Inhibition of TonEBP by SUMO (small ubiquitin‐like modifers) modification

Abstract

TonEBP is a transcriptional stimulator regulated by ambient tonicity. TonEBP is a key regulator for the development of the renal medulla and its function such as the urinary concentration and the protection of cells from the deleterious effects of high salt and urea. Here we report that TonEBP is post-translationally modified by SUMO. When HEK293 cells are cultured in hypertonic medium, we observe two slower bands with 20 and 40 kDa increments in molecular weight. The slow bands are reproduced with overexpressed TonEBP, which is further enhanced when SUMO and ubc9, the SUMO conjugating enzyme E2, are coexpressed. SUMO3 is most efficient and SUMO1 is least efficient in the modification. Analyses of site-directed TonEBP mutants reveal that K556 and K603 are SUMO modified in response to hypertonicity. Functional analyses using expression of a TonE-driven luciferase gene demonstrate that the SUMO modification inhibits TonEBP. A non-SUMO-modification mutant K556R/K603R is twice as active as the wild type (WT). Single mutants K556R and K603R show an intermediate activity. On the other hand, a SUMO3-TonEBP fusion mimicking constitutive SUMO modification is inactive. A hybrid molecule consisting of the SUMO3 fusion and the K556R/K603R mutation displays near normal activity. These data suggest that the stoichiometry of SMUO modification is a rheostat for the modulation of TonEBP in hypertonicity.