title: Emerging role of Akt substrate protein AS160 in the regulation of AQP2 translocation.
abstract: AS160, a novel Akt substrate of 160 kDa, contains a Rab GTPase-activating 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 knockdown 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 LY 294002 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/Akt pathways. siRNA-mediated AS160 knockdown significantly decreased total AS160 and phospho-AS160 expression. Immunocytochemistry 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 biotinylation assays of mpkCCDc14 cells with AS160 knockdown exhibited significantly higher AQP2 expression [150 ± 15% of control mpkCCDc14 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 higher AQP2 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 of AQP2 to the plasma membrane.
authors.affiliation: Dept. of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Dongin-dong 101, Taegu 700-422, South Korea.
authors.forename: Hyo-Young
authors.initials: HY
authors.lastname: Kim
authors.forename: Hyo-Jung
authors.initials: HJ
authors.lastname: Choi
authors.forename: Jung-Suk
authors.initials: JS
authors.lastname: Lim
authors.forename: Eui-Jung
authors.initials: EJ
authors.lastname: Park
authors.forename: Hyun Jun
authors.initials: HJ
authors.lastname: Jung
authors.forename: Yu-Jung
authors.initials: YJ
authors.lastname: Lee
authors.forename: Sang-Yeob
authors.initials: SY
authors.lastname: Kim
authors.forename: Tae-Hwan
authors.initials: TH
authors.lastname: Kwon
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000096:biosynthesis / Q000378:metabolism*; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D020690:GTPase-Activating Proteins / Q000502:physiology*; D020868:Gene Silencing; D007150:Immunohistochemistry; D007672:Kidney Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D034741:RNA, Small Interfering; D017483:Receptors, Vasopressin / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D051399:Aquaporin 2; D020690:GTPase-Activating Proteins; D034741:RNA, Small Interfering; D017483:Receptors, Vasopressin; C505647:Tbc1d4 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D003894:Deamino Arginine Vasopressin