| Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) is tightly regulated by cAMP-dependent phosphorylation, but the protein phosphatases (PP) that dephosphorylate and inactivate CFTR remain poorly understood. Differential regulation of single CFTR channels by protein phosphatase 2C (PP2C), protein phosphatase 2A (PP2A) and other phosphatases has been reported. The results suggest multiple protein phosphatases are required for complete CFTR deactivation, and that PP2C may be the primary phosphatase regulating CFTR in human airway and colonic epithelia, two major sites of disease.; The goal of this project was to identify PP2C isoforms that regulate CFTR. Six isoforms of PP2C reported previously in mouse were recently recloned in this laboratory with epitope tags, and their interaction with CFTR was assessed by co-immunoprecipitation. Preliminary results indicated that MB2.1, one of the PP2Cbeta isoforms, and MA3, one of the PP2Calpha isoforms, interact with CFTR. To overexpress these phosphatases, I developed two inducible systems in which MB2.1 or MA3 gene expression is controlled by a promoter that is activated upon treatment of cells with ponasterone A, an ecdysone analogue. Although specific PP2C inhibitors are not presently available, mutagenesis and enzyme assays have identified residues that are essential for its enzymatic activity. I made PP2Calpha and PP2Cbeta mutants which are predicted to act as dominant negative inhibitors, and have stably transfected them using the inducible gene expression system. Furthermore, I showed that MB2.1 and MA3 are functionally expressed in the inducible expression system and that site-directed mutagenesis inactivated their phosphatase activity as assayed using phosphatase p-nitrophenyl phosphate (pNPP) as a substrate. Baby Hamster Kidney (BHK) cells stably expressing CFTR + inducible MB2.1, MA3 or their dominant negative mutants, are presently being used in iodide efflux assays to study the downregulation of CFTR by MB2.1 and MA3. Based on results, we exclude PP2C beta regulating CFTR. However, if PP2C alpha is the phosphatase which dephosphorylates CFTR can not be tested because of iodide loading problem. |
