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Putative E3 ligases as candidates controlling BRASSINOSTEROID INSENSITIVE 2 (BIN2) kinase in Arabidopsis

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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 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 (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.

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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

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/MS:

Liquid chromatography tandem mass spectrometry

LRR:

Leucine-rich repeat

MALDI–TOF/TOF MS:

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry

MAX2:

MORE AXILLARY GROWTH 2

MS:

Murashige and Skoog

MyRF:

Myelin regulatory factor

PP2A:

Protein phosphatase 2A

PSA:

Pairwise sequence alignment

SCF:

Skp1/Cullin1/F-box protein

TIR1:

TRANSPORT INHIBITOR RESPONSE 1

TLR:

Toll-like receptor

TRAF6:

Tumor necrosis factor receptor-associated factor 6

β-TrCP:

β-Transducin repeat-containing protein

YFP:

Yellow fluorescent protein

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Acknowledgments

We would like to thank MS. Lee of the National Center for Inter-university Research Facilities (NCIRF) for assistance with the LC-MS experiments. This work was done with the financial assistance of Pro. Sunghwa Choe and grants from the National Research Foundation of Korea (NRF; Grant No. 2013R1A1A2059445 to Y.J.J).

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  1. School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, 08826, Korea

    Yu Jeong Jeong

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11816_2020_646_MOESM1_ESM.jpg

Supplementary file1 Fig S1. Identification of the BIN2 protein determined by LC-MS/MS analysis.a. Mass spectrum obtained from a doubly charged ion with monoisotopic m/z=538.8+2 (corresponding to LLQYSPSLR), m/z=617.3+2 (corresponding to MPPEAIDFASR), m/z=679.3+2 (corresponding to QEVAGSSPELVNK), m/z=617.3+2 (corresponding to VLGTPTREEIR), m/z=434.9+2 (corresponding to VLKHYSSANQR), and m/z=705.4+2 (corresponding to VVGTGSFGIVFQAK), present in protein band 1 in Fig. 2a. b. Peptide identification by LC-MS/MS analysis. * m/z: mass to charge ratio; MC #: miscleaved trypsin; RT: retention time on reverse phase C18 column; z: multiple charge number. c. The amino acid sequences of the identified Arabidopsis BIN2 protein. The peptides determined by MS/MS analysis are shown in red. (JPG 309 KB)

11816_2020_646_MOESM2_ESM.xlsx

Supplementary file2 Table S1. Pairwise alignment score list of the entire amino acid and nucleotide sequences of the Arabidopsis BRF1 and BRF2 homologs, determined using BLAST.(XLSX 33 KB)

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Jeong, Y.J. Putative E3 ligases as candidates controlling BRASSINOSTEROID INSENSITIVE 2 (BIN2) kinase in Arabidopsis. Plant Biotechnol Rep 14, 703–712 (2020). https://doi.org/10.1007/s11816-020-00646-1

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