Altered ubiquitination and stability of aquaporin-1 in hypertonic stress.
Aquaporin-1 (AQP1) water channel protein expression is increased by hypertonic 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 expression, suggesting basal proteasome-dependent degradation of the protein. Degradation by the proteasome is thought to be triggered by polyubiquitination of a target protein. To determine whether AQP1 is ubiquitinated, immunoprecipitation with anti-AQP1 antibodies was performed, and the resultant samples were probed by protein immunoblot for the presence of ubiquitin. Immunoblots 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 ubiquitination of AQP1, an effect that persisted through 24 h in hypertonic medium. Using metabolic labeling with [(35)S]methionine, the half-life of AQP1 protein under isotonic conditions was found to be <4 h. AQP1 protein half-life was markedly increased by exposure of cells to hypertonic medium. These observations provide evidence that aquaporins are a target for ubiquitination and proteasome-dependent degradation. Additionally, these studies demonstrate that reduced protein ubiquitination and increased protein stability lead to increased levels of AQP1 expression during hypertonic stress.
Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
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D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009097:Multienzyme Complexes / Q000187:drug effects; D009997:Osmotic Pressure; D046988:Proteasome Endopeptidase Complex; D014452:Ubiquitins / Q000378:metabolism*
C496021:Aqp1 protein, mouse; D020346:Aquaporins; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D014452:Ubiquitins; D051398:Aquaporin 1; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex