PMC

SGK1 phosphorylates WNK4 at serine-1169 and complexes with WNK4 in mammalian kidney cells. (a) SGK1 phosphorylates WNK4 at serine-1169. The indicated (+) GST-tagged, C-terminal fragments of WNK4 (residues 1127–1179) containing either the wild-type serine or substitution of alanine at position 1169 were purified and incubated with active or inactive SGK1 in the presence of [γ-³²P]ATP; labeling of WNK4 was detected after PAGE by autoradiography as described in Methods, and total protein was revealed by Coomassie staining. (b) WNK4 and SGK1 coimmunoprecipitate in HEK-293T cells. Indicated constructs were expressed in HEK-293 cells, and lysates were immunoprecipitated with anti-HA antibodies, fractionated, transferred to membranes, and probed with indicated antibodies as described in Methods. SGK1 was detected by anti-FLAG antibodies, and WNK4 was detected by anti-HA.

Identification of a conserved SGK1 regulatory site within WNK4 kinase. The structure of WNK4 is shown, including the location of its kinase domain (blue) and two coil domains (red); the sequence of a highly conserved SGK/Akt consensus phosphorylation motif is shown for mouse (Mus musculus), human (Homo sapiens), dog (Canis familiaris), and chicken (Gallus gallus). WNK4 proteins were aligned with Clustal W. The predicted site of SGK1 phosphorylation (serine-1169 in mouse WNK4) is indicated with an asterisk and in red.

WNK4_S1169D mutation alleviates inhibition of ROMK. cRNAs encoding the indicated proteins were injected into Xenopus oocytes, and Ba²⁺-sensitive whole-cell K⁺ currents were recorded. Cumulative results of Ba²⁺-sensitive whole-cell K⁺ currents measured at +40 mV are shown as a proportion of the ROMK control. A minimum of 19 oocytes were analyzed in each group. Indicated P values represent the results of two-tailed Student's t tests. Western blotting of oocyte lysates confirms the presence of WNK4-HA in the corresponding experimental groups.

WNK4_S1169D mutation alleviates inhibition of ENaC. cRNAs encoding the indicated proteins were injected into Xenopus oocytes, and amiloride-sensitive, whole-cell Na⁺ currents were recorded as described in Methods. Cumulative results of amiloride-sensitive currents measured at −100 mV are shown as a proportion of the ENaC control; mean ± SE for each group is shown. A minimum of 29 oocytes were studied in each group. The ENaC alone and wild-type WNK4 data are the same data reported in ref. ; data for all experimental conditions were collected from the same batches of oocytes concurrently. Indicated P values represent the results of two-tailed Student's t tests. Western blotting of oocyte lysates confirms the presence of WNK4-HA in the corresponding experimental groups.

WNK4 orchestrates differential responses to hypovolemia and hyperkalemia. WNK4 has at least three distinct functional states defined by the effects of wild-type WNK4, PHAII-mutant WNK4, and WNK4_S1169D on downstream targets. The effects at two of these targets, ROMK and ENaC, are shown. We propose that intravascular volume depletion (Low IV Volume) and hyperkalemia (High K⁺), which are associated with either aldosterone plus angiotensin II (aldo + AII) or aldosterone alone, respectively, have differential effects at downstream targets. At equilibrium, WNK4 inhibits both ENaC and ROMK. PHAII mutation defines a state that alleviates inhibition of ENaC while augmenting inhibition of ROMK, promoting a net increase in renal Na⁺ reabsorption without K⁺ loss. Hyperkalemia results in increased aldosterone signaling, phosphorylation of the SGK1 site, and loss of WNK4 inhibition of both ENaC and ROMK. This results in a net increase in K⁺ secretion via increases in both the electrical driving force for K⁺ secretion and K⁺ channel activity.