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
Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Japan.
Biochem Biophys Res Commun. 2015 Nov 13;467(2):229-34. doi: 10.1016/j.bbrc.2015.09.184. Epub 2015 Oct 3.
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.
无赖氨酸激酶（WNK）1、WNK4、kelch样蛋白3（KLHL3）和Cullin3的突变会导致一种遗传性高血压疾病——II型假性醛固酮增多症。WNK激活钠氯共转运体（NCC），增加肾脏对钠的重吸收。此外，KLHL3是Cullin3依赖性E3泛素连接酶的衔接蛋白，最近发现它与WNK结合，从而使其降解。胰岛素和血管加压素已被确定为WNK信号的强大激活剂。在本研究中，我们分别研究了胰岛素和血管加压素信号的关键下游底物Akt和蛋白激酶A（PKA）对KLHL3的影响。质谱分析显示KLHL3在S433处发生磷酸化。磷酸特异性抗体显示磷酸化的KLHL3与WNK4之间的结合存在缺陷。与S433是Akt和PKA磷酸化基序的组成部分这一事实一致，体外激酶分析表明Akt和PKA可以使KLHL3在S433处磷酸化，此前有报道称该位点可被蛋白激酶C（PKC）磷酸化。此外，PKA的代表性刺激剂毛喉素通过抑制导致WNK4降解的KLHL3效应，增加了人胚肾293（HEK293）细胞中KLHL3在S433处的磷酸化以及WNK4蛋白表达。胰岛素也增加了培养细胞中KLHL3在S433处的磷酸化。总之，我们发现Akt和PKA使KLHL3在S433处磷酸化，PKA对KLHL3的磷酸化抑制了WNK4的降解。这可能是胰岛素和血管加压素在生理上激活WNK信号的一种新机制。
