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 time-of-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 (BIN2^E264K)-HA, and BIN2KD (BIN2^K69R)-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.

糖原合酶激酶3（GSK3）样激酶BRASINOSTEROID-INSENSITIVE2（BIN2）是油菜素类固醇（BRs）信号传导中的主要负调节剂，通过与新型底物相互作用而参与多种植物信号传导途径，并在细胞，生长和发育调节。尽管BIN2的功能性研究包括BR信号传导相关的蛋白酶体介导的BIN2降解，但调节BIN2降解的分子调控机制及其相关蛋白质成分尚不完全清楚。这项研究旨在：i）从BL处理的BIN2-HA和bin2-6D-HA系的HA免疫沉淀（IP）中识别BIN2蛋白及其相互作用的伴侣，使用液相色谱串联质谱（LC-MS / MS）和基质辅助激光解吸/电离飞行时间/飞行时间质谱（MALDI-TOF / TOF MS），并且ii）表征BIN2与相互作用的伴侣蛋白。我们生成了组成型表达BIN2-HA，bin2-6D（BIN2^E264K）-HA和BIN2KD（BIN2 ^K69R）-HA构建体。IP标记HA标签的bin2-6D / BIN2蛋白，随后进行质谱分析，鉴定出F-box蛋白，含有LRR和FBD结构域的BR ASSINOSTEROID F- BOX 1（BRF1）和BRF2是与BIN2相互作用的蛋白。通过酵母双杂交分析的体外验证进一步证实了相互作用的蛋白质。这些结果，再加上BRF1和BRF2同源的系统发育和序列比对，采用多种方法，表明f-box蛋白BRF1和BRF2在调节BIN2中起冗余或重叠的作用。BIN2蛋白的稳定性可能受BRF1和BRF2的控制。