Abstract
By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C). Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein.
Highlights
Cystic fibrosis (CF) is caused by mutations affecting a polytopic integral membrane protein, termed Cystic Fibrosis Transmembrane-conductance Regulator (CFTR) functioning as a phosphorylation stimulated, ATP-dependent anion channel
We were impressed in particular by the observation that, several potential phosphoacceptor sites for CK2 are present in CFTR and some of these are readily phosphorylated by CK2 in vitro [9] none of these have been ever reported to become phosphorylated in vivo, in sharp contrast with potential PKA phosphoacceptor sites for many of which a good correlation has been shown to exist between in vitro and in vivo phosphorylation [5,6,7]
Considering on the one hand the stringent quality control experienced by CFTR, leading to the destruction of almost half of the newly synthesized protein, and, on the other, the role frequently played by CK2 mediated phosphorylation to commit proteins to degradation (e.g. [10,11,12]) a possibility would be that in its physiological environment CFTR phosphorylation by CK2 cannot be detected because it is immediately followed by CFTR fragmentation
Summary
Cystic fibrosis (CF) is caused by mutations affecting a polytopic integral membrane protein, termed Cystic Fibrosis Transmembrane-conductance Regulator (CFTR) functioning as a phosphorylation stimulated, ATP-dependent anion channel. In an attempt to disclose molecular events that may underlie deregulation of CFTR functionality and/or stability, attention has been focused on post-translational events, with special reference to phosphorylation These studies clearly demonstrated that multiple phosphorylation of the regulatory domain of CFTR by PKA and probably by other basophilic protein kinases such as PKC is essential to confer the full channel functionality to CFTR. The same residues phosphorylated in vitro by PKA [5] (and in some cases by PKC) were found to undergo phosphorylation in living cells [6,7], providing the clear-cut demonstration of the physiological occurrence of these events It was found in mutation studies that no single PKA site was dominant towards CFTR function and even a mutant with all PKA sites deleted retained some CFTR function
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