Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3

• Published in: Biochemical and biophysical research communications Vol. 467; no. 2; pp. 229 - 234
• Main Authors: Yoshizaki, Yuki, Mori, Yutaro, Tsuzaki, Yoshihito, Mori, Takayasu, Nomura, Naohiro, Wakabayashi, Mai, Takahashi, Daiei, Zeniya, Moko, Kikuchi, Eriko, Araki, Yuya, Ando, Fumiaki, Isobe, Kiyoshi, Nishida, Hidenori, Ohta, Akihito, Susa, Koichiro, Inoue, Yuichi, Chiga, Motoko, Rai, Tatemitsu, Sasaki, Sei, Uchida, Shinichi, Sohara, Eisei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13-11-2015
• Subjects: Akt; Amino Acid Sequence; Carrier Proteins - genetics; Carrier Proteins - metabolism; Colforsin - pharmacology; Cullin Proteins - genetics; Cullin Proteins - metabolism; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Gene Expression Regulation; HEK293 Cells; Humans; Insulin; Insulin - pharmacology; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; KLHL3; Minor Histocompatibility Antigens; Molecular Sequence Data; Phosphorylation - drug effects; PKA; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins c-akt - genetics; Proto-Oncogene Proteins c-akt - metabolism; Signal Transduction; Vasopressins - pharmacology; WNK Lysine-Deficient Protein Kinase 1; WNK4; WNK4; KLHL3; Akt; Insulin; PKA
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2015.09.184

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.
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•Phosphorylation of KLHL3 at S433 inhibits the binding between KLHL3 and WNK4.•PKA phosphorylates KLHL3 at S433, inhibiting KLHL3 effect for WNK4 degradation.•Akt and insulin phosphorylate KLHL3 at S433.