Figure 1. Phosphorylation does not impact ABI5 turnover and interactions with KEG E3 ligase. (A) Schematic representation of ABI5 showing the conserved regions, C1 - C4, and basic leucine zipper (bZIP) domain. Lysine (K) residues linked to ubiquitination (K344, bold and italics) and sumoylation (K391, bold and underlined) are highlighted. Phosphorylation sites within each conserved region (C1-C4) are shown in bold. Phosphoamino acids (serine [S] and threonine [T]) that were changed to alanine (A) or asparagine (D) are indicated (larger font and numbered). (B) Cell-free protein degradation assays. Phosphorylation sites in all 4 conserved domains (ABI5^AAAA and ABI5^DDDD) of ABI5 were mutated to A or D. Recombinant Flag-His tagged ABI5, ABI5^AAAA and ABI5^DDDD were incubated with protein extracts prepared from wild type Arabidopsis seedlings. Samples were taken at the indicated times and the level of ABI5 proteins determined by western blotting using His antibodies. Coomassie staining was used to confirm equal loading. (C) Yeast-2-hybrid experiments showing interactions between KEG and a series of ABI5 phosphomutants. Phosphorylation sites in the C2 (ABI5^S145A and ABI5^S145D), C4 (ABI5^S439A and ABI5^S439D), or all 4 conserved domains (ABI5^AAAA and ABI5^DDDD) of ABI5 were mutated to A or D. ABI5 cDNA constructs were fused to the GAL4 binding domain (BD) and KEG cDNA encoding for the ankyrin repeats (KEG^Ankyrin) was fused to the GAL4 activating domain (AD). Interaction was analyzed by growth on selection medium without Trp, Leu, His, and Ade (SD/-T/-L/-H/-A). ABI5^DDDD produced a high level of autoactivation. The pGADT7 and pGBKT7-Rec empty vectors were used as negative controls. (D-E) BiFC analysis in tobacco epidermal cells. Fluorescence indicates interactions between KEG^AA-YN (RING mutant lacking E3 ligase activity) and ABI5^AAAA-YC (D) or ABI5^DDDD-YC (E). Bars = 100 μm.