Light-dependent dimerisation in the N-terminal sensory module of cyanobacterial phytochrome 1

Phytochromes, photoreceptors controlling important physiological processes in plants and many prokaryotes, are photochromic biliproteins. The red-absorbing Pr ground state is converted by light into the farred-absorbing Pfr which can be photoconverted back to Pr. In plants at least Pfr is the physiologically active signalling state. Here, we show that the N-terminal photochromic module of Cph1 homodimerises reversibly and independently in Pr and Pfr, Pfr-dimers being significantly more stable. Implications for the mechanism of signal transduction are discussed.     

Dual-histidine kinases in basidiomycete fungi

Dual-histidine kinases (HKs) are complex hybrid HKs containing in a single polypeptide two HK transmitter modules (T) and two-response regulator received domains (R) that are combined in a TRTR geometry. In fungi, this protein family is limited to some particular species of the phylum Basidiomycota and absent in the other phyla. This study extends the investigation of dual-HKs to 80 fully sequenced genomes of basidiomycetes, analyzing their distribution, domain architecture and phylogenetic relationships. Moreover, similarly to dual-HKs of basidiomycetes, several species of bacteria were found that contain hybrid HKs with a TRTR domain architecture encoded in a single gene.     

Involvement of protein kinase C in the response of Neurospora crassa to blue light

As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa. Received: 4 December 1998 / Accepted: 21 May 1999     

Functional insights from the molecular modelling of a novel two-component system

Two-component systems (TCSs) are the major signalling pathway in bacteria and represent potential drug targets. Among the 11 paired TCS proteins present in Mycobacterium tuberculosis H37Rv, the histidine kinases (HKs) Rv0600c (HK1) and Rv0601c (HK2) are annotated to phosphorylate one response regulator (RR) Rv0602c (TcrA). We wanted to establish the sequence-structure-function relationship to elucidate the mechanism of phosphotransfer using in silico methods. Sequence alignments and codon usage analysis showed that the two domains encoded by a single gene in homologous HKs have been separated into individual open-reading frames in M. tuberculosis. This is the first example where two incomplete HKs are involved in phosphorylating a single RR. The model shows that HK2 is a unique histidine phosphotransfer (HPt)-mono-domain protein, not found as lone protein in other bacteria. The secondary structure of HKs was confirmed using "far-UV" circular dichroism study of purified proteins. We propose that HK1 phosphorylates HK2 at the conserved H131 and the phosphoryl group is then transferred to D73 of TcrA.     

phoR1, a gene encoding a new histidine protein kinase Myxococcus xanthus

A soil bacterium able to undergo multicellular development and a coordinated gliding in swarms, requires an accurate regulatory network of phosphorelay proteins. Inorganic phosphate is a limiting nutrient in soil and its importance in regulation is critical. As a step towards studying phosphate regulation and its influence in the developmental process in this bacterium, we screened a Myxococcus xanthus library for clones with phosphatase activity, and found four different ones. The deduced sequence of one of the cloned inserts is similar to that of the classic transmembrane histidine protein kinase of the sensor family of the two-component signal transduction systems with a high sequence similarity to the sensor kinase in the Pho regulon of Bacillus subtilis PhoR. This gene has been named phoR1 and its deduced amino acid sequence consists of 455 residues with a predicted molecular mass of 48.5 kDa. The M. xanthus PhoR1 deduced sequence contains all the characteristic histidine protein kinase motifs in the same order and with the same spacing. A hydropathy profile indicates two membrane-spanning segments located at the extreme N-terminus, according to the putative sensor role of this domain. A gene-disrupted mutant is unable to produce normal mature fruiting bodies and produces fewer spores. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Candida albicans histidine kinase Chk1p: Signaling and cell wall mannan

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.     

Structural characterization of the intra-membrane histidine kinase YbdK from Bacillus subtilis in DPC micelles

Bacterial histidine kinases (HKs) play a critical role in signal transduction for cellular adaptation to environmental conditions and stresses. YbdK from Bacillus subtilis is a 320-residue intra-membrane sensing HK characterized by a short input domain consisting of two transmembrane helices without an extracytoplasmic domain. While the cytoplasmic domains of HKs have been studied in detail, the intra-membrane sensing domain systems are still uncharacterized due to difficulties in handling the transmembrane domain. Here, we successfully obtained pure recombinant transmembrane domain of YbdK (YbdK-TM) from E. coli and analyzed the characteristics of YbdK-TM using nuclear magnetic resonance (NMR) and other biophysical methods. YbdK-TM was found to form homo-dimers in DPC micelles based on cross-linking assays and analytical ultracentrifugation analyses. We estimated the size of the YbdK-TM DPC complex to be 46 kDa using solution state NMR T₁/T₂ relaxation analyses in DPC micelles. These results provide information that will allow functional and structural studies of intra-membrane sensing HKs to begin.     

Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of Agrobacterium tumefaciens

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor. VirA also responds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential. To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes. In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein. Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars. Received: 14 December 1999 / Accepted: 10 April 2000     

The HOG1-like MAP kinase Sak1 of Botrytis cinerea is negatively regulated by the upstream histidine kinase Bos1 and is not involved in dicarboximide- and phenylpyrrole-resistance

In filamentous ascomycetes, HOG-like signal transduction cascades are involved in the resistance to hyper-osmotic conditions and to dicarboximides and phenylpyrroles. The histidine kinase (HK) Bos1 and the mitogen-activated protein kinase (MAPK) Sak1 are important for the adaptation to hyper-osmotic and oxidative stress, development, and pathogenicity in the phytopathogenic fungus Botrytis cinerea. However, bos1Δ and sak1Δ mutants created previously, also presented different phenotypes, especially the sak1Δ mutants were not resistant to high fungicide concentrations. Since both single mutants were constructed in different parental strains, phenotypic variations due to the genetic background might be suspected. In order to establish the relationship between both protein kinases, we analyzed Sak1 phosphorylation under the control of the Bos1 HK and we realized epistasis analysis between bos1Δ and sak1Δ mutations through the construction of isogenic single and double mutants. Our results show that Bos1 negatively regulates Sak1 phosphorylation and that Bos1 regulates certain phenotypes independently of Sak1. They include fungicide susceptibility, adaptation and conidiation on high neutral osmolarity.     

蛋白激酶B及其在磷脂酰肌醇3-激酶介导的信号转导中的作用

蛋白激酶Ｂ（ＰＫＢ）是原癌基因ｃ－ａｋｔ的表达产物,它参与由生长因子激活的经磷脂磷肌醇３－激酶（ＰＩ３Ｋ）介导的信号转导过程。与许多蛋白激酶相似,ＰＫＢ分子具有一特殊的ＡＨ／ＰＨ结构域（ＡＨ／ＰＨｄｏｍａｉｎ）,后者能介导信号分子间的相互作用。ＰＫＢ是ＰＩ３Ｋ直接的靶蛋白。ＰＩ３Ｋ产生的脂类第二信使ＰＩ－３,４,Ｐ２和ＰＩ－３,４,５－Ｐ３等均能与ＰＫＢ和磷酸肌醇依赖性蛋白激酶（ＰＤＫ）的ＡＨ／Ｐ     

Mutations in protein kinase subdomain X differentially affect MEKK2 and MEKK1 activity

MAPK/ERK kinase kinase 2 (MEKK2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of protein kinases. MAP3Ks are components of a three-tiered protein kinase pathway in which a MAP3K phosphorylates and activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates a mitogen-activated protein kinase (MAPK). We have previously identified residues within protein kinase subdomain X in the MAP3K, MEKK1, that are critical for its interaction with the MAP2K, MKK4, and MEKK1-induced MKK4 activation. We report here that kinase subdomain X also plays a critical role in MEKK2 activity. Select point mutations in subdomain X impair MEKK2 phosphorylation of the MAP2Ks, MKK7 and MEK5, abolish MEKK2-induced activation of the MAPKs, JNK1 and ERK5, and diminish MEKK2-dependent activation of an AP-1 reporter gene. Interestingly, the spectrum of mutations in subdomain X of MEKK2 that affects its activity is overlapping with but not identical to those that have effects on MEKK1. Thus, mutations in subdomain X differentially affect MEKK2 and MEKK1.     

Identification and characterization of KvgAS,a two-component system in Klebsiella pneumoniae CG43

A two-component system encoding gene cluster kvgAS that is present only in virulent Klebsiella pneumoniae CG43 was isolated and its sequence determined. RT-PCR and Southern analysis demonstrated that kvgAS is organized as an operon. No apparent effect of a kvgS deletion on bacterial virulence was observed in a mouse peritonitis model. In the presence of paraquat or 2,2-dipyridyl, the activity of kvgAS promoter in the kvgS mutant was found to be reduced to half of the level in the wild-type strain. The data suggest that the KvgAS system is autoregulated and plays a role in countering free radical stresses and sensing iron-limiting conditions.     

Genomic analysis of protein kinases,protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell-cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.     

Molecular cloning, expression and evolutionary analysis of the avian tyrosine kinase JAK1

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133 kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1–JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5 kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.     

Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies

Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D ¹⁵N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly α-helical. This method should be applicable to the NMR analysis of other transmembrane proteins.     

Protein-protein interaction of HP137 with histidine kinase HP244 does not contribute to flagellar regulation in Helicobacter pylori

Flagellar motility is essential for the ability of Helicobacter pylori to colonize the gastric mucosa. Expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors sigma54 and sigma28 and the anti-sigma28 factor FlgM. The protein-protein interaction map of H. pylori, which is based on a high-throughput two-hybrid screen (Rain et al., 2001. Nature 409, 211-215) indicated a protein-protein interaction between the gene product of ORF hp137 and both the histidine kinase HP244 and the flagellar hook protein HP908. We hypothesized that HP137 might be involved in a feedback regulatory mechanism controlling the activity of histidine kinase HP244. Here we demonstrate that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium Campylobacter jejuni.