Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum

F. S. Seibert, Xiu-Bao D Chang, A. A. Aleksandrov, D. M. Clarke, J. W. Hanrahan, J. R. Riordan

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl- channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed 32P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl- channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down- regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case. Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl- channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as ΔF508 do not generate Cl- active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascent chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containing vesicles into the plasma membrane as part of the mechanism of stimulation of chloride secretion has been reported, as has an influence of CFTR on the balance between endocytosis and exocytosis but these findings have not been universally confirmed.

Original languageEnglish (US)
Pages (from-to)275-283
Number of pages9
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1461
Issue number2
DOIs
StatePublished - Dec 6 1999

Fingerprint

Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Endoplasmic Reticulum
Phosphoproteins
Chemical activation
Cells
Proteolysis
Phosphopeptides
Cystic Fibrosis Transmembrane Conductance Regulator
Mutagenesis
Exocytosis
Cell membranes
Microsomes
Endocytosis
Phosphoric Monoester Hydrolases
Labeling
Chlorides
Phosphotransferases
Cell Membrane
Degradation

Keywords

  • CFTR
  • Chloride channel
  • Cystic fibrosis
  • Endoplasmic reticulum
  • Phosphorylation
  • Protein kinase A

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics

Cite this

Seibert, F. S., Chang, X-B. D., Aleksandrov, A. A., Clarke, D. M., Hanrahan, J. W., & Riordan, J. R. (1999). Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum. Biochimica et Biophysica Acta - Biomembranes, 1461(2), 275-283. https://doi.org/10.1016/S0005-2736(99)00163-7

Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum. / Seibert, F. S.; Chang, Xiu-Bao D; Aleksandrov, A. A.; Clarke, D. M.; Hanrahan, J. W.; Riordan, J. R.

In: Biochimica et Biophysica Acta - Biomembranes, Vol. 1461, No. 2, 06.12.1999, p. 275-283.

Research output: Contribution to journalArticle

Seibert, F. S. ; Chang, Xiu-Bao D ; Aleksandrov, A. A. ; Clarke, D. M. ; Hanrahan, J. W. ; Riordan, J. R. / Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum. In: Biochimica et Biophysica Acta - Biomembranes. 1999 ; Vol. 1461, No. 2. pp. 275-283.
@article{a06bd833e8464385818379b978302404,
title = "Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum",
abstract = "CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl- channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed 32P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl- channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down- regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case. Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl- channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as ΔF508 do not generate Cl- active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascent chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containing vesicles into the plasma membrane as part of the mechanism of stimulation of chloride secretion has been reported, as has an influence of CFTR on the balance between endocytosis and exocytosis but these findings have not been universally confirmed.",
keywords = "CFTR, Chloride channel, Cystic fibrosis, Endoplasmic reticulum, Phosphorylation, Protein kinase A",
author = "Seibert, {F. S.} and Chang, {Xiu-Bao D} and Aleksandrov, {A. A.} and Clarke, {D. M.} and Hanrahan, {J. W.} and Riordan, {J. R.}",
year = "1999",
month = "12",
day = "6",
doi = "10.1016/S0005-2736(99)00163-7",
language = "English (US)",
volume = "1461",
pages = "275--283",
journal = "Biochimica et Biophysica Acta - Biomembranes",
issn = "0005-2736",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum

AU - Seibert, F. S.

AU - Chang, Xiu-Bao D

AU - Aleksandrov, A. A.

AU - Clarke, D. M.

AU - Hanrahan, J. W.

AU - Riordan, J. R.

PY - 1999/12/6

Y1 - 1999/12/6

N2 - CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl- channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed 32P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl- channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down- regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case. Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl- channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as ΔF508 do not generate Cl- active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascent chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containing vesicles into the plasma membrane as part of the mechanism of stimulation of chloride secretion has been reported, as has an influence of CFTR on the balance between endocytosis and exocytosis but these findings have not been universally confirmed.

AB - CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl- channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed 32P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl- channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down- regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case. Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl- channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as ΔF508 do not generate Cl- active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascent chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containing vesicles into the plasma membrane as part of the mechanism of stimulation of chloride secretion has been reported, as has an influence of CFTR on the balance between endocytosis and exocytosis but these findings have not been universally confirmed.

KW - CFTR

KW - Chloride channel

KW - Cystic fibrosis

KW - Endoplasmic reticulum

KW - Phosphorylation

KW - Protein kinase A

UR - http://www.scopus.com/inward/record.url?scp=0032725782&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032725782&partnerID=8YFLogxK

U2 - 10.1016/S0005-2736(99)00163-7

DO - 10.1016/S0005-2736(99)00163-7

M3 - Article

C2 - 10581361

AN - SCOPUS:0032725782

VL - 1461

SP - 275

EP - 283

JO - Biochimica et Biophysica Acta - Biomembranes

JF - Biochimica et Biophysica Acta - Biomembranes

SN - 0005-2736

IS - 2

ER -