Abstract
AS160, a novel Akt substrate of 160 kDa, contains a Rab GTP aseactivating protein (GAP) domain. The present study examined the role of Akt and AS160 in aquaporin-2 (AQP2) trafficking. The main strategy was to examine the changes in AQP2 translocation in response to small interfering RNA (siRNA)-mediated AS160 knock down in mouse cortical collecting duct cells (M-1 cells and mpkCCDc14 cells).Short-term dDAVP treatment in M-1 cells stimulated phosphorylation of Akt (S473) and AS160, which was also seen in mpkCCDc14 cells. Conversely, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 diminished phosphorylation of Akt (S473) and AS160. Moreover, siRNA-mediated Akt1 knockdown was associated with unchanged total AS160 but decreased phospho-AS160 expression, indicating that phosphorylation of AS160 is dependent on PI3K/Aktpathways. siRNA-mediated AS160 knockdown significantly decreased total AS160 and phospho-AS160 expression. Immunocy to chemistry revealed that AS160 knockdown in mpkCCDc14 cells was associated with increased AQP2 density in the plasma membrane [135±3%of control mpkCCDc14 cells (n = 65), P < 0.05, n = 64] despite the absence of dDAVP stimulation. Moreover, cell surface biotinylationas says of mpkCCDc14 cells with AS160 knockdown exhibited significantly higher AQP2 expression [150 ± 15% of controlmpkCCDc14 cells (n = 3), P < 0.05, n = 3]. Taken together, PI3K/Akt pathways mediate the dDAVP-induced AS160 phosphorylation, and AS160 knockdown is associated with higherAQP2 expression in the plasma membrane. Since AS160 contains a GAP domain leading to a decrease in the active GTP-bound form of AS160 target Rab proteins for vesicle trafficking, decreased expression of AS160 is likely to play a role in the translocation ofAQP2 to the plasma membrane
Original language | English |
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Pages (from-to) | F151-F161 |
Journal | American Journal of Physiology - Renal Physiology |
Volume | 301 |
Issue number | 1 |
DOIs | |
State | Published - Jul 2011 |
Keywords
- Aquaporin
- Collecting duct
- Rab gtpase-activating protein
- Vasopressin