SUMMARYCystic fibrosis transmembrane conductance regulator (CFTR) anion channels are the regulated exit pathway in Cl -secretion by teleost mitochondria rich salt secreting (MR) cells of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry, immunogold transmission electron microscopy (TEM), and co-immunoprecipitation, using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase focal adhesion kinase (FAK) phosphorylated at Y407 (FAK pY407) are colocalized in the apical membrane and in subjacent membrane vesicles of MR cells. We showed previously that basolateral FAK pY407, unlike other FAK phosphorylation sites, is osmosensitive and dephosphorylates during hypotonic shock of epithelial cells (Marshall et al., 2008). In the present study, we found that hypotonic shock and the α 2 -adrenergic agonist clonidine (neither of which affects cAMP levels) rapidly and reversibly inhibit Cl -secretion by isolated opercular membranes, simultaneous with dephosphorylation of FAK pY407, located in the apical membrane. FAK pY407 is rephosphorylated and Cl -secretion rapidly restored by hypertonic shock as well as by forskolin and isoproterenol, which operate via cAMP and protein kinase A. We conclude that hormone mediated, cAMP dependent and osmotically mediated, cAMP independent pathways converge on a mechanism to activate CFTR and Cl -secretion, possibly through tyrosine phosphorylation of CFTR by FAK.
Focal adhesion kinase (FAK) is a tyrosine kinase with multiple Y sites and an ezrin/radixin/moesin binding site. Antibodies to the phosphorylated form of FAK at Y407 and Y587 are colocalized with the Cl− channel CFTR in the apical membrane of mitochondria‐rich (MR) cells of seawater‐acclimated euryhaline killifish opercular epithelium. Hypotonic treatment (80%, 224 mOsm/kg) causes rapid, reversible cell swelling (observed via confocal microscopy of living cells in situ), inhibition of Cl− secretion and dephosphorylation of FAK pY407 but no effect on FAK phosphorylated at Y576. Re‐phosphorylation of FAK at Y407 specifically in the apical membrane of MR cells was immunohistochemically detected following 10‐min treatments with forskolin (10 μM), 3‐isobutyl‐1‐methylxanthine (IBMX, 0.1 mM) and isoproterenol (1.0 μM), all known to increase cAMP and activate CFTR. There is parallel recovery of Cl− secretion by MR cells in the isolated epithelium, detected electrophysiologically. These results suggest involvement of phosphorylation of FAK pY407 but not pY576 in cell signaling pathways regulating the Cl− transport function of CFTR. (Supported by NSERC Canada)
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