JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro

Cell shrinkagephosphorylates and activates the Na-K-2Cl cotransporter (NKCC1),indicating the presence of a volume-sensitive protein kinase. Toidentify this kinase, extracts of normal and shrunken aorticendothelial cells were screened for phosphorylation of NKCC1 fusionproteins in an in-the-gel kinase assay. Hypertonic shrinkage activateda 46-kDa kinase that phosphorylated anNH₂-terminal fusion protein, withweaker phosphorylation of a COOH-terminal fusion protein. Thiscytosolic kinase was activated by both hypertonic and isosmoticshrinkage, indicating regulation by cell volume rather than osmolarity.Subsequent studies identified this kinase as c-JunNH₂-terminal kinase (JNK).Immunoblotting revealed increased JNK activity in shrunken cells; therewas volume-sensitive phosphorylation ofNH₂-terminal c-Jun fusion protein;immunoprecipitation of JNK from shrunken cells but not normal cellsphosphorylated NKCC1 in gel kinase assays; and treatment of cells withtumor necrosis factor, a known activator of JNK, mimicked the effect ofhypertonicity. We conclude that JNK is a volume-sensitive kinase inendothelial cells that phosphorylates NKCC1 in vitro. This is the firstdemonstration of a volume-sensitive protein kinase capable ofphosphorylating a volume-regulatory transporter.