Multistep regulation of autophagy by WNK1

Abstract

The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertensionrelated disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNAmediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phosphatidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phosphorylation, and activation of the kinase upstream of ULK1, the AMPactivated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related proline-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or -starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.T he with-no-lysine (K) (WNK) protein kinase family is an evolutionarily conserved, atypical group of serine/threonine kinases with the conserved ATP-binding lysine residue shifted to a different position within the kinase domain (1, 2). WNKs are the only kinases in the eukaryotic protein kinase superfamily with this unusual arrangement. This arrangement confers on them unique structural and functional properties (3). There are four WNK proteins in mammals, of which WNK1 is the largest, over 2,000 residues, and most widely expressed (4).The best-characterized function of WNKs is their binding and activation of downstream target kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK) (5-7). Once activated, OSR1 and SPAK phosphorylate and regulate downstream cation-chloride cotransporters of the SLC12 family (8-10). This WNK-SPAK/OSR1 pathway enables cells to adjust intracellular ions and cell volume in response to ion imbalances and osmotic stress (11). It is noteworthy that mutations in the regulatory components of the WNK pathway, including WNK1, WNK4, kelch-likes (KLHLs), and cullins, have been shown to cause increased expression of WNKs and ion reabsorption defects in kidney that lead to hypertension-related genetic diseases, such as Gordon's syndrome (pseudohypoaldosteronism II) (12-16). In addition, WNKs have been linked to the regulation of cell proliferation (17, 18), cell death (19), cell migration (20-23), endocytosis (24-27...

Secreted protein acidic and rich in cysteine (SPARC) induces cell migration and epithelial mesenchymal transition through WNK1/snail in non-small cell lung cancer

“…In humans, there are four WNK genes (WNK1, WNK2, WNK3, and WNK4) [ 40 , 41 ]. WNK kinases are involved in the enhancement of cell proliferation, migration, invasion, and autophagy [ 40 – 42 ]. Therefore, WNK1 is a potential regulator of SPARC-mediated cell migration.…”

Oncogenic role of MIR516A in human bladder cancer was mediated by its attenuating PHLPP2 expression and BECN1-dependent autophagy

“…However, the function of autophagy in human diseases, especially in various cancers' development, remains controversial. Studies suggest that autophagy has a protective effect on cancer by improving sensitivity to chemotherapy [16], whereas other studies indicate that autophagy promotes cancer development by inhibiting apoptosis [17][18][19]. PHLPP2 is a tumor suppressor by catalyzing the dephosphorylation of the AGC kinases.…”

OSR1 regulates a subset of inward rectifier potassium channels via a binding motif variant

“…The methods for immunofluorescence, SDS/PAGE, and immunoblotting are similar to those described in ref. 53 and are presented in detail in SI Appendix, Extended Methods.…”