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 DeltaF508 are phosphorylated in vitro by CK2 catalytic alpha-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and DeltaF508, 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 CK2alpha 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 beta-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 CK2alpha, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the DeltaF508 CFTR peptide. SPR analysis shows that NBD1 DeltaF508 interacts more tightly than NBD1 wt with the alpha-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the alpha- and the beta-subunits of CK2.

Figure 1

Phosphorylation of wild-type and ΔF508 mutated NBD1 by protein kinase CK2. In (A) 3 μg of murine and human NBD1 were incubated under conditions decribed in the Experimental Procedures with CK2α catalytic subunit either alone (lane 1) or previously combined with equimolar amounts of CK2β regulatory subunit in the absence (lane 2) or in the presence of 330 nM polylysine (lane 3). Only the autoradiograms are shown. In (B) and (C) the kinetics of phosphorylation by CK2 catalytic subunit and by CK2 holoenzyme, respectively, at increasing concentration of NBD1 wild type (○) and ΔF508 (●) are illustrated. The data represent the means obtained from experiments run in triplicate with SD never exceeding 15%.

Figure 2

MS analysis of NBD1 phosphorylation sites by protein kinase CK2. Phosphorylation of mouse wild-type NBD1 by CK2 holoenzyme in the presence of polylysine and elution of the radiolabeled bands from the SDS−PAGE gel were performed as described in the Experimental Procedures. (A) List of identified phosphopeptides. Experimental mass, charge state of the peptides, theoretical mass, and delta mass are listed with the peptide sequence. The position of the phospho residue is marked with an asterisk. Note the presence of several semitryptic peptides probably due to a contamination with chymotrypsin. (B) Annotated fragmentation spectra of the phosphopeptides HS*SDENNVSFSH (left panel) and HS*S*DENNVSFSH (right panel). For the monophosphorylated peptide the position of the phosphoresidue is clearly indicated by the transition y₁₀−y₁₁, while for the bisphosphorylated peptide are very clear the transitions y₉−y₁₀ and y₁₀−y₁₁ that indicate the positions of the two phosphoserines. Both spectra are dominated by the neutral losses of H₃PO₄ from the parent ion. The label Δ indicates the fragments originated by the neutral loss of phosphate.

Figure 3

Dose-dependent effect of NBD1-derived synthetic peptides on the phosphorylation of NBD1 by protein kinase CK2. Phosphorylation of wild-type murine NBD1 (3 μg) was performed by CK2 catalytic subunit (panel A) and by CK2 holoenzyme (panel B) as described in the Experimental Procedures in the presence of increasing concentration of the NBD1-derived wild-type GTIKENIIFGVSYDEYRYR (○) and ΔF508 mutated GTIKENIIGVSYDEYRYR (●) peptides. The insets show the corresponding autoradiograms. The arrow indicates the position of NBD1. The data represent the means obtained from experiments run in triplicate with SD never exceeding 15%.

Figure 4

Effect of variable substitutions within the NBD1-derived peptides on their stimulatory efficacy on NBD1 phosphorylation by CK2 holoenzyme. Murine NBD1 (3 μg) was phosphorylated by CK2 holoenzyme under conditions described in the Experimental Procedures in the presence of variably substituted peptides (160 μM) listed in (A). In (B) the SDS−PAGE corresponding autoradiograms are shown with arrows indicating the position of the NBD1 protein and of the autophosphorylated CK2β subunit, respectively. In (C) the quantitation of NBD1 radiolabeling with respect to the control in the absence of peptides (C) is reported as histograms. Numbering refers to the list reported in (A).

Figure 5

Variable modulation of CK2 holoenzyme (A) and CK2α (B) by wild-type and ΔF508 mutated NBD1 500−518 peptides. Evidence for an allosteric mechanism (C). Phosphorylation conditions by CK2α catalytic subunit and by the in vitro reconstituted holoenzyme are described in the Experimental Procedures. The substrate concentrations were 3.5, 5, 2.5, 0.5, and 100 μM for NBD1, calmodulin (CaM), inhibitor-2 of protein phosphatase 1 (I-2), HSP90, and peptide RRRADDSDDDDD, respectively. The data represent the means of at least three independent experiments with SD never exceeding 15%. (A) Effect of NBD1 peptides (80 μM) on the CK2 holoenzyme-mediated phosphorylation of the indicated substrates. (B) Effect of increasing concentrations of NBD1-derived peptides (as indicated) on CK2α catalytic subunit activity toward the indicated substrates. (C) Kinetic analysis of the mechanism of inhibition of CK2α by CFTR 500−518 ΔF508. Kinetics of the specific CK2 peptide substrate (RRRADDSDDDDD) phosphorylation by CK2α were either in the absence (◼) or in the presence of CFTR 500−518 ΔF508 peptide at either 10 μM (▲) or 30 μM (▼).

Figure 6

Positive cooperative effect of NBD1 phosphorylation by CK2 holoenzyme in the presence of wild-type and ΔF508 peptide. Phosphorylation condition at increasing concentration of the substrate and evaluation of the phosphate incorporated after SDS−PAGE were performed as described in the Experimental Procedures. The data represent the means obtained from experiments run in triplicate with SD never exceeding 20%. (A, B) Murine NBD1 either alone (control) or in the presence of wild-type and ΔF508 mutated peptides (80 μM). The corresponding autoradiograms are shown in (A). (C) Human NBD1 in the presence of wild-type and ΔF508 mutated synthetic peptides (80 μM). (D) Calmodulin in the presence of ΔF508 mutated peptide (80 μM).

Figure 7

SPR analysis of the interaction of mouse NBD1 wild type and ΔF508 with human CK2 subunits. Representative sensorgrams obtained as detailed in the Experimental Procedures by injection of 15 μM NBD1 wt and NBD1 ΔF508 over a sensor surface containing 1600 RU of immobilized CK2α (A) and of 10 μM NBD1 wt and NBD1 ΔF508 over a sensor surface containing 1660 RU of immobilized CK2β (B).

Figure 8

Effect of NBD1-derived synthetic peptides on the interaction between CK2α and CK2β subunits. 22 nM CK2α subunit was injected at a flow rate of 10 μL/min over a sensor surface containing 1660 RU of immobilized CK2β. The same amount of CK2α was injected after an incubation of 10 min with a fixed amount (5 μM) of CFTR peptide 506−518 ΔF (A) and its shortened derivative 509−518 (B). Similar results were obtained with different concentrations of CK2α. Injections of 5 μM peptide alone showed any RU alteration.