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Regulation of the Bovine Kidney Microsomal Chloride Channel p64 by p59 fyn , a Src Family Tyrosine Kinase*

J. Edwards,Shefalee Kapadia

Published 2000 in Journal of Biological Chemistry

ABSTRACT

p64 is a chloride channel of intracellular membranes which is present in regulated secretory vesicles. Mechanisms by which the p64 channel could be regulated are largely unknown. p59 fyn is a non-receptor tyrosine kinase of the Src family that has been implicated in a variety of intracellular signaling events. The N-terminal portion of p64 has several potential binding sites for Src family SH2 domains. In this paper, we demonstrate that p64 becomes tyrosine phosphorylated when co-expressed with p59 fyn in HeLa cells. We show that co-expression of p64 with p59 fyn renders p64 a ligand for the SH2 domain of p59 fyn and this SH2 binding is eliminated by treating p64 with alkaline phosphatase. Using site-directed mutagenesis, we find that tyrosine 33 in the p64 sequence is necessary for SH2 binding. We also characterized p64-p59 fyn interactions using native material from bovine kidney. We found that a small fraction of native kidney p64 can bind Fyn SH2 in vitro. Immunoprecipitation of p64 from solubilized kidney membranes yields a kinase activity with the same mobility by SDS-polyacrylamide gel electrophoresis as authentic bovine p59 fyn . Finally, we demonstrate that co-expression of p64 and p59 fyn in HeLa cells results in enhanced p64-associated chloride channel activity.

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