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

Abstract

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/glucocorticoidinduced kinase 1 (SGK1), and this inhibition is attenuated by the Srcfamily 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.W ith-no-lysine kinase 4 (WNK4) is expressed in the connecting tubule (CNT) and cortical collecting duct (CCD) (1, 2) and plays an important role in modulating the balance between renal K secretion and Na reabsorption (3-8). The effect of WNK4 on renal K secretion is partially mediated through inhibition of KCNJ1 (ROMK) channels in the CNT and in the CCD. ROMK inhibition is achieved by a stimulation of clathrinmediated endocytosis (1), an effect that is dependent on intersectin, a scaffold protein containing two Eps15 homology domains (9).Serum/glucocorticoid-induced kinase 1 (SGK1), a downstream mediator of aldosterone signaling, suppresses the inhibitory effect of WNK4 on ROMK channels through phosphorylation of WNK4 at Ser 1169 (2) and Ser 1196 (5). Both volume depletion and high K intake increase aldosterone and SGK1 levels (10). However, it is not clear why a high K intake or volume depletion modulates differently the effect of SGK1 on ROMK channels.Candidate regulators of differential ROMK expression in hyperkalemia and hypovolemia should be regulated in a potassiumdependent manner. One such protein is the protein tyrosine kinase c-Src, whose expression in renal cortex is reduced in states of high potassium intake (11). We have previously demonstrated a key role of c-Src in determining the effect of SGK1 on WNK4 (12). C-Src abolishes SGK1-induced phosphorylation of WNK4 and restores the inhibit...

Role of WNK4 and kidney-specific WNK1 in mediating the effect of high dietary K+ intake on ROMK channel in the distal convoluted tubule

“…In the present study, we have demonstrated that ROMK channel activity in the DCT2/CNT of WNK4 Ϫ/Ϫ mice was downregulated. WNK4 has been shown to inhibit ROMK channels in a variety of cells transfected with ROMK and WNK4 (7,9,15). In the mice expressing the pseudohypoaldosteronism type II (PHAII) mutant WNK4 (TgWNK4 PHAII mice), ROMK channel activity was significantly lower in the DCT2/CNT than the WT control, suggesting the role of WNK4 in inhibiting ROMK in vivo (28).…”

“…Although experiments performed in cell models support the role of WNK4 and KS-WNK1 in the regulation of ROMK (7,15,22), it is still not tested whether WNK4 and KS-WNK1 regulate ROMK channels in vivo. It is generally accepted that ROMK channels expressed in ASDN play a role in mediating K ϩ excretion under control conditions while HK intake-induced stimulation of renal K ϩ excretion is achieved by the activation of both ROMK and Ca 2ϩ -activated big-conductance K ϩ channels (BK) in the ASDN (5,8,16,18,21).…”

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor and NaCl transport mechanisms in the renal distal nephron

“…On the other hand, Src phosphorylates ROMK at Y337, which facilitates ROMK internalization (Lin et al 2004). ROMK is also known to be inhibited by serine threonine kinase WNK4 (With-No-Lysine 4) via kinase-independent mechanism (Kahle et al 2003), which acts in concert with Sgk1 and Src (Lin et al 2015).…”

ENaC and ROMK activity are inhibited in the DCT2/CNT of TgWnk4PHAII mice

“…We and others demonstrated that Wnk4 inhibited ROMK channels in vitro (7,10,12,30). Because ROMK plays a key role in mediating K ϩ secretion in aldosterone-sensitive distal nephron (6, 8), we examined ROMK channel activity by measuring TPNQ-sensitive K ϩ currents in the DCT2/CNT with perforated whole cell recording (5).…”