Evidence for serine/threonine and histidine kinase activity in the tobacco ethylene receptor protein NTHK2

Ethylene plays important roles in plant growth, development, and stress responses. Two ethylene receptors, ETR1 from Arabidopsis and NTHK1 from tobacco (Nicotiana tabacum), have been found to have His kinase (HK) activity and Ser/Thr kinase activity, respectively, although both show similarity to bacterial two-component HK. Here, we report the characterization of another ethylene receptor homolog gene, NTHK2, from tobacco. This gene also encodes a HK-like protein and is induced by dehydration and CaCl(2) but not significantly affected by NaCl and abscisic acid treatments. The biochemical properties of the yeast (Schizosaccharomyces pombe)-expressed NTHK2 domains were further characterized. We found that NTHK2 possessed Ser/Thr kinase activity in the presence of Mn(2+) and had HK activity in the presence of Ca(2+). Several lines of evidence supported this conclusion, including hydrolytic stability, phosphoamino acid analysis, mutation, deletion, and substrate analysis. These properties have implications in elucidation of the complexity of the ethylene signal transduction pathway and understanding of ethylene functions in plants.     

Roles of ethylene receptor NTHK1 domains in plant growth, stress response and protein phosphorylation

Ethylene receptors sense ethylene and regulate downstream signaling events. Tobacco ethylene receptor NTHK1, possessing Ser/Thr kinase activity, has been found to function in plant growth and salt-stress responses. NTHK1 contains transmembrane domains, a GAF domain, a kinase domain and a receiver domain. We examined roles of these domains in regulation of plant leaf growth, salt-stress responses and salt-responsive gene expressions using an overexpression approach. We found that the transgenic Arabidopsis plants harboring the transmembrane domain plus kinase domain exhibited large rosettes, had reduction in ethylene sensitivity, and showed enhanced salt sensitivity. The transgenic plants harboring the transmembrane domain plus GAF domain also showed larger rosettes. Truncations of NTHK1 affected salt-induced gene expressions. Transmembrane domain plus kinase domain promoted RD21A and VSP2 expression but decreased salt-induction of AtNAC2. The kinase domain itself promoted AtERF4 gene expression. The GAF domain itself enhanced Cor6.6 induction. Moreover, the NTHK1 functional kinase domain phosphorylated the HIS and ATP subdomains, and five putative phosphorylation sites were identified in these two subdomains. In addition, the salt-responsive element of the NTHK1 gene was in the transmembrane-coding region but not in the promoter region. These results indicate that NTHK1 domains or combination of them have specific functions in plant leaf growth, salt-stress response, gene expression and protein phosphorylation.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5＇-RACE方法克隆到烟草NTHK2的全长cDNA.其全长cDNA共有3 216bp,其中5'非编码区为509bp,3＇非编码区为427bp,编码区为2 280bp,编码产物为760个氨基酸.NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域;但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代.为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域.体外激酶分析表明,当有Mg2+存在的情况下NTHK2能够自我磷酸化.进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体,参与乙烯的信号传导过程.     

烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5′-ARCE方法克隆到烟草NTHK2的全长cDNA。其全长cDNA共有3216bp,其中5′非编码区为509bp,3′非编码区为427bp,编码区为2280bp,编码产物为760个氨基酸。NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域。但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代。为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域,体外激酶分析表明,当有Mg^2 存在的情况下NTHK2能够自我磷酸化。进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体。参与乙烯的信号传导过程。     

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.     

Spatial expression and characterization of a putative ethylene receptor protein NTHK1 in tobacco

A putative ethylene receptor gene NTHK1 encodes a protein with a putative signal peptide, three transmembrane segments, a putative histidine kinase domain and a putative receiver domain. The receiver domain was expressed in an Escherichia coli expression system, purified and used to generate polyclonal antibodies for immunohistochemistry analysis. The spatial expression of the NTHK1 protein was then investigated. We found that NTHK1 was abundant during flower and ovule development. It was also expressed in glandular hairs, stem, and in leaves that had been wounded. The NTHK1 gene was further introduced into the tobacco plant and we found that, in different transgenic lines, the NTHK1 gene was transcribed to various degrees. Upon ACC treatment, the etiolated transgenic seedlings showed reduced ethylene sensitivity when compared with the control, indicating that NTHK1 is a functional ethylene receptor in plants.     

Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum

In the model plant Arabidopsis, members of a family of two-componentsystem His kinase-like ethylene receptors have direct protein–proteininteractions with a single downstream Ser/Thr kinase CTR1. Thesecomponents of the ethylene signalling network found in Arabidopsisare conserved in the climacteric fruit tomato, but both theethylene receptors and CTR1-like proteins (LeCTRs) in tomatoare encoded by multigene families. Here, using a yeast two-hybridinteraction assay, it is shown that the tomato receptors LeETR1,LeETR2, and NEVER-RIPE (NR) can interact with multiple LeCTRs.In vivo protein localization studies with fluorescent taggedproteins revealed that the ethylene receptor NR was targetedto the endoplasmic reticulum (ER) when transiently expressedin onion epidermal cells, whereas the four LeCTR proteins werefound in the cytoplasm and nucleus. When co-expressed with NR,three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted thesame ER localization pattern in an NR receptor-dependent mannerbut not in the absence of NR. The receptor–CTR interactionswere confirmed by biomolecular fluorescence complementation(BiFC) showing that NR could form a protein complex with LeCTR1,3, and 4. This suggested that ethylene receptors recruit theseLeCTR proteins to the ER membrane through direct protein–proteininteraction. The receptor–CTR interactions and localizationobserved in the study reinforce the idea that ethylene receptorstransmit the signal to the downstream CTRs and show that a singlereceptor can interact with multiple CTR proteins. It remainsunclear whether the different LeCTRs are functionally redundantor have unique roles in ethylene signalling. Key words: BiFC, endoplasmic reticulum, Ser/Thr kinase, tomato ethylene signalling, two-component system His kinase     

Expression of tobacco ethylene receptor NTHK1 alters plant responses to salt stress

Ethylene has been regarded as a stress hormone involved in many stress responses. However, ethylene receptors have not been studied for the roles they played under salt stress condition. Previously, we characterized an ethylene receptor gene NTHK1 from tobacco, and found that NTHK1 is salt-inducible. Here, we report a further investigation towards the function of NTHK1 in response to salt stress by using a transgenic approach. We found that NTHK1 promotes leaf growth in the transgenic tobacco seedlings but affects salt sensitivity in these transgenic seedlings under salt stress condition. Differential Na+/K+ ratio was observed in the control Xanthi and NTHK1-transgenic plants after salt stress treatment. We further found that the NTHK1 transgene is also salt-inducible in the transgenic plants, and the higher NTHK1 expression results in early inductions of the ACC (1-aminocyclopropane-1-carboxylic acid) oxidase gene NtACO3 and ethylene responsive factor (ERF) genes NtERF1 and NtERF4 under salt stress. However, NTHK1 suppresses the salt-inducible expression of the ACC synthase gene NtACS1. These results indicate that NTHK1 regulates salt stress responses by affecting ion accumulation and related gene expressions, and hence have significance in elucidation of ethylene receptor functions during stress signal transduction.     

StoPK-1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response

The glycopeptide antibiotic-producing bacterium, Streptomyces toyocaensis NRRL 15009, has proteins phosphorylated on Ser, Thr, Tyr and His, implying the presence of a battery of associated kinases. We have identified the Ser/Thr protein kinase gene fragments stoPK-1, stoPK-2, stoPK-3 and stoPK-4 from S. toyocaensis NRRL 15009 by a polymerase chain reaction (PCR) strategy using oligonucleotide primers based on eukaryotic Ser/Thr and Tyr kinase sequences. One of these (stoPK-1) was subsequently cloned in its entirety from a 3.2 kb genomic BamHI fragment. stoPK-1 encodes a 642-amino-acid protein with a predicted N-terminal Ser/Thr kinase domain and a C-terminal coiled-coil region divided by a membrane-spanning region. Expression of StoPK-1 in Escherichia coli yielded a protein confined to the membrane fraction, which was found to be phosphorylated exclusively on Thr residues and could transfer phosphate to the model substrates myelin basic protein and histone H1. Both autophosphorylation and phosphoryl transfer could be inhibited by the flavanoid apigenin. Disruption of stoPK-1 with the apramycin resistance gene in the S. toyo-caensis chromosome resulted in changes in mycelial morphology and an increased sensitivity to the redox cycling agents paraquat and nitrofurantoin on glucose-containing media. Supplying stoPK-1 or the S. coelicolor homologue pkaF in trans could reverse this sensitivity, whereas a catalytically inactive mutant of stoPK-1 could not, indicating that kinase activity is essential for this phenotype. This suggests a link between this membrane-bound protein kinase in signalling pathways sensitive to oxidative stress and/or glucose metabolism. These results broaden the roles of Ser/Thr protein kinases in bacteria and underscore the diversity of signal transduction mechanisms available to respond to various stimuli.     

Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity

Tyrosine phosphorylation in plants could be performed only by dual-specificity kinases. Arabidopsis thaliana dual-specificity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His248 and Ser265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr208 in the TEY motif and Thr293 of the activation loop were converted to alanine. The conversion of Thr284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the first identification of a manganese dependent dual-specificity kinase and the importance of Thr208, Thr284, and Thr293 residues in the regulation of kinase activity.     

Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1

In Arabidopsis, ethylene is perceived by a receptor family consisting of five members, one of these being ETR1. The N-terminal half of ETR1 functions as a signal input domain. The C-terminal region of ETR1, consisting of a His kinase domain and a putative receiver domain, is likely to function in signal output. The role of the proposed signal output region in ethylene signaling was examined in planta. For this purpose, the ability of mutant versions of ETR1 to rescue the constitutive ethylene-response phenotype of the etr1-6;etr2-3;ein4-4 triple loss-of-function mutant line was examined. A truncated version of ETR1 that lacks both the His kinase domain and the receiver domain failed to rescue the triple mutant phenotype. A truncated ETR1 receptor that lacks only the receiver domain restored normal growth to the triple mutant in air, but the transgenic seedlings displayed hypersensitivity to low doses of ethylene. A mutation that eliminated His kinase activity had a modest effect upon the ability of the receptor to repress ethylene responses in air. These results demonstrate that the His kinase domain plays a role in the repression of ethylene responses. The potential roles of the receiver domain and His kinase activity in ethylene signaling are discussed.     

Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family

Receptors for the gaseous phytohormone ethylene show sequence similarity to bacterial two-component histidine kinases. These receptors are encoded by a multigene family that can be divided into subfamilies 1 and 2. It has been previously shown that a subfamily 1 Arabidopsis thaliana ethylene receptor, ETR1, autophosphorylates in vitro on a conserved histidine residue (1). However, sequence comparisons between the five ethylene receptor family members suggest that subfamily 2 members do not have all the motifs necessary for histidine kinase activity. Further, a tobacco subfamily 2 receptor, NTHK1, autophosphorylates on serines and threonines in vitro (2). Here we show that all five Arabidopsis ethylene receptor proteins autophosphorylate in vitro. We analyzed the nature of the phosphorylated amino acids by acid/base stability and bi-dimensional thin layer electrophoresis and demonstrated that unlike ETR1 all other ethylene receptors autophosphorylate predominantly on serine residues. ERS1, the only other subfamily 1 receptor, is able to phosphorylate on both histidine and serine residues in the presence of Mn2+. However, histidine autophosphorylation is lost when ERS1 is assayed in the presence of both Mg2+ and Mn2+, suggesting that this activity may not occur in vivo. Furthermore, mutation of the histidine residue conserved in two-component systems does not abolish serine autophosphorylation, eliminating the possibility of a histidine to serine phosphotransfer. Our biochemical observations complement the recently published genetic data that histidine kinase activity is not necessary for ethylene receptor function in plants and suggest that ethylene signal transduction does not occur through a phosphorelay mechanism.     

Protein kinase associated with ribosomes of streptomycetes

Protein kinases can be classified into two main superfamilies on the basis of their sequence similarity and substrate specificity. The protein His kinase superfamily which autophosphorylate a His residue, and superfamily Ser/Thr and Tyr protein kinases, which phosphorylate Ser, Thr or Tyr residues. During the last years genes encoding Ser/Thr protein kinases have been identified in several microorganisms. Phosphorylation of proteins on Ser/Thr residues can be involved in many functions of prokaryotic cells including cell differentiation, signal transduction and protein biosynthesis. Phosphorylation of prokaryotic protein-synthesizing systems showed that the phosphorylation of initiation and elongation factors is subject to alteration during cell differentiation or bacteriophage infection. Protein kinase associated with ribosomes of streptomycetes phosphorylate the elongation factor Tu and 11 ribosomal proteins even in bacteriophage-uninfected cells. After phosphorylation of ribosomal proteins, ribosomes lose about 30% of their activity at the translation of poly(U). Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

A two-component gene (NTHK1) encoding a putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco

The full-length of a two-component gene NTHK1 (Nicotiana tabacum histidine kinase-l) was isolated from tobacco (N. tabacum var. Xanthi) using a previously obtained NTHK1 cDNA fragment as a probe. Sequence analysis revealed that NTHK1 shared high homology with LeETR4 from tomato and encoded an ethylene- receptor homolog. The predicted NTHK1 protein had a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. The putative autophosphorylation site at His378 and the phosphate receiver site at Asp689 were also identified. By using the in situ hybridization technique, NTHK1 mRNA was detected during flower organ development. It is also highly expressed in the processes of pollen formation and embryo development. The expression of NTHK1 in response to wounding and other stresses was investigated using competitive RT-PCR. The results demonstrated that NTHK1 was inducible upon wounding (cutting). Floating of the cut leaf pieces in 0.5× MS, with shaking, led to a relatively rapid and strong expression. This phenomenon was confirmed by the in situ hybridization results. In addition to the up-regulation by wounding, NTHK1 expression was also induced following NaCl and PEG treatment, indicating a possible role for NTHK1 in multiple stress responses. Received: 28 June 2000 / Accepted: 1 August 2000     

Positive and negative regulation of type II TGF-beta receptor signal transduction by autophosphorylation on multiple serine residues.

The type II transforming growth factor-beta (TGF-beta) receptor Ser/Thr kinase (TbetaRII) is responsible for the initiation of multiple TGF-beta signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TbetaRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation which is essential for the activation of TbetaRII kinase activity, activation of TbetaRI and TGF-beta-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an intermolecular mechanism. Phosphorylation of Ser409 is essential for TbetaRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TbetaRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TbetaRII phosphorylation is regulated intricately and affects TGF-beta receptor signal transduction both positively and negatively.     

植物类受体激酶CrRLK1-L亚家族及其生物学功能

植物CrRLK1-L亚家族类受体激酶的胞外域具有新颖结构基序,但功能大都未知.该家族成员广泛存在于被子植物中,但在动物和微生物中不存在其同源物.CrRLK1-L家族成员相对较少,但组织表达非常广泛.它们定位于细胞质膜上,并且部分成员的定位还具有极性,这与其参与雌雄配子体的识别和受精作用密切相关.该家族成员普遍具有激酶活性,该活性对其功能的发挥至关重要.目前仅报道在拟南芥中参与助细胞与花粉的识别和调控营养组织的细胞伸长,但参与这些生物学过程的作用机制似乎独立于已知的信号通路之外,可能有自身独特的信号传导机制.所以对这一类具特有结构基序的类受体激酶基因的功能研究,将有助于解析植物特有生物学过程的分子作用机制,特别是在植物有性生殖过程中,合理利用这些分子开展育种实践对未来农业生产具有潜在的应用价值.     

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.