Interplay between phosphorylation and SUMOylation events determines CESTA protein fate in brassinosteroid signalling

Abstract

Brassinosteroids (BRs) are steroid hormones that are essential for plant growth. Responses to these hormones are mediated by transcription factors of the bri1-EMS suppressor 1/brassinazole resistant 1 subfamily, and BRs activate these factors by impairing their inhibitory phosphorylation by GSK3/shaggy-like kinases. Here we show that BRs induce nuclear compartmentalization of CESTA (CES), a basic helix-loop-helix transcription factor that regulates BR responses, and reveal that this process is regulated by CES SUMOylation. We demonstrate that CES contains an extended SUMOylation motif, and that SUMOylation of this motif is antagonized by phosphorylation to control CES subnuclear localization. Moreover, we provide evidence that phosphorylation regulates CES transcriptional activity and protein turnover by the proteasome. A coordinated modification model is proposed in which, in a BR-deficient situation, CES is phosphorylated to activate target gene transcription and enable further posttranslational modification that controls CES protein stability and nuclear dynamics.

Figure 1. K72 is a SUMOylation site that controls BR-induced CES nuclear body localization

(a) Fluorescence microscopic images of representative stomata of 14-day-old cpd × 35S:CES^wt-YFP/32 seedlings either untreated (upper panel) or treated with 1 μM BL for 2 h (lower panel). Scale bars: filter = 5 μm, magnified = 2 μm. (b) Structure of the CES protein with the SUMOylation motif underlined. Target lysine in red. (c) In vitro SUMOylation assays using the heterodimeric SUMO activating enzyme E1 and SUMO conjugating enzyme E2, SUM1 and SUM2 as modifiers, and assessing affinity purified, recombinant GST-CES^wt and GST-CES^K72R as substrate proteins. (d) Representative fluorescence microscopic images of stomata of 14-day-old plants of 35S:CES^wt-YFP (line 32, upper two rows) or 35S:CES^K72R-YFP (line 411, lower two rows) either untreated (upper row) or treated with 1 μM Brz for 24 h followed by 1 μM BL for 2 h (lower row).

Figure 2. Mutating serine 75 and 77 to alanine promotes SUMOylation and increases CES protein stability

(a) In vitro kinase assays with affinity purified recombinant CES^wt-GST or CES^S75A+S77A-GST protein, [γ-³²P]ATP and total plant extracts from arabidopsis flowers. (b) Confocal microscopic images showing CES^S75A+S77A-YFP localization in different tissues of two-week-old plants. Scale bars: hypocotyls: filter = 40 μm, magnified = 5 μm;: leave vasculature: filter = 40 μm, magnified = 5 μm; stomata: filter = 2 μm, magnified = 5 μm. (c) Immunoblotting of protein extracts of two-week old seedlings of the indicated lines using α-GFP antibody for CES-YFP detection. Upper panel: autoradiogram, lower panel: coomassie brilliant blue staining (CBB) as a loading control. (d) Detection of SUMOylated CES in vivo. CES was immuno-precipitated from arabidopsis protoplasts transiently expressing 35S:CES^S75A&S77A-Myc₍₆₎ (please note that CES tagged with Myc₍₆₎ is smaller as compared to CES tagged with YFP) using α-Myc antibody and detected by immunoblotting with an α-SUMO antibody.

Figure 3. T35 is a BIN2 target site that controls CES protein stability

(a) In vitro kinase assays using recombinant BIN2-GST and different GST-tagged CES variants. qM (quadruple mutant). CBB staining is shown as a loading control. (b) Representative fluorescence microscopic images of stomata of 14-day-old plants of 35S:CES^T35A-YFP (line 2, upper two rows) or 35S:CES^T35E-YFP (line 19, lower two rows) either untreated (upper rows) or treated for 24 h with 1 μM Brz followed by 2 h of 1 μM BL (lower rows). Scale bars: filter = 5 μm, magnified = 2 μm. (c) Immunoblotting of protein extracts of two-week old seedlings of the indicated lines using α-GFP antibody for CES-YFP detection. Upper panel: autoradiogram, lower panel: coomassie brilliant blue staining (CBB) as a loading control. (d) Immunoblotting of protein extracts of 7-day-old seedlings of 35S:CES^T35E-YFP/16 and 35S:CES^wt-YFP/32 in the presence or absence of MG132 (3 h treatment) using α-GFP antibody for CES-YFP detection. Bands were quantified with the image-quant software and the values are given. CBB staining is shown as a loading control.

Figure 4. CDPKs phosphorylate CES

(a) Sequence of the extended SUMOylation motif. The simple 1 (basic-XX-S/T) and simple 2 (S-X-R) CDPK recognition sites are highlighted. K72 in red, S75 and S77 in blue. (b) Fluorescence microscopic images of hypocotyls of 14-days-old seedlings of 35S:CES^wt-YFP/32 (upper panel) or 35S:CES^S75E+S77E-YFP/632 (lower panel), untreated or treated with 300 μM triflouperazine (TFP) for 1.5 h. Scale bars: CES^wt-YFP: filter = 20 μm, magnified = 5 μm; CES^S75E+S77E-YFP: filter = 20 μm, magnified = 5 μm. (c) In vitro kinase assays using recombinant, GST-tagged CES and CPK3, CPK4, CPK5, CPK6 and CPK11. CBB staining is shown as a loading control. (d) In vitro kinase assays with CPK3 using recombinant, GST-tagged CES mutant variants. CBB staining is shown as a loading control.

Figure 5. Phosphorylation regulates CES activity

(a) The LUC reporter construct shown was transiently expressed in protoplasts generated from haf bee1 bee3 plant either alone (0) or with the effectors CES, CES^S75A+S77A, CES^S75E+S77E, CES^K72R, CES^T35A or CES^T35E. The y-axis is fold increase in the ratio between the activities of firefly LUC (under control of the CYP718 promoter) and 35S promoter driven control Renilla LUC (Ren-LUC). The SD of three independent biological replicates is shown. (b) ChIP experiments with different plants expressing wild-type or mutant CES YFP reporter lines using an α-GFP antibody for CES-YFP precipitation. The enrichment of a G-box containing fragment of the CPD promoter in the antibody containing fraction (as compared to the control without antibody) was quantified by real-time PCR from immuno-precipitated samples. The SD of three biological replicates is shown.