Phosphorylation of focal adhesion kinase (FAK) at tyrosine 407 and integrin β1 regulate NKCC cotransporter in Fundulus heteroclitus

Abstract

Focal adhesion kinase (FAK) is a protein tyrosine kinase that functions in integrin-mediated signaling. We found that phosphorylation and dephosphorylation of FAK at tyrosine 407 is involved in NKCC functional regulation. In seawater killifish opercular membrane, we detected that FAK pY407 coimmunoprecipitated with NKCC. TEM immunogold double labeling revealed that FAK pY407 is closely colocalized with NKCC in basolateral membrane of chloride cells in seawater killifish opercular epithelium. Integrin β1 was also basolaterally colocalized with NKCC. Immunofluorescence of FAK pY407 rapidly diminished after treatment of membranes with clonidine (α-adrenergic receptor agonist, 3.2 μM) indicating that FAK was dephosphorylated by clonidine coincident with a rapid inhibition of Cl− secretion (measured as Isc) by isolated opercular membranes in Ussing chambers. The following serosal addition of protein tyrosine phosphatase inhibitor (4-methoxyphenacyl bromide, 700 μM) rapidly reversed clonidine inhibition. These results support the hypothesis that FAK, in an integrin signaling pathway, regulates the function of NKCC cotransporter in killifish opercular membrane involving a protein tyrosine phosphatase. (Supported by NSERC and CFI)