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SPAK is a substrate of both WNK4 and p40. In vitro in-solution kinase assays were performed with the indicated WNK4 proteins expressed and purified from HEK293 cells, using a GST fusion protein of kinase-inactive SPAK (D212A) as the substrate (predicted molecular mass, 88 kDa). Left: FL constructs of WT WNK4 and the indicated kinase-inactive or PHAII mutants. Right: FL WT WNK4 compared with a COOH-terminal fragment (594–1222) of WT WNK4 lacking the kinase domain.
