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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: Nat Cell Biol. 2011 Jan 23;13(2):124–131. doi: 10.1038/ncb2151

Figure 4. PP2A B’ subunit binds to the PEST domain of BZR1 to promote BZR1 dephosphorylation in vivo.

Figure 4

(a) Sequence of the region of BZR1 (aligned with the corresponding sequences of BZR2 and rice OsBZR1), containing the PP2A-binding domain and the bzr1-1D, bzs247 and bzs248 mutations (amino acid substitutions shown in boxes). (b) Yeast two-hybrid assay of PP2A binding by various fragments of BZR1 shown by box diagrams. Yeast growth on –AH medium indicates interaction between the BZR1 fragment and PP2AB’α. (c) Deletion of the PP2A-binding domain abolishes BR-induced BZR1 dephosphorylation in plants. Two-week old plants of a 35S::BZR1-YFP transgenic line expressing the wild type BZR1-YFP, or three independent transgenic lines expressing the mutant BZR1-YFP containing deletion of amino acids 232-251 (ΔPEST-A, -B, and -C lines), were treated with mock solution or 250 nM brassinolide (BL) for 1 hr. (d) The bzr1-1D mutation increases dephosphorylation of BZR1 in planta. Upper panel shows an immunoblot of BZR1/bzr1-1D protein extracted from wild type (BZR1) and bzr1-1D mutant plants. In each sample, the upper band is phosphorylated (pBZR1) and the lower band is unphosphorylated (BZR1) protein. Stain, Ponceau S staining shows equal loading. (e) PP2A binds more strongly to the bzr1-1D protein. MBP-BZR1 and MBP-bzr1-1D proteins were gel-blotted on a nitrocellulose membrane and probed sequentially with GST-PP2AB’α and anti-GST antibody. Lower panel shows Ponceau S staining of the gel blot. (f) From left to right are wild type, bzr1-1D, bzr1-1D bzs247 and bzr1-1D bzs248 seedlings grown in the dark on 2 μM BRZ for 5 days. Scale bar = 2.5 mm. (g) Anti-BZR1 immunoblot shows the phosphorylated BZR1 (pBZR1) and unphosphorylated BZR1 (BZR1) in the plants shown in panel f. (h) The bzs mutations reduce PP2A binding. Equal amounts of MBP, MBP-BZR1, MBP-bzr1-1D, and MBP-bzr1-1D containing bzs247 and bzs248 mutations were gel blotted to nitrocellulose membrane and probed with GST-PP2AB’α or GST-BIN2 and anti-GST antibody, and the blots were subsequently stained with Ponceau S (Stain). Full scans of immunoblots are shown in Supplementary Information, Fig. S9.