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Phosphorylation of the cystic fibrosis transmembrane conductance regulator
M R Picciotto1, J A Cohn, G Bertuzzi
1Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Abstract:
Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.
Insights
Cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate cystic fibrosis transmembrane conductance regulator (CFTR) through direct phosphorylation of its R-domain. This phosphorylation impacts epithelial chloride flux, crucial for cystic fibrosis treatment.
Area of Science:
- Molecular Biology
- Cellular Physiology
Background:
- Epithelial chloride flux is critical for physiological processes and is impaired in cystic fibrosis.
- The cystic fibrosis transmembrane conductance regulator (CFTR) protein plays a key role in regulating chloride ion transport.
- Phosphorylation is a key regulatory mechanism for many proteins, including ion channels.
Purpose of the Study:
- To investigate the specific protein kinases involved in the regulation of CFTR.
- To identify the phosphorylation sites on the CFTR protein.
- To elucidate the role of second messenger pathways in CFTR phosphorylation.
Main Methods:
- Expression and purification of a CFTR R-domain fragment (CF-2) from E. coli.
- In vitro phosphorylation assays using various protein kinases (PKA, PKC, PKG, CaM kinase I).
- Amino acid sequencing and peptide mapping to identify phosphorylation sites.
- In vivo studies using 32P-labeled T84 cells and immunoprecipitation.
Main Results:
- CFTR R-domain (CF-2) was phosphorylated by PKA, PKC, PKG, and CaM kinase I.
- Specific serine residues (e.g., 660, 700, 737, 813, 768, 795, 686, 790) were identified as phosphorylation sites for different kinases.
- In vitro phosphorylation of CFTR occurred at the same sites identified in CF-2.
- Agents activating PKA or PKC stimulated CFTR phosphorylation in T84 cells.
Conclusions:
- CFTR phosphorylation is mediated by multiple protein kinases, including PKA and PKC.
- Specific sites within the CFTR R-domain are targeted by these kinases.
- Regulation of CFTR function is likely achieved through direct phosphorylation by kinases activated by distinct second messenger pathways.