Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics

Abstract

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

Figure 1

Biotinylation of BZR1 by BIN2-TurboID and PP2AB′α-TurboID. A, Schematic diagram of proximity-dependent biotin labeling. B, Biotinylation of BZR1 by proximity labeling. The indicated TurboID (TbID) fusion proteins were co-expressed with BZR1-Myc in N. benthamiana leaves. Streptavidin pulldown and anti-GFP IP were performed using aliquots of the same protein extracts, and proteins were immunoblotted using antibodies shown on the right side. Ponceau S staining shows the loading of the input. Streptavidin pulled down 10.4-fold more BZR1-Myc than anti-GFP IP. Asterisks indicate phosphorylated BZR1. C, Comparison of BZR1 biotinylation by BIN2-TbID and AtSK41-TbID. D, Comparison of BZR1 biotinylation by PP2AB′α-TbID and PP2AB′ɛ-TbID.

Figure 2

The effect of biotin concentration on the efficiency of biotin labeling and affinity purification in Arabidopsis. A, Phenotypes of the wild-type (Col-0) and transgenic Arabidopsis overexpressing BIN2-YFP-TbID. Plants were grown for 4 weeks in soil. The scale bar is 1 cm. B, The effect of biotin concentration on TbID-mediated biotinylation. 14-d-old BIN2-YFP-TbID seedlings were treated with the indicated concentrations of biotin for 1 h. Total proteins were immunoblotted using streptavidin-HRP (SA-HRP). C, The effect of biotin concentration on biotinylation and streptavidin pulldown of BZR1. D, The effect of desalting on affinity purification of biotinylated proteins. BIN2-YFP-TbID seedlings were treated with 50 mM biotin for 3 h. Equal aliquots of protein extracts were desalted (+) or not desalted (−) before streptavidin pulldown. Input (1:300), flow through (FT, 1:300) and eluate after affinity pulldown (AP, 1:10) were separated by SDS-PAGE. Biotinylated proteins were probed with SA-HRP.

Figure 3

TurboID-based identification of BIN2-proximal proteins in Arabidopsis. A, Schematic diagram of the workflow of isotope labeling, proximity biotinylation (B) while BIN2 phosphorylates (P) its substrates, purification, fractionation by reverse phase chromatography (RP), and analysis on mass spectrometer (QE-HF) of BIN2-proximal proteins. B, Signal ratios between BIN2-YFP-TbID and YFP-YFP-TbID for proteins detected in two replicate experiments where isotopes were switched. Blue colored letter indicates previously reported BIN2 interactors. The dashed lines show a 3-fold cutoff ratio. C, Representative MS1 spectra show the enrichment of NPH3, PIN3, HERK1, PHOT1, and no enrichment of MCCA, by BIN2-YFP-TbID relative to the YFP-YFP-TbID control. Top panel: ¹⁴N: BIN2-YFP-TbID, ¹⁵N: YFP-YFP-TbID; Bottom panel: ¹⁴N: YFP-YFP-TbID, ¹⁵N: BIN2-YFP-TbID. Red and blue arrows point to the monoisotopic peaks of BIN2-YFP-TbID samples and YFP-YFP-TbID control, respectively.

Figure 4

Identification of bikinin-responsive phosphoproteins. A, Schematic diagram of phosphoproteomic analysis of bikinin responses using stable isotope labeling mass spectrometry. The seedlings were treated with mock or bikinin (30 µM) for 1 h. P and IMAC indicate phosphate and immobilized metal affinity chromatography, respectively. B, Representative MS1 spectra show the signals of phosphopeptides of BZR1 and BES1 in bikinin- (red arrow) and mock-treated (black arrow) samples. Top and bottom panels are repeats experiments with isotope reversed. C and D, Phosphorylation motifs enriched in the phosphopeptides decreased (C) or increased (D) by bikinin treatment.

Figure 5

Validation of BIN2 phosphorylation of putative substrate proteins. A, In vitro kinase assays of GST-BIN2 and MBP-fused substrates to test BIN2 phosphorylation of the BIN2 substrates. MBP-fused protein of At5g18610 (kinase-inactive form; K112R), At1g30320, At3g20250, At5g11710, At5g03040, At3g45780 (kinase-inactive form; D806N), At5g64330, At1g13020, At5g18230, At5g05970, At4g39680, or At1g16860 (N-terminal partial protein; 1-209 aa) was incubated with GST-BIN2 in a kinase buffer containing ³²P-γ-ATP. MBP-YFP and MBP-fused At3g09840 were used as negative controls. CBB, Coomassie Brilliant Blue. B, Inhibition of BIN2 phosphorylation by bikinin. Four MBP-fused proteins were incubated with GST-BIN2 in a kinase assay buffer without or with 15 μM bikinin. Single and double asterisks indicate the auto-phosphorylated GST-BIN2 and the substrate proteins phosphorylated by GST-BIN2, respectively. C, Venn diagram shows overlaps among BIN2-proximal proteins (BIN2-TurboID), bikinin-repressed, and BR-responsive phosphoproteins (Clark et al., 2021).

Figure 6

The BIN2 signaling network. A, The diagram of the BIN2 signaling network shows BIN2 substrates and their interactors. The filled nodes (circles and squares) represent BIN2 substrates identified as BIN2-proximal proteins that showed dephosphorylation upon bikinin treatment. The rectangle nodes indicate O-GlcNAcylated proteins. Clusters with specific biological functions are highlighted by different colors of edges. ER, Endoplasmic Reticulum, BR, Brassinosteroid, TGN, Trans-Golgi Network. B, Venn diagram shows the overlaps among the bikinin-sensitive phosphoproteins, BIN2-proximal phosphoproteins (BIN2-TbID), and O-GlcNAcylated proteins (Xu et al., 2017) in Arabidopsis. C, Venn diagram shows the overlaps among the phosphoproteins and O-GlcNAcylated proteins in synapse and GSK3 substrates in the hippocampus of mouse (Trinidad et al., 2012; Kaasik et al., 2013).