Fig. 5: HY5 can regulate the kinase activity of BIN2 independently of its transcriptional activity.
From: Modulation of BIN2 kinase activity by HY5 controls hypocotyl elongation in the light
a The attenuated interaction between BIN2 and mutated HY5 (HY5-M) in yeast. WT wild type. b In vitro pull-down assay showing attenuated interaction between BIN2 and HY5-M. c LCI assay showing attenuated interaction between BIN2 and HY5-M in N. tabacum leaves. The values measuring the interaction between HY5 and BIN2 were obtained by subtracting the interaction between HY5 and the cLUC empty vector. Error bar represents SD, n = 3. Asterisks indicate the P value of two-tailed Student’s t-test; ***P < 0.001. Experiments were performed three times with similar results. d Yeast one-hybrid assay showing HY5-M could bind to the EXP2 promoter in yeast similarly to HY5-WT. HY5 was fused with the activation domain (AD). The EXP2 promoter was inserted upstream of the LacZ reporter gene. e Schematic representation of various constructs used in the transient transfection assay in Arabidopsis protoplasts. LUC, firefly luciferase. REN, renilla luciferase. f Relative LUC expression level driven by EXP2pro in Arabidopsis protoplasts. The gene expression levels were quantified as the ratios of LUC/REN enzyme activities. Error bar represents SD of three independent transient transfections in protoplasts. n.s., not significant in two-tailed Student’s t-test. Experiments were performed three times with similar results. g In vitro kinase assay showing HY5-M could not enhance BIN2 activity. Control, no recombinant HY5 was added to the reaction. h The abundance of the dephosphorylated form of BZR1 in HA-HY5-M lines. Numbers below the bands indicated the relative band intensities of dBZR1. pBZR1, phosphorylation form of BZR1; dBZR1, dephosphorylation form of BZR1. HA-HY5 or HA-HY5-M were overexpressed in the hy5 mutant background. HSP was used as a loading control. i Hypocotyl lengths of HA-HY5/hy5 and HA-HY5-M/hy5, respectively, in the light. Seedlings were grown for 6 days in the light. Error bars represent SD, n ≥ 20. Asterisks indicate the P value of two-tailed Student’s t-test; ***P < 0.001. Experiments were performed three times with similar results. Source data are provided as a source data file.
