[en] Highlights: • Phosphorylation of SPAK at S387 and OSR1 at S339 enhances binding to MO25. • WNK1 kinase phosphorylates SPAK at S387 and OSR1 S339 in vitro. • In cells, SPAK S387 and OSR1 S339 phosphorylation involves WNK kinases. • MO25 mutants that compromise MO25-dependent activation of SPAK and OSR1 kinases identified. SPAK and OSR1 are two protein kinases that play important roles in regulating the function of numerous ion co-transporters. They are activated by two distinct mechanisms that involve initial phosphorylation at their T-loops by WNK kinases and subsequent binding to a scaffolding protein termed MO25. To understand this latter SPAK and OSR1 regulation mechanism, we herein show that MO25 binding to these two kinases is enhanced by serine phosphorylation in their highly conserved WEWS motif, which is located in their C-terminal domains. Furthermore, we show that this C-terminal phosphorylation is carried out by WNK kinases in vitro and involves WNK kinases in cells. Mutagenesis studies revealed key MO25 residues that are important for MO25 binding and activation of SPAK and OSR1 kinases. Collectively, this study provides new insights into the MO25-mediated activation of SPAK and OSR1 kinases, which are emerging as important players in regulating ion homeostasis.