RESUMO
A novel plant protein kinase, designated Brassica napus kinase 1 (BNK1), was isolated from a lambda-pistil cDNA library. The deduced BNK1 protein contains all eleven conserved subdomains of a kinase and encodes a functional serine/threonine protein kinase. Phylogenetic analysis of several plant protein kinase subfamilies showed that BNK1 is most closely related to the NAK subfamily of protein kinases. Genomic Southern blot analysis revealed that BNK1 is a single copy gene in the B. napus genome and does not appear to be a member of a multigene family. Expression studies revealed that the BNK1 transcript was ubiquitously expressed throughout the plant, with highest levels in stem and pistil tissues.
RESUMO
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.
Assuntos
Brassica/enzimologia , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Cisteína/genética , Cisteína/metabolismo , DNA Recombinante , Óperon Lac/genética , Dados de Sequência Molecular , Mutagênese , Mutação , Proteínas de Plantas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Tiorredoxinas/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast two-hybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.
Assuntos
Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Fosforilação , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
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.
