VH1/BRL2 receptor-like kinase interacts with vascular-specific adaptor proteins VIT and VIK to influence leaf venation

VH1/BRL2 is a receptor-like kinase of the BRI1 family with a role in vascular development. In developing Arabidopsis leaves it is expressed first in ground cells and then becomes restricted to provascular and procambial cells as venation forms. We isolated proteins interacting with the activated (phosphorylated) cytoplasmic domain of VH1/BRL2, and found that most belong to three processes: proteasome activity, vesicle traffic and intracellular signal transduction. Two adaptor proteins are included that we named VIT [VH1-interacting tetratricopeptide repeat (TPR)-containing protein] and VIK (VH1-interacting kinase), which are co-expressed in the same cells as VH1/BRL2 at two distinct time points in vein differentiation. Mutation of either adaptor or of VH1 results in vein pattern defects and in alterations in response to auxin and brassinosteroids. We propose that these two adaptors facilitate the diversification and amplification of a ligand signal perceived by VH1/BRL2 in multiple downstream pathways affecting venation.     

Perception of the novel MAMP eMax from different Xanthomonas species requires the Arabidopsis receptor-like protein ReMAX and the receptor kinase SOBIR

As part of their innate immune system plants carry a number of pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). In a recently published article¹ we described a novel, proteinaceous MAMP termed eMax (enigmatic MAMP of Xanthomonas) that derives from Xanthomonas and gets recognized by the receptor-like protein ReMAX (RECEPTOR OF eMax) of Arabidopsis thaliana. ReMAX has no ortholog in Nicotiana benthamiana and this species does not respond to eMax even when transformed with ReMAX. However, interfamily transfer of eMax perception was successful with a chimeric form of ReMAX where the C-terminal part of the protein was replaced by the corresponding part of the tomato RLP EIX2 (ETHYLENE INDUCING XYLANASE2). In this addendum we describe the difficulties with the purification and identification of the MAMP eMax and we present data demonstrating that functionality of ReMAX, much like that of related RLPs, depends on the presence of the receptor kinase SOBIR (SUPPRESSOR OF BIR1–1).     

A novel coiled-coil protein co-localizes and interacts with a calcium-dependent protein kinase in the common ice plant during low-humidity stress

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is induced transiently by salinity and water deficit stress and also McCPK1 undergoes dynamic subcellular localization changes in response to these same stresses. Here we have confirmed that low humidity is capable of causing a drastic change in McCPK1’s subcellular localization. We attempted to elucidate this phenomenon by isolating components likely to be involved in this process. McCAP1 (M. crystallinum CDPK adapter protein 1) was cloned in a yeast two-hybrid screen with a constitutively active McCPK1 as bait. We show that McCPK1 and McCAP1 can interact in the yeast two-hybrid system, in vitro, and in vivo as demonstrated by coimmunoprecipitation experiments from plant extracts. However, McCAP1 does not appear to be a substrate for McCPK1. DsRed–McCAP1 and EGFP–McCPK1 fusions colocalize in epidermal cells of ice plants exposed to low humidity. McCAP1 is homologous to a family of proteins in Arabidopsis with no known function. Computational threading analysis suggests that McCAP1 is likely to be an intermediate filament protein of the cytoskeleton.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

A high-throughput screen of cell-death-inducing factors in Nicotiana benthamiana identifies a novel MAPKK that mediates INF1-induced cell death signaling and non-host resistance to Pseudomonas cichorii

A high-throughput overexpression screen of Nicotiana benthamiana cDNAs identified a gene for a mitogen-activated protein kinase kinase (MAPKK) as a potent inducer of the hypersensitive response (HR)-like cell death. NbMKK1 protein is localized to the nucleus, and the N-terminal putative MAPK docking site of NbMKK1 is required for its function as a cell-death inducer. NbMKK1-mediated leaf-cell death was compromised in leaves where NbSIPK expression was silenced by virus-induced gene silencing. A yeast two-hybrid assay showed that NbMKK1 and NbSIPK physically interact, suggesting that NbSIPK is one of the downstream targets of NbMKK1. Phytophthora infestans INF1 elicitor-mediated HR was delayed in NbMKK1-silenced plants, indicating that NbMKK1 is involved in this HR pathway. Furthermore, the resistance of N. benthamiana to a non-host pathogen Pseudomonas cichorii was compromised in NbMKK1-silenced plants. These results demonstrate that MAPK cascades involving NbMKK1 control non-host resistance including HR cell death.     

Hrip1, a novel protein elicitor from necrotrophic fungus,Alternaria tenuissima,elicits cell death,expression of defence‐related genes and systemic acquired resistance in tobacco

Here, we report the identification, purification, characterization and gene cloning of a novel hypersensitive response inducing protein secreted by necrotrophic fungus, Alternaria tenuissima, designated as hypersensitive response inducing protein 1 (Hrip1). The protein caused the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis‐related events including DNA laddering. The protein‐encoding gene was cloned by rapid amplification of cDNA ends (RACE) method. The sequence analysis revealed that the cDNA is 495 bp in length and the open reading frame (ORF) encodes for a polypeptide of 163 amino acids with theoretical pI of 5.50 and molecular weight of 17 562.5 Da. Hrip1 induced calcium influx, medium alkalinization, activation of salicylic acid‐induced protein kinase and several defence‐related genes after infiltration in tobacco leaves. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of defence‐related genes was activated. After several days, systemic acquired resistance was also induced. The tobacco plant cells that perceived the Hrip1 generated a cascade of signals acting at local, short, and long distances, and caused the coordinated expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. Thus, Hrip1 represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance in necrotrophic fungi.     

Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3)

Threonine(668) (thr(668)) within the carboxy-terminus of the Alzheimer's disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr(668) is believed to regulate APP function and metabolism. Thr(668) precedes a proline, which suggests that it is targeted for phosphorylation by proline-directed kinase(s). We have investigated the ability of four major neuronally active proline-directed kinases, cyclin dependent protein kinase-5, glycogen synthase kinase-3 beta, p42 mitogen-activated protein kinase and stress-activated protein kinase-1b, to phosphorylate APPthr(668) and report here that SAPK1b induces robust phosphorylation of this site both in vitro and in vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.     

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

Isolation and characterization of a novel v-SNARE family protein that interacts with a calcium-dependent protein kinase from the common ice plant, <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis>

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydration-stress responsive. The enzyme also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system, we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2 in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin superfamily that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTI1a, an Arabidopsis v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma membrane along actin microfilaments of the cytoskeleton. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.     

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">OsBISERK1</Emphasis>, a gene encoding a leucine-rich repeat receptor-like kinase,during disease resistance responses in rice

A rice gene, OsBISERK1, encoding a protein belonging to SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) type of leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. The OsBISERK1 encodes a 624 aa protein with high level of identity to known plant SERKs. OsBISERK1 contains a hydrophobic signal peptide, a leucine zipper, and five leucine-rich repeat motifs in the extracellular domain; the cytoplasmic region carries a proline-rich region and a single transmembrane domain, as well as a conserved intracellular serine/threonine protein kinase domain. OsBISERK1 has a low level of basal expression in leaf tissue. However, expression of OsBISERK1 was induced by treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice, and also up-regulated after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during incompatible interaction between a blast-resistant rice genotype and M. grisea. The results suggest that OsBISERK1 may be involved in disease resistance responses in rice.     

Amyloid β protein activates PKC-δ and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

The increased accumulation of activated microglia containing amyloid β protein (Aβ) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Aβ. We investigated intracellular signaling in response to Aβ stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-δ but not that of PKC- or - is increased by stimulation of microglia with Aβ, with a striking tyrosine phosphorylation of PKC-δ. In microglia stimulated with Aβ, tyrosine phosphorylation of PKC-δ was evident at the membrane fraction without an overt translocation of PKC-δ. PKC-δ co-immunoprecipitated with MARCKS from microglia stimulated with Aβ. Aβ induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Aβ. Taken together with our previous observations that Aβ-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Aβ induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-δ signaling pathway in microglia.