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Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

Lin, Dao-Hong, Yue, Peng, Yarborough, Orlando, Scholl, Ute I., Giebisch, Gerhard, Lifton, Richard P., Rinehart, Jesse, Wang, Wen-Hui
Proceedings of the National Academy of Sciences of the United States of America 2015 v.112 no.14 pp. 4495-4500
absorption, animal models, hyperkalemia, phosphorylation, secretion, tyrosine
With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr ¹⁰⁹², Tyr ¹⁰⁹⁴, and Tyr ¹¹⁴³, and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr ¹⁰⁹² or Tyr ¹¹⁴³ to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4 Y¹⁰⁹²/¹⁰⁹⁴F. Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser ¹¹⁹⁶, an effect that was abrogated in the double mutant. WNK4 Y¹¹⁴³F inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4 Y¹¹⁴³F could not be reversed by SGK1. The failure to reverse WNK4 Y¹¹⁴³F-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4–PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4 Y¹¹⁴³F-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr ¹⁰⁹² and Tyr ¹¹⁴³ plays an important role in modulating the inhibitory effect of WNK4 on ROMK.