| WNK4 plays a key role in the regulation of blood pressure and electrolyte balance, as seen with the discovery that mutations in WNK4 cause a rare Mendelian form of human hypertension: Pseudohypoaldosteronism type II (PHAII). Research has made significant gains in identifying effectors downstream of WNK4, yet it remains poorly understood what molecular mechanism regulates WNK4 to achieve electrolyte balance. We used phosphoproteomics to elucidate the role of phosphorylation in regulating WNK4 modulation of NaCl reabsorption and K+ secretion, and how a PHAII mutation in WNK4 influences this regulation. We have learned that WNK4 extracted from the MDCK and HEK cell models is phosphorylated at 19 residues along its length, including the N- and C-terminal domains, and within the Aldosterone Switch domain, with most phosphorylation sites previously uncharacterized. There is a novel signature motif at sites phosphorylated by WNK1 and these sites in WNK4 show WNK1-dependent phosphorylation in HEK cells. Phosphomimetic mutations of WNK1 sites in WNK4 inhibit ROMK, while non-phosphorylation of these sites releases WNK4's inhibition of ROMK. Tyrosine kinase Src induces dynamic and coordinated phosphorylation of WNK4, in its N- and C-terminal domains, at specific sites not previously described to be affected by Src, and through WNK1-dependent phosphorylation. Additionally, PHAII elevates phosphorylation of WNK4 at specific sites without eliminating regulation through phosphorylation. These findings identify a number of phosphorylation elements within WNK4, and provide insight to phosphorylation as a mechanism regulating WNK4 function in renal electrolyte balance. Moreover, these findings indicate that the effects of the PHAII mutation are not explained by changes in phosphorylation, and imply that it remains unclear precisely how the PHAII mutation imparts its effects and what is the normal physiologic correlate of the PHAII mutation. |
