CFTR CHLORIDE CHANNEL ACTIVATION BY GENISTEIN - THE ROLE OF SERINE THREONINE PROTEIN PHOSPHATASES/

We have previously shown [B. Illek, H. Fischer, G. F. Santos, J. H. Wi ddicomhe, T. E. Machen, and W. W. Reenstra, Am. J. Physiol. 268 (Cell Physiol, 37): C886-C893, 1995] that genistein, a tyrosine kinase inhib itor, activates the cystic fibrosis transmembrane conductance regulato r (CFTR) chloride channel in NIH/3T3 cells that have been stably trans fected with an expression vector for the CFTR (NIH-CFTR cells). In thi s study, we present evidence suggesting that both genistein and the se rine/threonine protein phosphatase (PPase) inhibitor calyculin A activ ate the CFTR by inhibiting PPase activity. As measured by I-125 efflux , genistein and calyculin A stimulate the CFTR, to similar to 50% of t he maximal activity with forskolin. Neither agonist increases CFTR act ivity at saturating forskolin concentrations, but genistein and calycu lin A have an additive effect on CFTR activity. Forskolin, but neither genistein nor calyculin A, stimulates protein kinase A (PK/4) activit y. The PKA inhibitor H-89 inhibits CFTR activation and in vivo phospho rylation by all three agonists. Proteolytic digestion of in vivo phosp horylated CFTR suggests that tile CFTR is phosphorylated on the same s ites during stimulation with genistein and forskolin but on different sites during stimulation with calyculin A. The data suggest that genis tein and calyculin A inhibit different PPase activities, allowing CFTR , phosphorylation and partial stimulation, by a basal PKA activity.