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. 1997 Sep 30;94(20):11055-60.
doi: 10.1073/pnas.94.20.11055.

Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

S M Travis  1 , H A Berger, M J Welsh
Affiliations

Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

S M Travis et al. Proc Natl Acad Sci U S A. .

Abstract

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Calpha is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Calpha and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Calpha inactivated CFTR Cl- channels; reactivation required readdition of kinase. Finally, coexpression of PP2Calpha with CFTR in epithelia reduced the Cl- current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

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Figures

Figure 3
Figure 3
Expression, purification, and properties of recombinant PP2Cα. (A) Coomassie blue-stained SDS/10% polyacrylamide gel showing proteins in uninduced and induced bacteria and PP2Cα purified by nickel-affinity chromatography. (B) Effects of Mg2+ or Mn2+ on phosphatase activity. Activity was measured with casein as the substrate under standard assay conditions except the total concentration of Mg2+ or Mn2+ was buffered with sodium citrate. Data are mean ± SEM of triplicate determinations. (C) Properties of PP2Cα activity. Phosphatase activity of PP2Cα and PP2A toward casein was measured under standard assay conditions in the absence and presence of 100 μM sodium orthovanadate or 10 μM okadaic acid. The incubation time and enzyme concentration were chosen to give incomplete dephosphorylation of casein in the absence of inhibitor; in the control samples, PP2A dephosphorylated casein 36%, and PP2Cα dephosphorylated it 55%. Data are mean ± SEM of triplicate determinations.
Figure 1
Figure 1
Effect of okadaic acid on CFTR Cl− current. Primary cultures of human airway epithelia (Left) or T84 cells (Right) were grown on permeable supports; apical membrane current was recorded after the basolateral membrane was permeabilized with S. aureus α-toxin. Apical membrane Cl− current was activated by 100 μM cAMP during time indicated by the bars, in the absence (A and D) or in the continuous presence (B and E) of 10 μM okadaic acid. For experiments with okadaic acid, permeabilized monolayers were preincubated with the drug for 30 min. C and F show mean ± SEM of Cl− current in the presence of cAMP and after its removal; n = 4. “Wash” refers to the period after removal of cAMP. Currents were determined as the average of at least the last 1 min of the intervention.
Figure 2
Figure 2
Products of RT-PCR of PP2Cα from human airway and T84 cells. RNA was reverse transcribed from mRNA by using a PP2Cα-specific primer, then amplified with two additional PP2Cα-specific primers. The presence and absence of RT is indicated.
Figure 4
Figure 4
PP2Cα dephosphorylation of CFTR and an R domain peptide in vitro. Substrates were phosphorylated with PKA and [γ-32P]ATP as described in the text. (A) CFTR was incubated without PP2Cα (lane 1), with PP2Cα (lane 2), or with PP2Cα and 10 μM okadaic acid (lane 3). In lane 1, the two glycosylated forms of CFTR are apparent. (B) R1 was incubated with PP2Cα for the time indicated. Samples were analyzed by SDS/PAGE and autoradiography.
Figure 5
Figure 5
Effect of PP2Cα on CFTR Cl− current in excised membrane patches. (A) Time course of current in an excised, inside-out membrane patch from HeLa cells transiently expressing CFTR. Current had been activated by PKA and ATP. ATP (1 mM), PKA (75 nM), and PP2Cα (0.1 unit/ml) were present in the cytosolic solution during times indicated by bars. (B) Average current in the absence or presence of PP2Cα, determined as the average of at least the last 1 min of that intervention (n = 5). Asterisk indicates a significant difference in current in the absence and presence of PP2Cα (P < 0.00001).
Figure 6
Figure 6
Effect of coexpressing PP2Cα and CFTR on Cl− current in FRT epithelia. (A) Current activated with 500 μM cpt-cAMP in FRT epithelia expressing CFTR, or CFTR plus PP2Cα. (B) Current activated by 500 μM cpt-cAMP or 10 μM forskolin in FRT expressing CFTR, CFTR plus PP2Cα, or CFTR plus β-galactosidase (control) expressed as percent of current activated in FRT expressing only CFTR. Asterisk indicates a significant difference in current compared with FRT expressing CFTR alone (P < 0.00001, n = 8). (C) Time course of current inactivation. Current was recorded as a percent of current measured 1 min prior to removal of 500 μM cpt-cAMP (time zero). Data are mean (thick line) and SEM (thin lines), n = 8.
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