Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated Cl- channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial Cl- secretion. Previous studies have shown that cAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR Cl- channels. We asked whether other kinases would phosphorylate CFTR in vitro and activate CFTR Cl- channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca(2+)-independent and Ca(2+)-dependent isoforms of protein kinase C (PKC) activated the CFTR Cl- channel. Consistent with this finding, PKC also phosphorylated CFTR in vitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR Cl- channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in vitro. However, cGMP failed to increase the apical membrane Cl- permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR Cl- channels. These results suggest that if cGK phosphorylates CFTR in vivo, it does so at sites not involved in CFTR Cl- channel activation. Because cAMP-dependent activation of CFTR Cl- channels and Cl- secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR Cl- channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR Cl- channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in vitro. Our data indicate that CFTR Cl- channels can be phosphorylated and activated by PKA as well as by Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC and can be dephosphorylated and thus inactivated by PP2A.
Highlights
From the Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa52242
Previous studies have shown that CAMP-dependent protein kinase (PKA)can phosphorylate and activateCFTR C1- chan- Cystic fibrosis (CF)‘ is a genetic disease characterized by nels
CGMP failed toincreasetheapical membrane C1- Subsequent studies showed that cAMP agonists activated permeability in human airway epithelia, and addition CFTR C1- channels in heterologous cell expression systems of cGMP, ATP, and cGMP-dependent protein kinase (cGK) failed to activate CFTR C1- [8,9,10]
Summary
Protein Kinase C-Fig. 1, A and B, shows the effect of mm dish were solubilized in 1ml of lysis buffer: 50 mM Tris-HC1, pH adding PKC to an excised, cell-free patch from a 3T3 fibro-. CFTR C1- current activated by PKC alone or by PKC plus PKArequired the continuous presence of ATP on periments,CFTR wassolubilized, immunoprecipitated,andphos- the cytosolic surface; we have previously shown that CFTR phorylated by PKA as described above except that the phosphorylation reaction was conducted a t 30 "C for 30 min in 400 p1 of buffer containing 50 mM PIPES, pH 6.8, 10 mM MgCI,, 100 pg/ml bovine serumalbumin, 100 p M ATP, 5 Ci/mmol [y-"PIATP,and 75 nM PKA catalytic subunit. C1- channel activity requires ATP in a process independent of phosphorylation [13].Fig. 1B shows that when we removed PKC, PKA, DiC8, and ATP, current returned to near baseline values. During these experiments,we frequently assessed volume of 12 MM PKI and cooling the tube on ice; this was sufficent the current-voltage relationship t,o check that the PKC-acti-
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