Focal adhesion kinase and beta1 integrin regulation of Na+, K+, 2Cl- cotransporter in osmosensing ion transporting cells of killifish, Fundulus heteroclitus.
Focal adhesion kinase (FAK), also known as PYK2, is a tyrosine kinase that functions in integrin-mediated signaling in mechanosensitive cells but its role in osmosensing cells is unknown. Antibodies directed against phosphorylated FAK, whose epitopes are conserved among vertebrates, were used to follow phosphorylation patterns in an osmosensing ion secreting epithelium, the killifish (Fundulus heteroclitus) opercular membrane. At the electron microscopic level, a unique combination of integrin beta1, the phosphorylated form of FAK at tyrosine 407 (pY407) and Na(+), K(+), 2Cl(-) cotransporter (NKCC1) were all colocalized only on the basolateral membrane in chloride cells. The three proteins were also coimmunoprecipitated with each other in isotonic conditions, suggesting an osmosensing complex involving the three proteins. Only FAK pY407 was sensitive to hypotonic shock and became dephosphorylated with hypotonic shock, while FAK pY576 in the apical membrane and pY861 in cell-cell adhesions were insensitive to hypotonicity. NKCC1 contributes to NaCl secretion in seawater and previous reports showed that hypotonic shock (-60 mOsm/kg) rapidly inhibits Cl(-) secretion. These results indicate that chloride cells respond to hypotonic shock using integrin beta1 as an osmosensor that is connected to dephosphorylation of FAK pY407 which leads to NKCC1 deactivation in the basolateral membrane and the inhibition of NaCl secretion by these epithelial cells.
Marshall
W S
WS
Department of Biology, St. Francis Xavier University, P.O. Box 5000, Antigonish, Nova Scotia, Canada B2G 2W5. bmarshal@stfx.ca
Katoh
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F
Main
H P
HP
Sers
N
N
Cozzi
R R F
RR
D000818:Animals; D004848:Epithelium / Q000648:ultrastructure; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D023781:Fundulidae / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D019012:Integrin beta1 / Q000378:metabolism*; D017136:Ion Transport; D007552:Isotonic Solutions; D046529:Microscopy, Electron, Transmission; D019000:Phosphotyrosine / Q000378:metabolism; D021381:Protein Transport; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D014882:Water-Electrolyte Balance
D019012:Integrin beta1; D007552:Isotonic Solutions; D028021:Sodium-Potassium-Chloride Symporters; D019000:Phosphotyrosine; D051416:Focal Adhesion Protein-Tyrosine Kinases