The influence of SRC-family tyrosine kinases on Na,K-ATPase activity in lens epithelium

Purpose: 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.