title: Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.
abstract: Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal.
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yoshizaki
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Mori
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Tsuzaki
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Takayasu
authors.initials: T
authors.lastname: Mori
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Nomura
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Wakabayashi
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Daiei
authors.initials: D
authors.lastname: Takahashi
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Moko
authors.initials: M
authors.lastname: Zeniya
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Kikuchi
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Araki
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Ando
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Isobe
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Nishida
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Ohta
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Susa
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Inoue
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Motoko
authors.initials: M
authors.lastname: Chiga
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Tatemitsu
authors.initials: T
authors.lastname: Rai
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Sei
authors.initials: S
authors.lastname: Sasaki
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Uchida
authors.affiliation: Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan. Electronic address: esohara.kid@tmd.ac.jp.
authors.forename: Eisei
authors.initials: E
authors.lastname: Sohara
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014667:Vasopressins / Q000494:pharmacology; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; C489837:KLHL3 protein, human; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D014667:Vasopressins; D005576:Colforsin; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human; C433806:WNK4 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Akt; Insulin; KLHL3; PKA; WNK4