REGULATION OF CFTR BY PROTEIN PHOSPHATASE 2B AND PROTEIN-KINASE-C

The activity of the CFTR Cl- channel is dependent on its phosphorylati on status set by kinases and phosphatases. We report here that protein phosphatase 2B (PP2B) and protein kinase C (PKC) are potential regula tors of the cystic fibrosis conductance regulator (CFTR). Treating CFT R-expressing 3T3 cells with either of the two specific PP2B blockers c yclosporin A (CsA, 1 mu M) or deltamethrin (DM, 30 nM) caused rapid ac tivation of CFTR in cell-attached patches. As determined by noise anal ysis of multi channel patches, DM-or CsA-activated CFTR displayed gati ng kinetics comparable to those of forskolin-activated CFTR. After act ivation of CFTR by blocking PP2B, CFTR still inactivated. CFTR-mediate d currents were, on average, 6.1 times larger when cells were stimulat ed by forskolin during PP2B block compared to stimulation by forskolin alone. This suggests that, in CFTR-expressing 3T3 cells, a phosphoryl ation site of CFTR is regulated by cellular PKA, PP2B and another phos phatase. However, in the epithelial cell lines Calu-3 and HT-29/B6, Cs A and DM had no effect on CFTR activity in both cell-attached patch-cl amp and transepithelial experiments. In contrast, when exogenous PP2B was added to patches excised from 3T3 or Calu-3 cells, PKA-activated C FTR currents were quickly inactivated. This indicates that free exogen ous PP2B can inactivate CFTR in patches from both cell types. We propo se that in order to reg regulate CFTR in an intact cell, PP2B may requ ire a selective subcellular localization to become active. When excise d patches were PKC-phosphorylated, the gating kinetics of CFTR were si gnificantly different from those of PKA-phosphorylated CFTR. Addition of PP2B also inactivated PKC-activated CFTR showing the indiscriminate dephosphorylation of different phosphorylation sites by PP2B.