Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole

Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis tra nsmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined th e possible role of phosphatases in stimulation by these drugs using patch-c lamp and biochemical methods. Genistein inhibited the spontaneous rundown o f channel activity that occurs after membrane patches are excised from cAMP -stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [P-32]P O4 release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR c hannels, but, in marked contrast to genistein, it did inhibit all four prot ein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was obser ved with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)-p-Br-t, a stereoisomer of Br-t that does not inhibit alkal ine phosphatases. Br-t appeared to act exclusively through phosphatases sin ce it did not affect CFTR channels in patches that had low apparent endogen ous phosphatase activity (i.e., those lacking spontaneous rundown). We conc lude that genistein and Br-t act through different mechanisms. Genistein st imulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibit ing a membrane-associated protein phosphatase (probably PP2C) that presumab ly allows basal phosphorylation to accumulate.