Modulation of Protein Kinase CK2 Activity by Fragments of CFTR Encompassing F508 May Reflect Functional Links with Cystic Fibrosis Pathogenesis

Abstract

Deletion of F508 in the first nucleotide binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator protein (CFTR) is the commonest cause of cystic fibrosis (CF). Functional interactions between CFTR and CK2, a highly pleiotropic protein kinase, have been recently described which are perturbed by the F508 deletion. Here we show that both NBD1 wild type and NBD1 ΔF508 are phosphorylated in vitro by CK2 catalytic α-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and ΔF508, the phosphorylated residues are S422 and S670, while phosphorylation of S511 could not be detected. Accordingly, peptides encompassing the 500−518 sequence of CFTR are not phosphorylated by CK2; rather they inhibit CK2α catalytic activity in a manner which is not competitive with respect to the specific CK2 peptide substrate. In contrast, 500−518 peptides promote the phosphorylation of NBD1 by CK2 holoenzyme overcoming inhibition by the β-subunit. Such a stimulatory efficacy of the CFTR 500−518 peptide is dramatically enhanced by deletion of F508 and is abolished by deletion of the II507 doublet. Kinetics of NBD1 phosphorylation by CK2 holoenzyme, but not by CK2α, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the ΔF508 CFTR peptide. SPR analysis shows that NBD1 ΔF508 interacts more tightly than NBD1 wt with the α-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the α- and the β-subunits of CK2.

Contribution of Casein Kinase 2 and Spleen Tyrosine Kinase to CFTR Trafficking and Protein Kinase A-Induced Activity

“…Furthermore, here we show that this effect of pharmacological CK2 inhibition applies not only to CFTR function as a chloride channel but also to the processing of wt CFTR. Indeed, our results show that CK2 activity is also essential for the successful processing (and membrane trafficking) of CFTR: this may involve direct phosphorylation of CFTR by CK2, previously shown to occur at S422 (26), or this effect may involve other targets of CK2 that relate to CFTR-associated proteins.…”

“…Previous observations evidenced that CK2 was able only to phosphorylate CFTR peptides corresponding to the sequence PGTIKENIIFGVSY 512 D EYRYR, provided that residue Y512 was replaced with phosphotyrosine (26), strongly suggesting the potential for hierarchical phosphorylation, i.e., an interaction between CK2 and SYK at S511, the CK2 consensus, depending on a negative charge at the adjacent tyrosine (26,27).…”

“…Subsequently, in vitro data suggested that a serine at position 422 within NBD1 was phosphorylated by CK2 with the surprising finding that the most likely candidate site at S511 near F508 was not labeled (26). Apart from this, there is only one preliminary report of another potential CK2 motif in the C-terminal end of CFTR (T1471) located within an acidic cluster (25).…”

Protein Kinase CK2 in Health and Disease

“…they found that peptides encompassing the 500 -518 sequence of CFTR promote the phosphorylation of NBD1 by the holoenzyme in an F508-dependent manner while inhibiting its phosphorylation by the isolated CK2a. Of note, these peptides also perturb the interaction between the a and b subunits of CK2 [55]. Although the mechanism by which NBD1 interacts with CK2 is not fully understood, this rather complex CK2-CFTR association is the first described DF508-dependent CFTR-kinase interaction that provides a functional link in the most frequent form of cystic fibrosis.…”

How druggable is protein kinase CK2?

“…It has been recently reported that the CK2 activity is modulated by specific fragments of the CFTR protein and it could reflect a functional link between CK2 and cystic fibrosis pathogenesis. 132 …”