Altered ubiquitination and stability of aquaporin-1 in hypertonic stress

Aquaporin-1 (AQP1) water channel protein expression is increased by hyperto nic stress. The contribution of changes in protein stability to hypertonic induction of AQP1 have not been described. Incubation of BALB/c fibroblasts spontaneously expressing AQP1 with proteasome inhibitors increased AQP1 ex pression, suggesting basal proteasome-dependent degradation of the protein. Degradation by the proteasome is thought to be triggered by polyubiquitina tion of a target protein. To determine whether AQP1 is ubiquitinated, immun oprecipitation with anti-AQP1 antibodies was performed, and the resultant s amples were probed by protein immunoblot for the presence of ubiquitin. Imm unoblots demonstrated ubiquitination of AQP1 under control conditions that increased after treatment with proteasome inhibitors (MG132, lactacystin). Exposure of cells to hypertonic medium for as little as 4 h decreased ubiqu itination of AQP1, an eff ect that persisted through 24 h in hypertonic med ium. Using metabolic labeling with [S-35]methionine, the half-life of AQP1 protein under isotonic conditions was found to be <4 h. AQP1 protein half-l ife was markedly increased by exposure of cells to hypertonic medium. These observations provide evidence that aquaporins are a target for ubiquitinat ion and proteasome-dependent degradation. Additionally, these studies demon strate that reduced protein ubiquitination and increased protein stability lead to increased levels of AQP1 expression during hypertonic stress.