The Cul4‐DDB1‐WDR3/WDR6 Complex Binds SPAK and OSR1 Kinases in a Phosphorylation‐Dependent Manner

SPAK and OSR1 are two protein kinases that play critical roles in regulating ion homeostasis. They are activated under osmotic stress through phosphorylation by their upstream WNK kinases at a conserved threonine site on their T‐loops. Additionally, WNK kinases phosphorylate SPAK and OSR1 at a highly conserved serine residue on their S‐motif, the function of which remains elusive. Using affinity pull down and mass spectrometry, we identified the E3 ubiquitin ligase complex Cullin 4‐DDB1‐WDR3/WDR6 as a binder to OSR1 kinase in a SPAK/OSR1 S‐motif phosphorylation‐dependent manner. This binding was found to be compromised by S‐motif phosphorylation following osmotic stress. Using proteasomal and neddylation inhibitors, we subsequently showed that OSR1 ubiquitylation was abolished under osmotic stress when its S‐motif is phosphorylated. These results provide the first example of an E3 ubiquitin ligase system that binds the OSR1 kinase and, thus, links the CRL4 complex to ion homeostasis. The protein kinases SPAK and OSR1 kinase become activated under osmotic stress and consequently phosphorylate a series of ion co‐transporters. Herein, we show that, under resting conditions, SPAK and OSR1 bind the Cul4‐DDB1‐WDR3/6 complex and this binding is compromised by osmotic stress. This finding links this ubiquitylation system to SPAK/OSR1‐regulated ion homeostasis.