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• W2023292433 abstract "Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTRHis10) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni2+-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTRHis10 after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel. Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTRHis10) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni2+-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTRHis10 after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel. cystic fibrosis transmembrane conductance regulator cAMP-dependent protein kinase type 1 phosphatase type 2 phosphatase baby hamster kidney nitrilotriacetic acid glutathione S-transferase dithiobis(sulfosuccinimidyl propionate) polyacrylamide gel electrophoresis regulatory dithiothreitol E2, E3, first, second, and third elution fractions wild type CFTR1 is a tightly regulated chloride channel expressed in epithelial and other cell types (1Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79 (suppl.): 77-107Crossref Scopus (369) Google Scholar). CFTR channels become deactivated within ∼10 s after membrane patches are excised from cAMP-stimulated Chinese hamster ovary cells into bath solution containing ATP at 37 °C (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar). Similar results are obtained using Chinese hamster ovary and airway epithelial cell lines at 20 °C except the deactivation requires ∼100 s (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar). Channel rundown is caused by dephosphorylation of PKA sites, because it does not occur if the PKA catalytic subunit is present in the bath solution, and deactivated channels can be restimulated by exposure to PKA (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar, 4Berger H.A. Anderson M.P. Gregory R.J. Thompson S. Howard P.W. Maurer R.A. Mulligan R. Smith A.E. Welsh M.J. J. Clin. Invest. 1991; 88: 1422-1431Crossref PubMed Scopus (183) Google Scholar). These results indicate that at least one of the phosphatases regulating CFTR is membrane-delimited.The phosphatases that control CFTR have not yet been identified definitively at the molecular level. CFTR is regulated by PKA phosphorylation on multiple serine residues (5Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 6Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar, 7Chang X.-B. Tabcharani J.A. Hou Y.-X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar). Serine/threonine protein phosphatases are classified according to their functional properties: Type 1 phosphatases (PP1) dephosphorylate the β subunit of phosphorylase kinase and are inhibited by thermostable protein inhibitors 1 and 2. Type 2 phosphatases (PP2) dephosphorylate the α subunit of phosphorylase kinase and are insensitive to protein inhibitors 1 and 2 (8Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (401) Google Scholar). Among the type 2 protein phosphatases, PP2A is distinguished by its sensitivity to okadaic acid and calyculin A, PP2B by its requirement for Ca2+ and calmodulin and sensitivity to inhibitors such as deltamethrin, and PP2C by its requirement for relatively high (mM) levels of Mg2+.Several phosphatases dephosphorylate full-length CFTR or recombinant R domain protein in vitro (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 9Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar, 10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar), but this does not establish their physiological role because all are usually present in intact cells. The specificity of protein phosphatases depends more on proximity than on the sequence of the target phosphoprotein, hence the importance of determining if CFTR is normally associated with a particular phosphatase.Functional studies implicate PP2C as a CFTR phosphatase in Chinese hamster ovary, baby hamster kidney (BHK), and airway epithelial cells. CFTR rundown is relatively insensitive to okadaic acid and does not require Ca2+ or calmodulin (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar) but is inhibited by low Mg2+ (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). PP2C is the most potent deactivator of CFTR channels among the exogenous phosphatases tested, and like the endogenous (i.e. membrane-associated) CFTR phosphatase in these cell types, exogenous PP2C inhibits the channel by decreasing the rate at which it enters open bursts. None of the purified phosphatases deactivate CFTR channels completely (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar), consistent with the notion that CFTR is regulated by multiple phosphatases (12Hwang T.-C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar). Indeed, 5–10% of the activity remains even when patches are exposed to both PP2C and PP2A simultaneously, suggesting they may not be the only players (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar).Rapid deactivation of CFTR channels in excised patches has led to speculation that CFTR and its phosphatase(s) may be physically associated within a regulatory complex (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar, 3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 13Becq F. Biochim. Biophys. Acta. 1996; 1286: 53-63Crossref PubMed Scopus (32) Google Scholar). In this study we test this possibility in unstimulated BHK cells using co-immunoprecipitations and chemical cross-linking. The results indicate that CFTR is closely associated with PP2C but not PP1, PP2A, or PP2B.DISCUSSIONThe present results demonstrate that PP2C is present in the membrane fraction of BHK cells and is closely associated with CFTR. We confirmed that phosphorylated CFTR is a good substrate for PP2C and found that the susceptibility of bacterially expressed GST-R domain fusion protein to different phosphatases is similar to that of full-length CFTR.Rapid rundown of CFTR channel activity in excised membrane patches results from dephosphorylation of PKA sites; it can be reversed by exposure to the PKA catalytic subunit and does not occur if patches are excised into bath solution containing PKA (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar). The association of CFTR with PP2C observed in the present work may be a widespread phenomenon because rundown with similar characteristics has been observed in many other cell types, including those from tissues affected in cystic fibrosis (e.g. pancreatic ducts (23Gray M.A. Greenwell J.R. Argent B.E. J. Membr. Biol. 1988; 105: 131-142Crossref PubMed Scopus (159) Google Scholar), human colon (17Tabcharani J.A. Low W. Elie D. Hanrahan J.W. FEBS Lett. 1990; 270: 157-164Crossref PubMed Scopus (131) Google Scholar), and human airway cells (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 24Haws C. Finkbeiner W.E. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1994; 266: L502-L512Crossref PubMed Google Scholar).Functional studies of excised patches indicate a major role for membrane-delimited PP2C but do not exclude regulation by PP2A and other phosphatases in situ. Deactivation of CFTR in excised patches is relatively insensitive to okadaic acid and calyculin A, independent of Ca2+ and calmodulin, and inhibited by lowering free magnesium concentration (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar, 3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Transepithelial studies are consistent with a predominant role of PP2C, because deactivation of the short-circuit current after removing forskolin or 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate is unaffected by the PP1 and PP2A inhibitors okadaic acid (10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar) or calyculin A (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Although co-expression of PP2Cα with CFTR in Fisher rat thyroid cells has little effect on the rate of chloride current deactivation after cAMP washout, it inhibits the cAMP-stimulated current by ∼70% (10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar). The isoform of PP2C that regulates CFTR remains to be established. The antibody used in this study would be expected to recognize α and known splice variants of the β isoform because of their homology but not the more distantly related γ isoform (25Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (47) Google Scholar).PP2A regulates CFTR in some cell types, particularly cardiac and sweat ducts (12Hwang T.-C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar, 26Reddy M.M. Quinton P.M. Am. J. Physiol. 1996; 270: C474-C480Crossref PubMed Google Scholar), and we confirmed that PP2C and PP2A are both more effective in dephosphorylating full-length CFTR and recombinant R domain peptide compared with equimolar PP1 or PP2B. However PP2C was more abundant in the membrane fraction from BHK cells, and no association between CFTR and PP2A was detected by immunoprecipitation or chemical cross-linking. By contrast, a specific monoclonal antibody against CFTR (M3A7 (27Kartner N. Riordan J.R. Methods Enzymol. 1998; 292: 629-652Crossref PubMed Scopus (24) Google Scholar)) co-precipitated PP2C but did not bring down PP1 or PP2A. Polyclonal anti-PP2C antibody co-precipitated CFTR, whereas antibodies against PP1, PP2A, and PP2B did not. The association between PP2C and CFTR must be relatively strong, because immunoprecipitations were carried out in the presence of detergent (1% Triton X-100).After exposure of cell lysates to the cross-linker DTSSP and purification by nickel chelate chromatography, CFTRHis10appeared in high molecular mass complexes that also contained PP2C but not PP1, PP2A, or PP2B. Control experiments with untransfected BHK cells or cells expressing CFTR without a polyhistidine tail (data not shown) confirmed that PP2C binding to Ni2+-NTA-agarose requires CFTRHis10. The association between CFTR and PP2C does not require strong phosphorylation of CFTR, because all experiments were carried out using cells that had not been stimulated with cAMP.A stable complex that includes CFTR and PP2C would presumably increase the efficiency of CFTR dephosphorylation. Such targeting of PP2C to particular substrates has not been reported previously, although other protein phosphatases form stable complexes with their substrates. For example, PP2B is associated with a neuronal potassium channel (28Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 1: 4572-4579Crossref Google Scholar) and PP2A is targeted to the microtubule-associated protein tau 32 (29Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar).The monoclonal anti-CFTR antibody M3A7 used to co-precipitate PP2C in this study has been extensively characterized (27Kartner N. Riordan J.R. Methods Enzymol. 1998; 292: 629-652Crossref PubMed Scopus (24) Google Scholar) and did not recognize proteins in control (untransfected) BHK lysates. The polyclonal antibody raised against PP2C was relatively specific but did recognize an unidentified 30-kDa protein, which may be another PP2C isoform.Adding a polyhistidine tag to CFTR did not grossly alter its structure because CFTRHis10 had normal Cl− channel activity when incorporated into planar bilayers. 2D. Dahan, unpublished observation. Also, specific association of PP2C was observed with both wild-type and histidine-tagged CFTR. Any alterations in structure would seem more likely to disrupt than create specific interactions between the proteins, although that possibility cannot be formally excluded. CFTR and PP2C-like phosphatase activities were both present in the membrane fraction, and the Mg2+ dependence of channel rundown in excised patches provides further independent evidence that PP2C and CFTR are normally co-localized at the plasma membrane (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Thus the selective co-precipitation and cross-linking were not artifacts of lysing the cells but likely reflect their co-localization under physiological conditions.Close association of CFTR with PP2C could help explain the robust down-regulation of its channel activity in vivo when PKA stimulation is removed. The phosphatase-regulating CFTR has been suggested as a target for pharmacotherapies aimed at increasing (i.e. restoring) CFTR activity in cystic fibrosis, therefore these observations may have relevance to the development of therapies for cystic fibrosis (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 30Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (712) Google Scholar, 31Becq F. Verrier B. Chang X.-B. Riordan J.R. Hanrahan J.W. J. Biol. Chem. 1996; 271: 16171-16179Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A phosphatase inhibitor might be useful for stimulating mutant channels that reach the plasma membrane when used alone or as an adjunct therapy to increase the efficacy of other treatments such as drugs that improve CFTR processing or gene therapies. Regardless, ion channel rundown is a common phenomenon in patches excised from neuronal, cardiac, and other cell types. It will be interesting to learn if PP2C also forms regulatory complexes with other ion channels besides CFTR and mediates their deactivation. CFTR1 is a tightly regulated chloride channel expressed in epithelial and other cell types (1Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79 (suppl.): 77-107Crossref Scopus (369) Google Scholar). CFTR channels become deactivated within ∼10 s after membrane patches are excised from cAMP-stimulated Chinese hamster ovary cells into bath solution containing ATP at 37 °C (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar). Similar results are obtained using Chinese hamster ovary and airway epithelial cell lines at 20 °C except the deactivation requires ∼100 s (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar). Channel rundown is caused by dephosphorylation of PKA sites, because it does not occur if the PKA catalytic subunit is present in the bath solution, and deactivated channels can be restimulated by exposure to PKA (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar, 4Berger H.A. Anderson M.P. Gregory R.J. Thompson S. Howard P.W. Maurer R.A. Mulligan R. Smith A.E. Welsh M.J. J. Clin. Invest. 1991; 88: 1422-1431Crossref PubMed Scopus (183) Google Scholar). These results indicate that at least one of the phosphatases regulating CFTR is membrane-delimited. The phosphatases that control CFTR have not yet been identified definitively at the molecular level. CFTR is regulated by PKA phosphorylation on multiple serine residues (5Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 6Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar, 7Chang X.-B. Tabcharani J.A. Hou Y.-X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar). Serine/threonine protein phosphatases are classified according to their functional properties: Type 1 phosphatases (PP1) dephosphorylate the β subunit of phosphorylase kinase and are inhibited by thermostable protein inhibitors 1 and 2. Type 2 phosphatases (PP2) dephosphorylate the α subunit of phosphorylase kinase and are insensitive to protein inhibitors 1 and 2 (8Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (401) Google Scholar). Among the type 2 protein phosphatases, PP2A is distinguished by its sensitivity to okadaic acid and calyculin A, PP2B by its requirement for Ca2+ and calmodulin and sensitivity to inhibitors such as deltamethrin, and PP2C by its requirement for relatively high (mM) levels of Mg2+. Several phosphatases dephosphorylate full-length CFTR or recombinant R domain protein in vitro (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 9Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar, 10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar), but this does not establish their physiological role because all are usually present in intact cells. The specificity of protein phosphatases depends more on proximity than on the sequence of the target phosphoprotein, hence the importance of determining if CFTR is normally associated with a particular phosphatase. Functional studies implicate PP2C as a CFTR phosphatase in Chinese hamster ovary, baby hamster kidney (BHK), and airway epithelial cells. CFTR rundown is relatively insensitive to okadaic acid and does not require Ca2+ or calmodulin (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar) but is inhibited by low Mg2+ (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). PP2C is the most potent deactivator of CFTR channels among the exogenous phosphatases tested, and like the endogenous (i.e. membrane-associated) CFTR phosphatase in these cell types, exogenous PP2C inhibits the channel by decreasing the rate at which it enters open bursts. None of the purified phosphatases deactivate CFTR channels completely (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar), consistent with the notion that CFTR is regulated by multiple phosphatases (12Hwang T.-C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar). Indeed, 5–10% of the activity remains even when patches are exposed to both PP2C and PP2A simultaneously, suggesting they may not be the only players (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Rapid deactivation of CFTR channels in excised patches has led to speculation that CFTR and its phosphatase(s) may be physically associated within a regulatory complex (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar, 3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 13Becq F. Biochim. Biophys. Acta. 1996; 1286: 53-63Crossref PubMed Scopus (32) Google Scholar). In this study we test this possibility in unstimulated BHK cells using co-immunoprecipitations and chemical cross-linking. The results indicate that CFTR is closely associated with PP2C but not PP1, PP2A, or PP2B. DISCUSSIONThe present results demonstrate that PP2C is present in the membrane fraction of BHK cells and is closely associated with CFTR. We confirmed that phosphorylated CFTR is a good substrate for PP2C and found that the susceptibility of bacterially expressed GST-R domain fusion protein to different phosphatases is similar to that of full-length CFTR.Rapid rundown of CFTR channel activity in excised membrane patches results from dephosphorylation of PKA sites; it can be reversed by exposure to the PKA catalytic subunit and does not occur if patches are excised into bath solution containing PKA (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar). The association of CFTR with PP2C observed in the present work may be a widespread phenomenon because rundown with similar characteristics has been observed in many other cell types, including those from tissues affected in cystic fibrosis (e.g. pancreatic ducts (23Gray M.A. Greenwell J.R. Argent B.E. J. Membr. Biol. 1988; 105: 131-142Crossref PubMed Scopus (159) Google Scholar), human colon (17Tabcharani J.A. Low W. Elie D. Hanrahan J.W. FEBS Lett. 1990; 270: 157-164Crossref PubMed Scopus (131) Google Scholar), and human airway cells (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 24Haws C. Finkbeiner W.E. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1994; 266: L502-L512Crossref PubMed Google Scholar).Functional studies of excised patches indicate a major role for membrane-delimited PP2C but do not exclude regulation by PP2A and other phosphatases in situ. Deactivation of CFTR in excised patches is relatively insensitive to okadaic acid and calyculin A, independent of Ca2+ and calmodulin, and inhibited by lowering free magnesium concentration (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar, 3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Transepithelial studies are consistent with a predominant role of PP2C, because deactivation of the short-circuit current after removing forskolin or 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate is unaffected by the PP1 and PP2A inhibitors okadaic acid (10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar) or calyculin A (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Although co-expression of PP2Cα with CFTR in Fisher rat thyroid cells has little effect on the rate of chloride current deactivation after cAMP washout, it inhibits the cAMP-stimulated current by ∼70% (10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar). The isoform of PP2C that regulates CFTR remains to be established. The antibody used in this study would be expected to recognize α and known splice variants of the β isoform because of their homology but not the more distantly related γ isoform (25Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (47) Google Scholar).PP2A regulates CFTR in some cell types, particularly cardiac and sweat ducts (12Hwang T.-C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar, 26Reddy M.M. Quinton P.M. Am. J. Physiol. 1996; 270: C474-C480Crossref PubMed Google Scholar), and we confirmed that PP2C and PP2A are both more effective in dephosphorylating full-length CFTR and recombinant R domain peptide compared with equimolar PP1 or PP2B. However PP2C was more abundant in the membrane fraction from BHK cells, and no association between CFTR and PP2A was detected by immunoprecipitation or chemical cross-linking. By contrast, a specific monoclonal antibody against CFTR (M3A7 (27Kartner N. Riordan J.R. Methods Enzymol. 1998; 292: 629-652Crossref PubMed Scopus (24) Google Scholar)) co-precipitated PP2C but did not bring down PP1 or PP2A. Polyclonal anti-PP2C antibody co-precipitated CFTR, whereas antibodies against PP1, PP2A, and PP2B did not. The association between PP2C and CFTR must be relatively strong, because immunoprecipitations were carried out in the presence of detergent (1% Triton X-100).After exposure of cell lysates to the cross-linker DTSSP and purification by nickel chelate chromatography, CFTRHis10appeared in high molecular mass complexes that also contained PP2C but not PP1, PP2A, or PP2B. Control experiments with untransfected BHK cells or cells expressing CFTR without a polyhistidine tail (data not shown) confirmed that PP2C binding to Ni2+-NTA-agarose requires CFTRHis10. The association between CFTR and PP2C does not require strong phosphorylation of CFTR, because all experiments were carried out using cells that had not been stimulated with cAMP.A stable complex that includes CFTR and PP2C would presumably increase the efficiency of CFTR dephosphorylation. Such targeting of PP2C to particular substrates has not been reported previously, although other protein phosphatases form stable complexes with their substrates. For example, PP2B is associated with a neuronal potassium channel (28Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 1: 4572-4579Crossref Google Scholar) and PP2A is targeted to the microtubule-associated protein tau 32 (29Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar).The monoclonal anti-CFTR antibody M3A7 used to co-precipitate PP2C in this study has been extensively characterized (27Kartner N. Riordan J.R. Methods Enzymol. 1998; 292: 629-652Crossref PubMed Scopus (24) Google Scholar) and did not recognize proteins in control (untransfected) BHK lysates. The polyclonal antibody raised against PP2C was relatively specific but did recognize an unidentified 30-kDa protein, which may be another PP2C isoform.Adding a polyhistidine tag to CFTR did not grossly alter its structure because CFTRHis10 had normal Cl− channel activity when incorporated into planar bilayers. 2D. Dahan, unpublished observation. Also, specific association of PP2C was observed with both wild-type and histidine-tagged CFTR. Any alterations in structure would seem more likely to disrupt than create specific interactions between the proteins, although that possibility cannot be formally excluded. CFTR and PP2C-like phosphatase activities were both present in the membrane fraction, and the Mg2+ dependence of channel rundown in excised patches provides further independent evidence that PP2C and CFTR are normally co-localized at the plasma membrane (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Thus the selective co-precipitation and cross-linking were not artifacts of lysing the cells but likely reflect their co-localization under physiological conditions.Close association of CFTR with PP2C could help explain the robust down-regulation of its channel activity in vivo when PKA stimulation is removed. The phosphatase-regulating CFTR has been suggested as a target for pharmacotherapies aimed at increasing (i.e. restoring) CFTR activity in cystic fibrosis, therefore these observations may have relevance to the development of therapies for cystic fibrosis (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 30Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (712) Google Scholar, 31Becq F. Verrier B. Chang X.-B. Riordan J.R. Hanrahan J.W. J. Biol. Chem. 1996; 271: 16171-16179Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A phosphatase inhibitor might be useful for stimulating mutant channels that reach the plasma membrane when used alone or as an adjunct therapy to increase the efficacy of other treatments such as drugs that improve CFTR processing or gene therapies. Regardless, ion channel rundown is a common phenomenon in patches excised from neuronal, cardiac, and other cell types. It will be interesting to learn if PP2C also forms regulatory complexes with other ion channels besides CFTR and mediates their deactivation. The present results demonstrate that PP2C is present in the membrane fraction of BHK cells and is closely associated with CFTR. We confirmed that phosphorylated CFTR is a good substrate for PP2C and found that the susceptibility of bacterially expressed GST-R domain fusion protein to different phosphatases is similar to that of full-length CFTR. Rapid rundown of CFTR channel activity in excised membrane patches results from dephosphorylation of PKA sites; it can be reversed by exposure to the PKA catalytic subunit and does not occur if patches are excised into bath solution containing PKA (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar). The association of CFTR with PP2C observed in the present work may be a widespread phenomenon because rundown with similar characteristics has been observed in many other cell types, including those from tissues affected in cystic fibrosis (e.g. pancreatic ducts (23Gray M.A. Greenwell J.R. Argent B.E. J. Membr. Biol. 1988; 105: 131-142Crossref PubMed Scopus (159) Google Scholar), human colon (17Tabcharani J.A. Low W. Elie D. Hanrahan J.W. FEBS Lett. 1990; 270: 157-164Crossref PubMed Scopus (131) Google Scholar), and human airway cells (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 24Haws C. Finkbeiner W.E. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1994; 266: L502-L512Crossref PubMed Google Scholar). Functional studies of excised patches indicate a major role for membrane-delimited PP2C but do not exclude regulation by PP2A and other phosphatases in situ. Deactivation of CFTR in excised patches is relatively insensitive to okadaic acid and calyculin A, independent of Ca2+ and calmodulin, and inhibited by lowering free magnesium concentration (2Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (453) Google Scholar, 3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Transepithelial studies are consistent with a predominant role of PP2C, because deactivation of the short-circuit current after removing forskolin or 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate is unaffected by the PP1 and PP2A inhibitors okadaic acid (10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar) or calyculin A (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Although co-expression of PP2Cα with CFTR in Fisher rat thyroid cells has little effect on the rate of chloride current deactivation after cAMP washout, it inhibits the cAMP-stimulated current by ∼70% (10Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar). The isoform of PP2C that regulates CFTR remains to be established. The antibody used in this study would be expected to recognize α and known splice variants of the β isoform because of their homology but not the more distantly related γ isoform (25Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (47) Google Scholar). PP2A regulates CFTR in some cell types, particularly cardiac and sweat ducts (12Hwang T.-C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar, 26Reddy M.M. Quinton P.M. Am. J. Physiol. 1996; 270: C474-C480Crossref PubMed Google Scholar), and we confirmed that PP2C and PP2A are both more effective in dephosphorylating full-length CFTR and recombinant R domain peptide compared with equimolar PP1 or PP2B. However PP2C was more abundant in the membrane fraction from BHK cells, and no association between CFTR and PP2A was detected by immunoprecipitation or chemical cross-linking. By contrast, a specific monoclonal antibody against CFTR (M3A7 (27Kartner N. Riordan J.R. Methods Enzymol. 1998; 292: 629-652Crossref PubMed Scopus (24) Google Scholar)) co-precipitated PP2C but did not bring down PP1 or PP2A. Polyclonal anti-PP2C antibody co-precipitated CFTR, whereas antibodies against PP1, PP2A, and PP2B did not. The association between PP2C and CFTR must be relatively strong, because immunoprecipitations were carried out in the presence of detergent (1% Triton X-100). After exposure of cell lysates to the cross-linker DTSSP and purification by nickel chelate chromatography, CFTRHis10appeared in high molecular mass complexes that also contained PP2C but not PP1, PP2A, or PP2B. Control experiments with untransfected BHK cells or cells expressing CFTR without a polyhistidine tail (data not shown) confirmed that PP2C binding to Ni2+-NTA-agarose requires CFTRHis10. The association between CFTR and PP2C does not require strong phosphorylation of CFTR, because all experiments were carried out using cells that had not been stimulated with cAMP. A stable complex that includes CFTR and PP2C would presumably increase the efficiency of CFTR dephosphorylation. Such targeting of PP2C to particular substrates has not been reported previously, although other protein phosphatases form stable complexes with their substrates. For example, PP2B is associated with a neuronal potassium channel (28Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 1: 4572-4579Crossref Google Scholar) and PP2A is targeted to the microtubule-associated protein tau 32 (29Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). The monoclonal anti-CFTR antibody M3A7 used to co-precipitate PP2C in this study has been extensively characterized (27Kartner N. Riordan J.R. Methods Enzymol. 1998; 292: 629-652Crossref PubMed Scopus (24) Google Scholar) and did not recognize proteins in control (untransfected) BHK lysates. The polyclonal antibody raised against PP2C was relatively specific but did recognize an unidentified 30-kDa protein, which may be another PP2C isoform. Adding a polyhistidine tag to CFTR did not grossly alter its structure because CFTRHis10 had normal Cl− channel activity when incorporated into planar bilayers. 2D. Dahan, unpublished observation. Also, specific association of PP2C was observed with both wild-type and histidine-tagged CFTR. Any alterations in structure would seem more likely to disrupt than create specific interactions between the proteins, although that possibility cannot be formally excluded. CFTR and PP2C-like phosphatase activities were both present in the membrane fraction, and the Mg2+ dependence of channel rundown in excised patches provides further independent evidence that PP2C and CFTR are normally co-localized at the plasma membrane (11Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). Thus the selective co-precipitation and cross-linking were not artifacts of lysing the cells but likely reflect their co-localization under physiological conditions. Close association of CFTR with PP2C could help explain the robust down-regulation of its channel activity in vivo when PKA stimulation is removed. The phosphatase-regulating CFTR has been suggested as a target for pharmacotherapies aimed at increasing (i.e. restoring) CFTR activity in cystic fibrosis, therefore these observations may have relevance to the development of therapies for cystic fibrosis (3Becq F. Jensen T.J. Chang X.-B. Savoia A. Rommens J.M. Tsui L.-C. Buchwald M. Riordan J.R. Hanrahan J.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9160-9164Crossref PubMed Scopus (146) Google Scholar, 30Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (712) Google Scholar, 31Becq F. Verrier B. Chang X.-B. Riordan J.R. Hanrahan J.W. J. Biol. Chem. 1996; 271: 16171-16179Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A phosphatase inhibitor might be useful for stimulating mutant channels that reach the plasma membrane when used alone or as an adjunct therapy to increase the efficacy of other treatments such as drugs that improve CFTR processing or gene therapies. Regardless, ion channel rundown is a common phenomenon in patches excised from neuronal, cardiac, and other cell types. It will be interesting to learn if PP2C also forms regulatory complexes with other ion channels besides CFTR and mediates their deactivation." @default.
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• W2023292433 title "Association of Cystic Fibrosis Transmembrane Conductance Regulator and Protein Phosphatase 2C" @default.
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