Altered ubiquitination and stability of aquaporin-1 in hypertonic stress

Abstract

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 BALB͞c 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.A quaporin-1 (AQP1) is a water channel protein expressed in many tissues, whose regulation is known to be complex (1). Age-and tissue-specific patterns of AQP1 expression have been shown for kidney, lung, brain, and eye (2, 3). Corticosteroids induce AQP1 in rat lung in vivo (3) and an erythroleukemia cell line in vitro (4); the AQP1 proximal promoter contains classical glucorticoid response elements to which activity has been localized. AQP1 expression is induced by hypertonic stress in kidney cells lines (5, 6), as well as in BALB͞c fibroblasts in culture and in rat in vivo (L.K., unpublished observations). A potential role for posttranslational events in the regulation of AQP1 expression has not been described.It is increasingly apparent that posttranslational events play an active role in determining the level of protein expression in a cell. Two major protein degradative pathways are known to operate in mammalian cells, the 26S proteasome (7), and cysteine proteases, including cytoplasmic calcium-dependent calpains (8) and lysosomal acidic cathepsins (9). The 76-aa protein ubiquitin plays a key role in proteasome-mediated protein degradation, because polyubiquitination of a target protein is a necessary signal for processing by the proteasome (7). A limited number of membrane transport proteins have been demonstrated to be ubiquitinated (10). Ubiquitination has not been shown for any member of the aquaporin family of proteins.The studies reported here examine the role of ubiquitination and protein degradation in hypertonic induction of AQP1 e...

Short-Term Functional Adaptation of Aquaporin-1 Surface Expression in the Proximal Tubule, a Component of Glomerulotubular Balance

“…40 AQP1 availability also has elsewhere been shown to increase on the inhibition of its proteasomal degradation. 41 However, in the same context, hypertonic stress was shown to reduce ubiquitination of AQP1, leading to increased half-life of AQP1; like in our results, ubiquitin bands ranged between 50 and 60 kDa. 41 The regulatory role of varying protein stability has also been shown for AQP2.…”

“…41 However, in the same context, hypertonic stress was shown to reduce ubiquitination of AQP1, leading to increased half-life of AQP1; like in our results, ubiquitin bands ranged between 50 and 60 kDa. 41 The regulatory role of varying protein stability has also been shown for AQP2. 42 Altered ubiquitination, as observed in our setting of stimulated AQP1-OKCs, could have contributed as well to part of the changes seen in the microperfused tubules.…”

Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog

“…Indeed, treatment of spi2a-transfected RAW264.7 cells with proteasome inhibitors had no effect on the accumulation of spi2a-ISG15 conjugates (J. A. Hamerman, L. A. Schroeder and A. Aderem, unpublished observations), whereas this accumulates ubiquitin-protein conjugates (47,48). These conditions have also been shown to have no effect on the half-life of total ISG15 conjugates in a lung carcinoma cell line while producing significant effects on total protein degradation (J. Narasimhan and A. Haas, unpublished observations).…”

Role of proneuregulin 1 cleavage and human epidermal growth factor receptor activation in hypertonic aquaporin induction

“…Reaction was quenched by the addition of 20 mM Tris (pH 7.5) for 15 min on ice. Monolayers were then washed twice with ice-cold PBS and lysed (25) for immunoprecipitation as described (26).…”