The disease cystic fibrosis results from reduced epithelial Cl-permeability due to mutations in the gene encoding the cystic fibrosis transmembrane conductances regulator (CFTR) Cl- channel. CFTR channels, like all other members of the family of ATP-binding cassette (ABC) transporters, incorporates two nucleotide binding domains (NBDs) but also includes a unique regulatory R domain containing more than 10 consensus sites for phosphorylation by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). In cells with normal CFTR channels, receptor-mediated activation of PKA causes phosphorylation of several R-domain serines, permitting channel opening and closing via cycles involving ATP hydrolysis. We have recently obtained strong evidence that ATP hydrolysis energizes the conformational change that opens the channel gate, and that the degree of phosphorylation of a channel is one of the determinants of how long the gate stays open. Our working hypothesis is that phosphorylation of particular serines controls, independently, the function of the two NBDs. In a strongly phosphorylated channel with both NBDs functional, then hydrolysis of ATP at one NBD opens the channel, whereupon a second ATP can bind at the other NBD and in so doing can stabilize the open conformation. Hydrolysis of that second ATP then abolishes the stabilization, prompting channel closure. We have also hypothesized that distinct cellular phosphatases differentially dephosphorylate the various phosphoserines. Hence, in the cell, activation or inhibition of specific phosphatases could contribute to the complex mechanisms that regulate channel gating.
The aim of this project is to learn which serines are phosphorylated under which experimental condition, with the goal of eventually discerning the exact role of each phosphoserine in orchestrating the function of the individual channel domains. The approach is to correlate biochemical information on phosphorylation with precise assays of function at the single channel level. Traditionally, identification of phosphorylated serines has been accomplished by tryptic digestion followed by 2-D phosphopeptide mapping and either direct sequencing or comparison with appropriate (serine-alanine) mutant peptides. The spectacular initial results obtained using mass spectrometry to identify phosphorylated serines in phosphopeptides, however indicate that this will be our method of choice. The method has already shown (a) that Ser 768 is both the residue most readily phosphorylated at limiting ATP levels and the residue most readily dephosphorylated by purified phosphatase 2A, (b) that Ser 737 is also readily phosphorylated and that its phosphorylation likely alters the conformation of the R domain, and (c) that Ser 670, not previously shown to be a target of PKA, is indeed phosphorylated by PKA in vitro. These early results suggest obvious targets for site-specific mutation and detailed functional analysis. We are currently carrying out very detailed MS studies of both phosphorylation and dephosphorylation (with a number of different phosphatases) in order to more ccompletely define costs of chanel conducatance via the R-domain. This work involves integrated use of a variety of proceudres including MALDI-TOF-MS, MALDI-ITMS and LC-ESI-ITMS/MS. The stage is now set for detailed parallel functional and biochemical studies, in both wild- type and mutant CFTR channels, of the roles of specific phosphoserines in single-channel gating and of their sensitivities to selective phosphatases.
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