Elucidating the regulation of SPAK and OSR1 kinases by ubiquitylation

The WNK-SPAK/OSR1 signalling pathway is a master regulator of ion homeostasis. The WNK kinases, whose mutations cause an inherited form of hypertension, activate SPAK and OSR1 kinases via phosphorylation. Upon phosphorylation by WNK kinases, SPAK and OSR1 kinases, consequently, phosphorylate an array of sodium, potassium and chloride ion co-transporters leading to either their activation or inhibition. WNK kinases phosphorylate SPAK and OSR1 kinases on their N-terminal threonine-rich region (termed the T-loop) as well as their serine-rich region (termed the S-motif). Although T-loop phosphorylation of SPAK and OSR1 kinases is known to activate these kinases, the function of their S-motif phosphorylation remains unclear. This project was aimed to unravel the function of SPAK and OSR1 S-motif phosphorylation by WNK kinases. \nUsing peptide pull-down assays that employed SPAK and OSR1 peptide sequences derived from their S-motifs, followed by mass spectrophotometry analysis, the E3 ubiquitin ligases Cullin 4A and 4B (CUL 4A and B) were identified as novel binders of SPAK and OSR1 S-motif. Additionally, the adaptor protein DDB1, which is known to form a complex with CUL4A/B, was also identified along with two other proteins known as WDR3 and WDR6. This binding of these proteins to SPAK and OSR1 kinases was subsequently verified using pull-down assays of overexpressed proteins in cells as well as immunoprecipitation of the endogenous proteins and followed by Western blotting. The results showed that these proteins bind SPAK and OSR1 kinases when unphosphorylated on their S-motif, while phosphorylation on these sites by WNK-kinases compromises the binding. \nGiven that CUL4A/B E3 ubiquitin ligases ubiquitylate their protein substrates, overexpressed SPAK and OSR1 kinases, along with the proteasome inhibitor, MG132, and the neddylation inhibitor MLN4924 were used to investigate whether SPAK and OSR1 kinases are ubiquitylated by the CUL4A/B-DDB1-WDR3/6 complex. The results indicated that SPAK and OSR1 are constitutively ubiquitylated under resting conditions, whereas under osmotic stress when SPAK and OSR1 kinases are phosphorylated by WNK kinases, the ubiquitylation of these proteins is significantly reduced. \nTogether, the data presented in this work represents the first example of an E3 ubiquitin ligase system that binds SPAK and OSR1 kinases. Critically, it provides a new molecular insight that links the CUL4A/B-mediated protein ubiquitylation to ion homeostasis and the regulation of blood pressure through SPAK and OSR1 kinases.