title: The role of 70-kDa heat shock protein in dDAVP-induced AQP2 trafficking in kidney collecting duct cells.
abstract: It has been reported that several proteins [heat shock protein 70 (Hsp70 and Hsc70), annexin II, and tropomyosin 5b] interact with the Ser(256) residue on the COOH terminus of aquaporin-2 (AQP2), where vasopressin-induced phosphorylation occurs for mediating AQP2 trafficking. However, it remains unknown whether these proteins, particularly Hsp70, play a role in AQP2 trafficking. Semiquantitative immunoblotting revealed that renal expression of AQP2 and Hsp70 was significantly increased in water-restricted or dDAVP-infused rats. In silico analysis of the 5'-flanking regions of AQP2, Hsp70-1, and Hsp70-2 genes revealed that transcriptional regulator binding elements associated with cAMP response were identified at both the Hsp70-1 and Hsp70-2 promoter regions, in addition to AQP2. Luciferase reporter assay demonstrated the significant increase of luminescence after dDAVP stimulation (10(-8) M, 6 h) in the LLC-PK1 cells transfected with luciferase vector containing 1 kb of the 5'-flanking region of Hsp70-2 gene. Hsp70-2 protein expression was also increased in mpkCCDc14 cells treated by dDAVP in a concentration-dependent manner. Cell surface biotinylation analysis demonstrated that forskolin (10(-5) M, 15 min)-induced AQP2 targeting to the apical plasma membrane was significantly attenuated in the mpkCCDc14 cells with Hsp70-2 knockdown. Moreover, forskolin-induced AQP2 phosphorylation (Ser(256)) was not significantly induced in the mpkCCDc14 cells with Hsp70-2 knockdown. In contrast, Hsp70-2 knockdown did not affect the dDAVP-induced AQP2 abundance. In addition, siRNA-directed knockdown of Hsp70 significantly decreased cell viability. The results suggest that Hsp70 is likely to play a role in AQP2 trafficking to the apical plasma membrane, partly through affecting AQP2 phosphorylation at Ser(256) and cell viability.
authors.affiliation: Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, South Korea.
authors.forename: Eui-Jung
authors.initials: EJ
authors.lastname: Park
authors.forename: Jung-Suk
authors.initials: JS
authors.lastname: Lim
authors.forename: Hyun Jun
authors.initials: HJ
authors.lastname: Jung
authors.forename: Eunjung
authors.initials: E
authors.lastname: Kim
authors.forename: Ki-Hwan
authors.initials: KH
authors.lastname: Han
authors.forename: Tae-Hwan
authors.initials: TH
authors.lastname: Kwon
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000096:biosynthesis / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D007008:Hypokalemia / Q000503:physiopathology; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009994:Osmolar Concentration; D010766:Phosphorylation; D021381:Protein Transport; D051381:Rats; D014554:Urination; D014868:Water Deprivation / Q000502:physiology
chemical_list: D051399:Aquaporin 2; D018840:HSP70 Heat-Shock Proteins; C490435:Hspa2 protein, rat; D003894:Deamino Arginine Vasopressin