Regulation of the bovine kidney microsomal chloride channel p64 by p59fyn, a Src family tyrosine kinase.
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.
Edwards
J C
JC
Renal Division, Department of Medicine, St. Louis University and the St. Louis Veterans Affairs Medical Center, St. Louis, Missouri 63106, USA. John.Edwards3@med.va.gov
Kapadia
S
S
D000818:Animals; D002417:Cattle; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D016296:Mutagenesis; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
C489932:CLCNKA protein, human; D018118:Chloride Channels; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases