Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.
Highlights
The cystic fibrosis transmembrane conductancereg- ity of this channel is regulated by the combined action of ulator (CFTR) plays a central role in transepithelial protein kinases, primarily protein kinase A [7,8,9,10] and the ion transport by acting as a tightly regulated apical interaction of ATP with specific nucleotide binding folds [11]. chloride channel
The cystic fibrosis transmembrane conductance regulator, the product of the gene mutated in patients with cystic fibrosis (CF’; 1-3), is anepithelial anion channelinvolved in both the secretion and reabsorption ofC1- [4,5]
No combination of PKA phosphorylation sites which we have studied was critical for the response, and significant activation was still observed when mutagenesis was used to eliminate all 10 strong consensus sites (-R/KR/KXS/T-) in the molecule, The abbreviations used are: CF, cystic fibrosis; CFTR, cystic implying that additional unidentified sitesnot adhering fibrosis transmembrane conductance regulator; CHO, Chinese ham- strictly to thisconsensus may contribute to theactivation. ster ovary; Me2S0, dimethyl sulfoxide; IBMX, 3-isobutyl-I-methylxanthine; PCR,polymerase chain reaction; PKA, proteinkinase A PKC, protein kinase C; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid; DHFR, dihydrofolate reductase; bp, base In VitroMutagenesis of CFTR cDNA-We have inserted the coding pair(s)
Summary
Patch-clump Studieosf CFTR-expressing Cells-CHO cells expressing wild-type or mutant CFTR were cultured on glass coverslips a t 37 "Cin 5% COZ for 1-4 days before use. I- Efflux and Cl- Channel Activity-Fig. 3A shows that the simultaneous conversionof serines at positions 660, 737,795, subunit. Ecause we were initially surprisedat the amouonft activity remaining in view of the report of Cheng et al [8], the portion of the CFTR sequence containing thefour mutations was amplified by PCR using DNA from the same CHO cells employed in the functional assay. Phorylation a t these sites is a consequence of the negative charges on the phosphoryl groups, we constructed an analogous multiple mutant witghlutamic acid replacingthe serines a t these four positions (4SE). This apparently did not result in constitutive channel activity (Fig. 4F). Single channel recording did not reveal anyqualitative differencesbetween the gating behaviorof wild-type CFTR and4SA; channels showed voltage-dependent flickering and a wavelike pattern of activ-
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