Serum and Glucocorticoid-induced Kinase (SGK) 1 and the Epithelial Sodium Channel Are Regulated by Multiple with No Lysine (WNK) Family Members

Charles J Heise,Bing-E Xu,Staci L Deaton,Seung-Kuy Cha,Chih-Jen Cheng,Svetlana Earnest,Samarpita Sengupta,Yu-Chi Juang,Steve Stippec,Yingda Xu,Yingming Zhao,Chou-Long Huang,Melanie H Cobb

doi:10.1074/jbc.m110.103432

Abstract

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.

Highlights

The subsequent discovery that WNK1 and WNK4 are genetically linked to a rare type of hypertension, pseudohypoaldosteronism type 2 (PHA2) [4], demonstrated the importance of WNK function in man and an implicit significance of the sensitivity of WNK1 kinase activity to osmotic stress
Because questions remain about the abilities of WNKs to regulate this pathway, in this study we have examined the effects of the four WNK family members on SGK1 and ENaC activities using the same reconstituted system, and we have further explored some of their biochemical properties
Activation of SGK1 and ENaC by WNK Family Members— We compared activation of SGK1 and ENaC by WNK1 in the HEK293 cell line, the same line used for ENaC reconstitution below

Summary

EXPERIMENTAL PROCEDURES

Materials—Plasmids encoding mouse SGK1, rat WNK1 and WNK2, and human WNK3 and WNK4 fragments were as described [2, 34, 35]. Cells were harvested in isotonic lysis buffer containing 1% Triton X-100 and phosphatase and protease inhibitors as described [37]. Plasmids were transfected into the cells which were harvested 48 h later. In vitro kinase assays of immunoprecipitates were performed as described using either recombinant Nedd or crosstide as substrates [15]. Recombinant proteins used as substrates or for in vitro kinase assays were purified from Escherichia coli strain BL21 using standard protocols. Cells were cotransfected with plasmids encoding ␣, ␤, and ␥ ENaC subunits, fragments of each of WNK isoforms corresponding to WNK1(1– 491) (see Fig. 1A), enhanced green fluorescent protein, and with or without wild type SGK1, kinase-dead SGK1, wild type Nedd, siRNA for SGK1, siRNA for Nedd, or control oligonucleotides (200 nM) as indicated. Multiple comparisons were determined using one-way analysis of variance followed by Tukey’s multiple comparison tests. p values Ͻ 0.05 and 0.01 were considered significant for single and multiple comparisons, respectively

RESULTS

We then determined which other

DISCUSSION