Abstract
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.