A simple in vitro method to measure autophosphorylation of protein kinases

Taylor, Isaiah; Seitz, Kati; Bennewitz, Stefan; Walker, John C.

doi:10.1186/1746-4811-9-22

Cited by 26 publications

(35 citation statements)

References 12 publications

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“…These residues are well conserved in the A. thaliana HAE, HSL1 and HSL2 receptors and their orthologs in other species (Supplementary Figure S2A ). This includes the residue corresponding to S861 of HAE, which is subject to autophosphorylation (Taylor et al, 2013 ), and several residues predicted to be targets of Ser/Thr kinases (e.g., by using PhosPhAt http://phosphat.uni-hohenheim.de/ ) (Durek et al, 2010 ). However, in loops between helices positions of putative phosphorylation sites differ in HSL2 receptors compared to HAE/HSL1.…”

Section: Resultsmentioning

confidence: 99%

Conservation of the abscission signaling peptide IDA during Angiosperm evolution: withstanding genome duplications and gain and loss of the receptors HAE/HSL2

et al. 2015

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The peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which signals through the leucine-rich repeat receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2), controls different cell separation events in Arabidopsis thaliana. We hypothesize the involvement of this signaling module in abscission processes in other plant species even though they may shed other organs than A. thaliana. As the first step toward testing this hypothesis from an evolutionarily perspective we have identified genes encoding putative orthologs of IDA and its receptors by BLAST searches of publically available protein, nucleotide and genome databases for angiosperms. Genes encoding IDA or IDA-LIKE (IDL) peptides and HSL proteins were found in all investigated species, which were selected as to represent each angiosperm order with available genomic sequences. The 12 amino acids representing the bioactive peptide in A. thaliana have virtually been unchanged throughout the evolution of the angiosperms; however, the number of IDL and HSL genes varies between different orders and species. The phylogenetic analyses suggest that IDA, HSL2, and the related HSL1 gene, were present in the species that gave rise to the angiosperms. HAE has arisen from HSL1 after a genome duplication that took place after the monocot—eudicots split. HSL1 has also independently been duplicated in the monocots, while HSL2 has been lost in gingers (Zingiberales) and grasses (Poales). IDA has been duplicated in eudicots to give rise to functionally divergent IDL peptides. We postulate that the high number of IDL homologs present in the core eudicots is a result of multiple whole genome duplications (WGD). We substantiate the involvement of IDA and HAE/HSL2 homologs in abscission by providing gene expression data of different organ separation events from various species.

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Section: Resultsmentioning

confidence: 99%

Conservation of the abscission signaling peptide IDA during Angiosperm evolution: withstanding genome duplications and gain and loss of the receptors HAE/HSL2

et al. 2015

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“…Pro‐Q diamond phosphoprotein gel stains and subsequent Sypro Ruby stains were performed according to the manufacturer’s recommendations (Invitrogen, Life Technologies, Carlsbad, CA, USA; Taylor et al. , ). TGX stain‐free gels were used for all other protein analyses (Bio‐Rad, #456‐8085), and total protein was visualized using the stain‐free method or with Coomassie Brilliant Blue (CBB).…”

Section: Methodsmentioning

confidence: 99%

Kinase activity of SOBIR1 and BAK1 is required for immune signalling

Burgh

Postma

Robatzek

et al. 2019

Molecular Plant Pathology

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Summary Leucine‐rich repeat‐receptor‐like proteins (LRR‐RLPs) and LRR‐receptor‐like kinases (LRR‐RLKs) trigger immune signalling to promote plant resistance against pathogens. LRR‐RLPs lack an intracellular kinase domain, and several of these receptors have been shown to constitutively interact with the LRR‐RLK Suppressor of BIR1‐1/EVERSHED (SOBIR1/EVR) to form signalling‐competent receptor complexes. Ligand perception by LRR‐RLPs initiates recruitment of the co‐receptor BRI1‐Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) to the LRR‐RLP/SOBIR1 complex, thereby activating LRR‐RLP‐mediated immunity. We employed phosphorylation analysis of in planta ‐produced proteins, live cell imaging, gene silencing and co‐immunoprecipitation to investigate the roles of SOBIR1 and BAK1 in immune signalling. We show that Arabidopsis thaliana ( At ) SOBIR1, which constitutively activates immune responses when overexpressed in planta , is highly phosphorylated . Moreover, in addition to the kinase activity of SOBIR1 itself, kinase‐active BAK1 is essential for At SOBIR1‐induced constitutive immunity and for the phosphorylation of At SOBIR1. Furthermore, the defence response triggered by the tomato LRR‐RLP Cf‐4 on perception of Avr4 from the extracellular pathogenic fungus Cladosporium fulvum is dependent on kinase‐active BAK1. We argue that, in addition to the trans‐autophosphorylation of SOBIR1, it is likely that SOBIR1 and BAK1 transphosphorylate, and thereby activate the receptor complex. The signalling‐competent cell surface receptor complex subsequently activates downstream cytoplasmic signalling partners to initiate RLP‐mediated immunity.

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“…The cell lysates were boiled and centrifuged. The supernatant was separated by 10% SDS-PAGE and stained sequentially with Pro-Q Diamond (Invitrogen) and Coomassie Blue Silver as described previously (Taylor et al, 2013).…”

Section: Membrane Protein Isolation Immunoprecipitation Western Blomentioning

confidence: 99%

Scanning for New BRI1 Mutations via TILLING Analysis

et al. 2017

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The identification and characterization of a mutational spectrum for a specific protein can help to elucidate its detailed cellular functions. BRASSINOSTEROID INSENSITIVE1 (BRI1), a multidomain transmembrane receptor-like kinase, is a major receptor of brassinosteroids in Arabidopsis (). Within the last two decades, over 20 different mutant alleles have been identified, which helped to determine the significance of each domain within BRI1. To further understand the molecular mechanisms of BRI1, we tried to identify additional alleles via targeted induced local lesions in genomes. Here, we report our identification of 83 new point mutations in, including nine mutations that exhibit an allelic series of typical phenotypes, from subtle to severe morphological alterations. We carried out biochemicalanalyses to investigate possible mechanisms of these mutations in affecting brassinosteroid signaling. A number of interesting mutations have been isolated via this study. For example, , the only weak allele identified so far with a mutation in the activation loop, showed reduced autophosphorylation activity., a subtle allele with a mutation in the extracellular portion, disrupts the interaction of BRI1 with its ligand brassinolide and coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1. , with a mutation in the extracellular portion, is a subtle defective mutant. Surprisingly, root inhibition analysis indicated that it is largely insensitive to exogenous brassinolide treatment. In this study, we found that possesses kinase activity in vivo, clarifying a previous report arguing that kinase activity may not be necessary for the function of BRI1. These data provide additional insights into our understanding of the early events in the brassinosteroid signaling pathway.

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A simple in vitro method to measure autophosphorylation of protein kinases

Cited by 26 publications

References 12 publications

Conservation of the abscission signaling peptide IDA during Angiosperm evolution: withstanding genome duplications and gain and loss of the receptors HAE/HSL2

Conservation of the abscission signaling peptide IDA during Angiosperm evolution: withstanding genome duplications and gain and loss of the receptors HAE/HSL2

Kinase activity of SOBIR1 and BAK1 is required for immune signalling

Scanning for New BRI1 Mutations via TILLING Analysis

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