The influence of SRC-family tyrosine kinases on Na,K-ATPase activity in lens epithelium.
OBJECTIVE
Na,K-adenosine triphosphatase (ATPase) is essential for the regulation of cytoplasmic ion concentrations in lens cells. Earlier studies demonstrated that tyrosine phosphorylation by Lyn kinase, a Src-family member, inhibits Na,K-ATPase activity in porcine lens epithelium. In the present study, experiments were conducted to compare the ability of other Src-family kinases (Fyn, Src, and Lck) and Fes, a non-Src-family tyrosine kinase, to alter Na,K-ATPase activity.


METHODS
Membranes prepared from porcine lens epithelium were incubated with partially purified tyrosine kinases in buffer containing 1 mM adenosine triphosphate (ATP). ATP hydrolysis in the presence and absence of ouabain was used to measure Na,K-ATPase activity. Western blot analysis was used to examine phosphotyrosine-containing proteins and tyrosine kinase expression.


RESULTS
Fyn reduced Na,K-ATPase activity by approximately 30%. In contrast, Src caused a approximately 38% increase of Na,K-ATPase activity. Na,K-ATPase activity in membrane material treated with Lck or Fes was not significantly altered, even though Lck and Fes treatment induced robust tyrosine phosphorylation. Added exogenously, each tyrosine kinase induced a different pattern of membrane protein tyrosine phosphorylation. As judged by immunoprecipitation, Src, Fyn, Lyn, and Lck elicited tyrosine phosphorylation of the Na,K-ATPase alpha1 protein. Src, Fyn, Lyn, Lck, and Fes were each detectable in the epithelium by Western blot.


CONCLUSIONS
The results indicate considerable variation in the Na,K-ATPase activity response of lens epithelium to different tyrosine kinases. This could perhaps explain why inhibition of Na,K-ATPase activity is reported to be caused by tyrosine phosphorylation in some tissues, whereas stimulation of Na,K-ATPase activity is observed in other tissues.
Bozulic
Larry D
LD
Department of Biochemistry, University of Louisville, School of Medicine, KY 40292, USA.
Dean
William L
WL
Delamere
Nicholas A
NA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D006868:Hydrolysis; D007908:Lens, Crystalline / Q000187:drug effects* / Q000201:enzymology; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D014443:Tyrosine; D010042:Ouabain; D000255:Adenosine Triphosphate; D019061:src-Family Kinases; D000254:Sodium-Potassium-Exchanging ATPase