Molecular characterization of two highly homologous receptor-like kinase genes, RLK902 and RKL1, in Arabidopsis thaliana

Receptor-like kinases (RLKs) constitute a large family of signal perception molecules. We characterized two highly homologous RLK genes, RLK902 and RKL1, in Arabidopsis. RLK902 and RKL1 showed a 75% amino acid sequence identity over their entire regions. In the RLK902 pro::GUS transgenic lines, GUS activity was strong in the root tips, lateral root primordia, stipules, and floral organ abscission zones, while the RKL1 promoter activity was dominant in the stomata cells, hydathodes and trichomes of young rosette leaves, and floral organ abscission zones. Neither the rlk902 mutant line, rkl1 mutant line nor rlk902/rkl1 double-knockout mutant line showed any significant phenotypes under normal growth conditions. These results suggest that RLK902 and RKL1 might mediate the signal transduction pathway in which at least one other complementary signaling pathway to these two RLKs might exist.     

Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase.

To determine potential targets of the S locus receptor kinase (SRK) during the Brassica self-incompatibility response, a yeast two-hybrid library was screened with the SRK-910 protein kinase domain. Two thioredoxin-h-like clones, THL-1 and THL-2, were found to interact specifically with the SRK-910 protein kinase domain and not to interact with the protein kinase domains from the Arabidopsis receptor-like protein kinases (RLK) RLK4 and RLK5. The interaction between THL-1 and the SRK-910 protein kinase domain was confirmed using coimmunoprecipitation experiments with fusion proteins produced in Escherichia coli. THL-1 has thioredoxin activity based on an insulin reduction assay, and THL-1 is weakly phosphorylated by the SRK-910 protein kinase domain. THL-1 and THL-2 are both expressed in a variety of tissues but show some differences in steady state mRNA levels, with THL-2 being preferentially expressed in floral tissues. This indicates a more general biological function for these thioredoxins in addition to a potential role as effector molecules in the self-incompatibility signal cascade.     

Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: implications for transmembrane signaling in plants

The kinase interaction (KI) domain of kinase-associated protein phosphatase (KAPP) interacts with the phosphorylated form of an Arabidopsis thaliana receptor-like protein kinase (RLK). The KI domain may recruit KAPP into an RLK-initiated signaling complex. To examine additional roles that this domain may play in plant signal transduction, a search was conducted for other KI domain-containing proteins. One gene was isolated which encodes a KI domain, the maize homolog of KAPP. To test whether the maize KI domain associates with other maize proteins, it was used as a probe in a protein–protein interaction cloning strategy. A new maize RLK, K I domain i nteracting k inase 1 (KIK1), was identified by its interaction with the maize KI domain. The maize KI domain and the KIK1 kinase domain association required phosphorylation of the kinase. This work establishes that the KI domain phosphorylation-dependent signaling mechanism is present in both monocots and dicots. Additionally, it was determined that both the maize and Arabidopsis KI domains interact with several but not all of the active RLKs assayed. These multiple associations imply that KAPP may function in a number of RLK-initiated signaling pathways.     

Plant receptor-like serine threonine kinases: roles in signaling and plant defense

Plants are hosts to a wide array of pathogens from all kingdoms of life. In the absence of an active immune system or combinatorial diversifications that lead to recombination-driven somatic gene flexibility, plants have evolved different strategies to combat both individual pathogen strains and changing pathogen populations. The receptor-like kinase (RLK) gene-family expansion in plants was hypothesized to have allowed accelerated evolution among domains implicated in signal reception, typically a leucine-rich repeat (LRR). Under that model, the gene-family expansion represents a plant-specific adaptation that leads to the production of numerous and variable cell surface and cytoplasmic receptors. More recently, it has emerged that the LRR domains of RLK interact with a diverse group of proteins leading to combinatorial variations in signal response specificity. Therefore, the RLK appear to play a central role in signaling during pathogen recognition, the subsequent activation of plant defense mechanisms, and developmental control. The future challenges will include determinations of RLK modes of action, the basis of recognition and specificity, which cellular responses each receptor mediates, and how both receptor and kinase domain interactions fit into the defense signaling cascades. These challenges will be complicated by the limited information that may be derived from the primary sequence of the LRR domain. The review focuses upon implications derived from recent studies of the secondary and tertiary structures of several plant RLK that change understanding of plant receptor function and signaling. In addition, the biological functions of plant and animal RLK-containing receptors were reviewed and commonalities among their signaling mechanisms identified. Further elucidated were the genomic and structural organizations of RLK gene families, with special emphasis on RLK implicated in resistance to disease and development.     

Phosphoprotein and phosphopeptide interactions with the FHA domain from Arabidopsis kinase-associated protein phosphatase

FHA domains are phosphoThr recognition modules found in diverse signaling proteins, including kinase-associated protein phosphatase (KAPP) from Arabidopsis thaliana. The kinase-interacting FHA domain (KI-FHA) of KAPP targets it to function as a negative regulator of some receptor-like kinase (RLK) signaling pathways important in plant development and environmental responses. To aid in the identification of potential binding sites for the KI-FHA domain, we predicted (i) the structure of a representative KAPP-binding RLK, CLAVATA1, and (ii) the functional surfaces of RLK kinase domains using evolutionary trace analysis. We selected phosphopeptides from KAPP-binding Arabidopsis RLKs for in vitro studies of association with KI-FHA from KAPP. Three phosphoThr peptide fragments from the kinase domain of CLV1 or BAK1 were found to bind KI-FHA with KD values of 8-20 microM, by NMR or titration calorimetry. Their affinity is driven by favorable enthalpy and solvation entropy gain. Mutagenesis of these three threonine sites suggests Thr546 in the C-lobe of the BAK1 kinase domain to be a principal but not sole site of KI-FHA binding in vitro. The brassinosteroid receptor BRI1 and KAPP are shown to associate in vivo and in vitro. Further genetic studies indicate that KAPP may be a negative regulator of the BRI1 signaling transduction pathway. 15N-Labeled KI-FHA was titrated with the GST-BRI1 kinase domain and monitored by NMR. BRI1 interacts with the same 3/4, 4/5, 6/7, 8/9, and 10/11 recognition loops of KI-FHA, with similar affinity as the phosphoThr peptides.     

The RLK/Pelle family of kinases

The RLK/Pelle class of proteins kinases is composed of over 600 members in Arabidopsis. Many of the proteins in this family are receptor-like kinases (RLK), while others have lost their extracellular domains and are found as cytoplasmic kinases. Proteins in this family that are RLKs have a variety of extracellular domains that drive function in a large number of processes, from cell wall interactions to disease resistance to developmental control. This review will briefly cover the major subclasses of RLK/Pelle proteins and their roles. In addition, two specific groups on RLKs will be discussed in detail, relating recent findings in Arabidopsis and how well these conclusions have been able to be translated to agronomically important species. Finally, some details on kinase activity and signal transduction will be addressed, along with the mystery of RLK/Pelle members lacking kinase enzymatic activity.     

Molecular characterisation and regulation of a Nicotiana tabacum S-domain receptor-like kinase gene induced during an early rapid response to lipopolysaccharides

The isolation, characterization and regulation of the first lipopolysaccharide (LPS)-responsive S-domain receptor-like kinase (RLK) in Nicotiana tabacum are reported. The gene, corresponding to a differentially expressed LPS-responsive EST, was fully characterised to investigate its involvement in LPS-induced responses. The full genomic sequence, designated Nt-Sd-RLK, encodes for a S-domain RLK protein containing conserved modules (B-lectin-, S- and PAN-domains) reported to function in mediating protein-protein and protein-carbohydrate interactions in its extracellular domain, as well as the molecular architecture to transduce signals intracellularly through a Ser/Thr kinase domain. Phylogenetic analysis clustered Nt-Sd-RLK with S-domain RLKs induced by bacteria, wounding and salicylic acid. Perception of LPS induced a rapid, bi-phasic response in Nt-Sd-RLK expression with a 17-fold up-regulation at 3 and 9h. A defence-related W-box cis element was found in the promoter region of Nt-Sd-RLK and the transient induction of Nt-Sd-RLK in cultured cells by LPS exhibited a pattern typical of early response defence genes. Nt-Sd-RLK was also responsive to salicylic acid induction and was expressed in differentiated leaf tissue, where LPS elicited local as well as systemic up-regulation. The results contribute new knowledge about the potential role that S-domain RLKs may play within interactive signal transduction pathways associated with immunity and defence.     

Protein phosphatase 2C (PP2C) function in higher plants

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.     

Abiotic stress-inducible receptor-like kinases negatively control ABA signaling in Arabidopsis

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.     

大豆类受体蛋白激酶基因GmRLCK的克隆及初步分析

植物类受体蛋白激酶(receptor-likeproteinkinase,RLK)在高等植物生长发育和环境刺激的信号传导中起着重要的作用。本文报告了一个新的大豆类受体蛋白激酶基因的全长cDNA克隆及对其基因结构和功能的初步分析。研究表明该基因序列编码的蛋白包含一个跨膜域、一个具有丝氨酸/苏氨酸激酶活性的胞内域和一个缺少N-末端信号肽的胞外域。采用生物信息学方法分析表明,该基因与一些拟南芥菜类受体蛋白激酶基因具有很高的相似性,这些激酶N-末端都缺少信号序列,属于植物胞质类受体激酶(receptor-likecytoplasmickinase,RLCK)亚家族。因此命名该大豆基因为GmRLCK(GenBankAccessionNo.AY687390)。对GmRLCK激酶域中磷酸化可能性较高的位点进行了预测。RT-PCR的结果表明,GmRLCK在大豆子叶、根、花以及豆荚中都有较高的表达,而在胚根、茎和成熟叶片中的表达相对较弱。进化分析表明GmRLCK与一些衰老相关的植物类受体蛋白激酶具有较近的亲缘关系。     

Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis

To understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen- and salicylic acid (SA)-induced DNA-binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W-box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor-like protein kinases (RLK), each consisting of an N-terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C-terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W-box elements in its promoter region were recognized by both purified WRKY proteins and SA-induced W-box binding activities from SA-treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein-binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen- and SA-induced W-box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti-microbial activities, but also genes encoding proteins with regulatory functions.     

BIK1 cooperates with BAK1 to regulate constitutive immunity and cell death in Arabidopsiss

In Arabidopsis,both the membrane-anchored receptor-like kinase(RLK) BAK1 and the receptor-like cytoplasmic kinase(RLCK) BIK1 are important mediators of transmembrane signal transduction that regulate plant development and immunity.However,little attention has been paid to their genetic association.This study found the bak1 bik1 double mutant of Arabidopsis displayed a severe dwarfism phenotype due to constitutive immunity and cell death in developing plants.These data suggest that BIK1 cooperates with BAK1 to regulate constitutive immunity and cell death.     

Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system

The yeast two-hybrid system was used to further characterize the interactions between the Brassica S receptor kinase (SRK) and three putative substrates, ARC1 and the two thioredoxin h proteins, THL1 and THL2. Interactions were generally detectable with kinase domains of both Class I and Class II SRKs. Chimeric constructs were made between the SRK₉₁₀ kinase domain and the non-interacting Arabidopsis RLK5 kinase domain. Only one chimeric construct, SRR2, interacted with THL1 and THL2, while none of the chimeras were able to interact with ARC1. SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being derived from the SRK₉₁₀ kinase domain and was the only chimeric construct that retained kinase activity. Deletion or substitution of a conserved cysteine at the N-terminal end of the SRK₉₁₀ kinase domain resulted in loss of interaction with THL1 and THL2, while the addition of this cysteine to a related receptor kinase, SFR1, conferred the ability to interact with the thioredoxin h proteins. In addition, substitution of the cysteines in the THL1 active site abolished the interaction. Lastly, the two Arabidopsis thioredoxin h clones most closely related to THL1 and THL2 were found to interact with the SRK kinase domains. Thus, the nature of the interaction of the thioredoxin h clones with SRK involves the reducing activity of these proteins and is restricted to the class of thioredoxin h proteins which have the variant CPPC active site.     

The juxtamembrane domains of Arabidopsis CERK1, BAK1, and FLS2 play a conserved role in chitin‐induced signaling

Arabidopsis thaliana CERK1 is an essential receptor‐like kinase in the chitin signal transduction pathway. The juxtamembrane (JM) domain of CERK1 regulates the kinase activity of this receptor. Here we demonstrate that the JM domains of LysM‐RLKs, CERK1, and OsCERK1 play a functionally conserved role in the activation of chitin signaling in Arabidopsis. The C‐termini of the JM domains of both CERK1 and OsCERK1 are indispensable for their function. Moreover, after replacing the JM domain of CERK1 with that of the nonhomologous RLK, BAK1 (CJBa) or FLS2 (CJFl), the chimeric CERK1 receptors maintained their ability to activate chitin signaling in Arabidopsis. Interestingly, the heterologous expression of CJBa and CJFl did not induce cell death in Nicotiana benthamiana leaves. These results suggest that the JM domains of CERK1, BAK1, and FLS2 play a conserved role in chitin signaling via a mechanism not related to sequence homology.     

Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.     

A cytoplasmic coiled-coil domain is required for histidine kinase activity of the yeast osmosensor, SLN1

The yeast histidine kinase, Sln1p, is a plasma membrane-associated osmosensor that regulates the activity of the osmotic stress MAP kinase pathway. Changes in the osmotic environment of the cell influence the autokinase activity of the cytoplasmic kinase domain of Sln1p. Neither the nature of the stimulus, the mechanism by which the osmotic signal is transduced nor the manner in which the kinase is regulated is currently clear. We have identified several mutations located in the linker region of the Sln1 kinase (just upstream of the kinase domain) that cause hyperactivity of the Sln1 kinase. This region of histidine kinases is largely uncharacterized, but its location between the transmembrane domains and the cytoplasmic kinase domain suggests that it may have a potential role in signal transduction. In this study, we have investigated the Sln1 linker region in order to understand its function in signal transduction and regulation of Sln1 kinase activity. Our results indicate that the linker region forms a coiled-coil structure and suggest a mechanism by which alterations induced by osmotic stress influence kinase activity by altering the alignment of the phospho-accepting histidine with respect to the catalytic domain of the kinase.