Our studies have explored the role of protein kinase CK2 in CFTR regulation. We demonstrate that inhibition of CK2 closes the CFTR Cl− channel. The consistency of our results using mammalian cells and Xenopus oocytes expressing recombinant CFTR and epithelial cells expressing endogenous CFTR (including an intact, perfused pancreatic duct) argues that the CK2-CFTR interaction is a general feature of CFTR-expressing cells. In addition, we established that deletion of F508 disrupts the interaction of this kinase with CFTR.
Because CK2 is an essential early gene in development with over three hundred in vivo
], dominant negative, inhibitory or knockdown studies inevitably impact on multiple cellular pathways making interpretation of results problematic given that CK2 may control 10% of all phosphorylated proteins. The constitutive ‘always-on’ activity of CK2 makes it necessary to apply site-directed mutagenesis to both the kinase itself (creating an inhibitor-insensitive mutant) and the putative target as discussed below. We complemented this approach with experiments using specific, competitive peptides to disrupt the local association of CK2 and CFTR. We find that the KENIIF (wild-type) peptide inhibits wild-type CFTR Cl−
channel function in oocytes. We undertook these studies because TBB is itself an anion; at high concentrations TBB is an open-channel blocker of the CFTR Cl−
channel (Z Xu, J-H Chen and DN Sheppard, unpublished observations). One possible interpretation of the peptide data is that KENIIF can sequester CK2 away from wild-type CFTR, and that CK2 is essential for normal CFTR function. Our hypothesis that ΔF508 abrogates CK2 binding is strengthened by the finding that the KENII (ΔF508) peptide cannot inhibit CFTR function, suggesting that it cannot sequester CK2 (but see alternative allosteric regulatory loop arguments below). The contrast in CFTR sensitivity to TBB using the cell-attached and the excised patch configurations shown in is striking. These data suggest a protein-protein interaction between CK2 and CFTR in the intact cell.
CK2 is a very complex kinase whose physiological regulators are unknown. In the present work, we show that when CFTR fails to traffic to the apical membrane of CF nasal epithelial cells, CK2 is absent from the apical membrane. This result is reminiscent of our recent studies of the interaction between CK2 and ENaC [58
]. In that study, we found that the transition between cytosolic and membrane-bound CK2 is dependent on the phosphorylation status of ENaC. Of note, when both recognised CK2 targets in ENaC were mutated to non-phosphorylatable alanines (αβS631AγT599A-ENaC), no CK2 migrated to the membrane despite abundant amounts in the cell interior [58
]. Moreover, other studies have described complex interactions between CK2 and the cytoskeleton (for review, see [59
]). Thus, trafficking of CK2 is not a simple matter of diffusion from the cytosol to a molar excess of target protein in the cell membrane. In another study [60
], we identified a related phenomenon for membrane local effects when investigating CFTR regulation by AMPK. In results reminiscent of those observed with ENaC [58
], adding millimolar quantities of the AMPK activator AMP to the cytosol failed to induce CFTR channel closure [60
]. By contrast, when we manipulated local AMP concentrations using pharmacological tools to prevent AMP formation at the cell membrane, CFTR Cl−
channels opened promptly [60
]. For these and other reasons (see below), we believe that adding complex, multi-subunit kinases (e.g. AMPK and CK2) to the solution bathing the intracellular face of excised inside-out membrane patches from cells expressing CFTR is unlikely to yield meaningful results. Our studies began with the notion that S511 in the F508 region of NBD1 is a potential target serine for phosphorylation by CK2 because the CFTR sequence S511
YDE has the appropriate acid residues C-terminal of the S511. However, when Pagano et al. [27
] investigated interactions between CK2 and NBD1 of murine CFTR in vitro
, no phosphorylation of S511 was identified despite it being an excellent potential CK2 target. Instead, CK2 phosphorylated S422, S423 and S670; S422 and S670 are conserved in the human sequence [27
]. We have also been unable to demonstrate phosphorylation of S511 either using radiolabelled ATP in the presence of recombinant CK2 and purified human NBD1 or by applying a site specific antibody for phosphoserine-511 to recombinant CFTR (KJ Treharne and A Mehta, unpublished observations). Nevertheless, the present results using intact cells and a variety of assays suggest strongly that some form of functional interaction occurs between CK2 and the F508 region of CFTR. However, further work is required to resolve why S511 appears to be important in conferring CK2 sensitivity on CFTR when expressed in the Xenopus
oocyte system, but has no major impact on the single-channel behaviour of CFTR in excised inside-out membrane patches (). Pagano et al. [27
] suggest that this region of CFTR confers the first ever described allosteric regulation towards the CK2 holoenzyme which alters when F508 is missing and when S511 is mutated. They also suggest that this region might inhibit the catalytic site of CK2 should CK2α be present without its β subunit. Thus, the simplest interpretation of our data is that when a patch of cell membrane is excised a local feedback loop involving CK2 is disrupted. Given that CK2 controls so many processes at the cell membrane, including PKA [61
] and the cytoskeleton [59
], this idea seems to be a reasonable working hypothesis.
Using biochemical assays, Pagano et al. [27
] also analysed the interplay between the KENIIF and KENII peptides, NBD1, CK2α and CK2β. In their in vitro
phosphorylation experiments, Pagano et al. [27
] applied peptides corresponding to the F508 region of CFTR and measured the activation status of CK2. They found that these peptides inhibited the phosphorylation of the β subunit of CK2, which is known to be essential for the activation of the holoenzyme, yet the same peptides enhanced the phosphorylation of NBD1 and could even modulate CK2 targeting towards physiologically important substrates such as calmodulin, inhibitor-2 of protein phosphatase 1 and heat shock protein 90 [27
]. The authors state that “the sequence around F508 represents, with respect to CK2, at the same time a docking site and an allosteric effector, whose dual function is increased by F508 deletion.” The complexity of their discoveries using purified proteins and in vitro
biochemical approaches makes direct comparisons with our physiological data difficult. In particular, the data shown in demonstrate that S511D-CFTR is insensitive to TBB inhibition even though S511 is not a CK2 target. Clearly, there may be other regions outside the NBDs that might be CK2 targets such as T1471 near the C-terminus of CFTR [62
], but their functional significance remains to be determined. Nevertheless, our data when combined with those of Pagano et al. [27
] suggest that the S511 region has a regulatory function in the reciprocal interaction of CK2 with CFTR such that CK2 controls CFTR and, in turn, CFTR controls CK2.
What might be the pathophysiological consequences of ΔF508-induced CK2 mislocalisation away from the apical membrane of epithelial cells? Some of the cellular processes reported to be defective in CF are dependent upon CK2. For example, polyamines, such as spermidine, which are stimulators of CK2 activity, are increased in CF [63
], suggesting a compensatory reaction to the loss of CK2 function. CFTR maturation is dependent on its association with calnexin in the ER [66
]; this chaperone is phosphorylated by CK2 [67
]. The residence time for ΔF508-CFTR at the apical membrane is reduced due to abnormal endocytic cycling [68
] and coating and uncoating of such vesicles is a CK2-dependent process [70
]. As discussed above, CK2 is targeted to the cell membrane by ENaC [57
], whose function is disrupted in CF by unknown mechanisms. Interestingly, the CF antigen, which is over-expressed in both CF patients and model systems, is a regulator of CK2 kinase activity [71
]. Thus, some of the reported targets of CK2 [26
] include proteins controlling unexplained aspects of CF disease [74
]. Overall, our proposed role for CK2 is consistent with published data on the cell biological consequences of the commonest CFTR mutation.
One approach to CF therapy aims to rescue defective processing and trafficking of ΔF508-CFTR [76
]. Our data suggest that CK2-dependent regulation of ΔF508-CFTR might still be defective under conditions where ΔF508-CFTR has been induced to traffic to the apical membrane, consistent with published data showing that restoration of trafficking fails to fully restore CFTR-mediated Cl−
]. Outside the CF field, understanding the CK2-CFTR interaction might generate new drugs for the treatment of secretory diarrhoeas because inhibitors of CFTR, selected for their impact on transepithelial ion transport, have been proposed as treatments for cholera [78
In conclusion, the data in this paper suggest not only that CK2 is a novel regulator of CFTR function, but also that CK2-dependent regulation of ΔF508-CFTR is dysfunctional. Although the ΔF508 mutation is responsible for most cases of CF, further work will be needed to determine whether other pathogenic mutations in CFTR act by a CK2-related mechanism. Our data suggest a radically different explanation for the commonest CF defect that links the deletion of F508 with defective function of a pleiotropic kinase that controls diverse pathways currently thought to lie outside CF pathophysiology. Our work complements the data of Pagano et al. [27
], who suggest that the F508 region of CFTR, in isolation, can regulate CK2 activity towards specific targets, including NBD1, itself. Here, we present the corollary, regulation of the CFTR Cl−
channel by CK2, a kinase with hundreds of potential targets that acts dominantly over PKA regulation.