Cell volume regulation in metazoans involves phosphorylation of TSC22D proteins.

Intracellular water and ion homeostasis are fundamental determinants of cell volume and macromolecular crowding. WNK kinases are central sensors of osmotic stress, yet how their activity is spatiotemporally regulated remains unclear. Here, we identify that TSC22D and NRBP protein families are WNK-dependent substrates. TWN bodies-phase condensates containing TSC22Ds, WNKs, and NRBPs-act as signaling hubs that couple intracellular crowding to kinase activation and volume control. In mice, knocking out Tsc22d2 results in a sub-lethal phenotype, with smaller embryos and reduced cell size. Structural modeling and mutagenesis reveal that TSC22Ds scaffold NRBP to gate WNK activation, while phosphorylation of NRBP1 at T232 influences pathway output. Hyperphosphorylation of intrinsically disordered regions in TSC22D proteins alters the material properties of TWN bodies, promoting their dissolution and driving cell volume recovery. Together, these findings support a model of phosphorylation-controlled condensate dynamics as a central mechanism governing WNK-dependent osmoregulatory signaling.