Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na+/H+ exchangers

The signaling pathways by which cell volume regulates ion transporters, e.g . Na+/H+ exchangers (NHEs), and affects cytoskeletal organization are poorl y understood, We have previously shown that shrinkage induces tyrosine phos phorylation in CHO cells, predominantly in an 85-kDa band. To identify volu me-sensitive kinases and their substrates, we investigated the effect of hy pertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src, Fyn activation was also observed in nystatin-permea bilized cells, where shrinkage cannot induce intracellular alkalinization, In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1, PPI, a selective Src family inhibitor, strongly reduc ed the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stress-elicited phosphorylation as cortac tin, an 85-kDa actin-binding protein and well known Src family substrate. C ortactin phosphorylation was triggered by shrinkage and not by changes in o smolarity or pH(i) and was abrogated by PP1, Hyperosmotic cortactin phospho rylation was reduced in Fyn-deficient fibroblasts but remained intact in Sr c-deficient fibroblasts, To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3, Thus, membe rs of the Src family are volume-sensitive enzymes that may participate in t he shrinkage-related reorganization of the cytoskeleton but are probably no t responsible for the osmotic regulation of NHE.