavage, i.e., multimerization of whole ribozyme molecules with their components or the reaction products. B:LS provides a convenient model for studying such interactions, since the mode of component binding allows generation of 1B:2LS and 2B:1LS complexes as well as complexes with the cleavage products. Nondenaturing PAGE was used to study the factors affecting association and dissociation of the ribozyme components. The possibility of interactions between the RNA components of the cis and trans ribozymes was demonstrated experimentally. It was shown that the ribozyme is capable of multimerization when LS is in excess over B and that the cleavage products are not significantly involved in this process. The results suggest intermolecular interactions for the cleavage of the natural cis ribozyme.     

Interaction network of core ABA signaling components in maize

Key message

We defined a comprehensive core ABA signaling network in monocot maize, including the gene expression, subcellular localization and interaction network of ZmPYLs, ZmPP2Cs, ZmSnRK2s and the putative substrates.

Abstract

The phytohormone abscisic acid (ABA) plays an important role in plant developmental processes and abiotic stress responses. In Arabidopsis, ABA is sensed by the PYL ABA receptors, which leads to binding of the PP2C protein phosphatase and activation of the SnRK2 protein kinases. These components functioning diversely and redundantly in ABA signaling are little known in maize. Using Arabidopsis pyl112458 and snrk2.2/3/6 mutants, we identified several ABA-responsive ZmPYLs and ZmSnRK2s, and also ZmPP2Cs. We showed the gene expression, subcellular localization and interaction network of ZmPYLs, ZmPP2Cs, and ZmSnRK2s, and the isolation of putative ZmSnRK2 substrates by mass spectrometry in monocot maize. We found that the ABA dependency of PYL-PP2C interactions is contingent on the identity of the PP2Cs. Among 238 candidate substrates for ABA-activated protein kinases, 69 are putative ZmSnRK2 substrates. Besides homologs of previously reported putative AtSnRK2 substrates, 23 phosphoproteins have not been discovered in the dicot Arabidopsis. Thus, we have defined a comprehensive core ABA signaling network in monocot maize and shed new light on ABA signaling.

     

Interaction of Btk and Akt in B cell signaling

Reactive oxygen species (ROS) or reactive oxygen intermediates (ROIs) mediate complex signaling involving multiple pathways. In this report, we demonstrate for the first time that endogenous Bruton's tyrosine kinase (Btk) and Akt can interact with each other in DT40 chicken B cells and human Nalm6 B cells and that this interaction is inducible following H2O2 stimulation. This interaction is supported by visualizing the co-localization of Btk and Akt in the perinuclear region and membrane ruffles in COS-7 cells. We have also shown the involvement of phosphatidylinositol 3-kinase (PI 3-K) and Btk in the phosphorylation of Akt following stimulation by hydrogen peroxide (H2O2). Interestingly, Akt phosphorylation was found in the presence of Btk even in the absence of oxidative stress. In addition, we have investigated the involvement of PI 3-K in the MAPKs and ERK and JNK phosphorylation, in the presence or absence of Btk. Phosphorylation of both ERK and JNK increased when the PI 3-K pathway was inhibited and both pathways were modulated positively by Btk. Taken together, based on the study of endogenous conditions, we show the novel interaction of Btk and Akt in H2O2 signaling in B cells.     

Whole-genome analysis of Oryza sativa reveals similar architecture of two-component signaling machinery with Arabidopsis

The two-component system (TCS), which works on the principle of histidine-aspartate phosphorelay signaling, is known to play an important role in diverse physiological processes in lower organisms and has recently emerged as an important signaling system in plants. Employing the tools of bioinformatics, we have characterized TCS signaling candidate genes in the genome of Oryza sativa L. subsp. japonica. We present a complete overview of TCS gene families in O. sativa, including gene structures, conserved motifs, chromosome locations, and phylogeny. Our analysis indicates a total of 51 genes encoding 73 putative TCS proteins. Fourteen genes encode 22 putative histidine kinases with a conserved histidine and other typical histidine kinase signature sequences, five phosphotransfer genes encoding seven phosphotransfer proteins, and 32 response regulator genes encoding 44 proteins. The variations seen between gene and protein numbers are assumed to result from alternative splicing. These putative proteins have high homology with TCS members that have been shown experimentally to participate in several important physiological phenomena in plants, such as ethylene and cytokinin signaling and phytochrome-mediated responses to light. We conclude that the overall architecture of the TCS machinery in O. sativa and Arabidopsis thaliana is similar, and our analysis provides insights into the conservation and divergence of this important signaling machinery in higher plants.