Putative E3 ligases as candidates controlling BRASSINOSTEROID INSENSITIVE 2 (BIN2) kinase in Arabidopsis
- PDF / 1,279,311 Bytes
- 10 Pages / 595.276 x 790.866 pts Page_size
- 106 Downloads / 191 Views
Plant Biotechnology Reports https://doi.org/10.1007/s11816-020-00646-1
ORIGINAL ARTICLE
Putative E3 ligases as candidates controlling BRASSINOSTEROID INSENSITIVE 2 (BIN2) kinase in Arabidopsis Yu Jeong Jeong1 Received: 13 October 2020 / Revised: 1 November 2020 / Accepted: 4 November 2020 © Korean Society for Plant Biotechnology 2020
Abstract The Glycogen synthase kinase 3 (GSK3)-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2), a major negative regulator in the Brassinosteroids (BRs) signaling, is involved in a variety of plant signaling pathways by interacting with novel substrates and plays a major role in cellular, growth and developmental regulation. Despite BIN2 functional studies including BR signaling related proteasome-mediated BIN2 degradation, the molecular regulating mechanisms and it’s related the protein component for regulating BIN2 degradation has not been completely known. This study aimed to i) identify BIN2 protein and its interacting partner from HA-Immunoprecipitation (IP) of the BL-treated BIN2-HA and bin2-6D-HA lines, using liquid chromatography tandem mass spectrometry (LC–MS/MS) and matrix-assisted laser desorption/ionization timeof-flight/time-of-flight mass spectrometry (MALDI–TOF/TOF MS) and ii) characterize relationships between BIN2 and interacting partner proteins. We generated transgenic plants constitutively expressing BIN2-HA, bin2-6D ( BIN2E264K)-HA, and BIN2KD (BIN2K69R)-HA construct. IP of the HA-tagged bin2-6D/BIN2 protein followed by mass spectrometry identified F-box protein, BRASSINOSTEROID F-BOX 1 (BRF1) and BRF2 containing the LRR and FBD domain, as interacting proteins with BIN2. Validation in vitro by yeast two-hybrid analyses further confirmed the interacting protein. These results, together with phylogeny and sequence alignments of BRF1 and BRF2 homologs multiple methods, suggest that f-box protein BRF1 and BRF2 play redundant or overlapping roles in regulating BIN2. It is likely that the BIN2 protein stability is controlled by BRF1 and BRF2. Keywords GSK3-like kinase · E3 ligases · BRASSINOSTEROID F-BOX 1 · BRF1 · BRF2 · MALDI–TOF/TOF MS Abbreviations ASK ARABIDOPSIS SKP1-LIKE BES1 BRI1-EMS-SUPPRESSOR1 BIN2 BRASSINOSTEROID INSENSITIVE 2 BL Brassinolide BR Brassinosteroid BRF1 BRASSINOSTEROID F-BOX 1 BRF2 BRASSINOSTEROID F-BOX 2 BRI1 BRASSINOSTEROID INSENSITIVE 1 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11816-020-00646-1) contains supplementary material, which is available to authorized users. * Yu Jeong Jeong [email protected] 1
School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Korea
BRZ Brassinazole BSK1 BR-SIGNALING KINASE 1 BSU1 BRI1 SUPPRESSOR 1 BZR1 BRASSINAZOLE-RESISTANT1 COI1 CORONATINE INSENSITIVE 1 EBF1/2 EIN3 BINDINGF-BOX PROTEIN 1/2 EIN3 ETHYLENE-INSENSITIVE 3 GSK Glycogen synthase kinase 3/ SHAGGY-like kinase GSK3β Glycogen synthase kinase 3β HA Hemagglutinin IP Immunoprecipitation JAZ JASMONATEZIM DOMAIN LC–MS/
Data Loading...
