Research ArticleBiochemistry

A posttranslational modification code for CFTR maturation is altered in cystic fibrosis

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Science Signaling  01 Jan 2019:
Vol. 12, Issue 562, eaan7984
DOI: 10.1126/scisignal.aan7984
  • Fig. 1 Identification of CFTR PTR hotspots.

    (A) PTMs identified for wild-type CFTR (below) or ∆F508 CFTR (above) isolated from human bronchial epithelial cell lines 16HBE41o and CFBE41o were mapped onto the CFTR amino acid sequence (center). Phosphorylation is indicated in green, methylation in black, ubiquitination in lilac/purple, and O-glycosylation in orange. PTM hotspots are indicated in yellow, transmembrane domains are shown in dark blue, and the R region and NBDs are labeled. (B) PTM spectrum of the tryptic peptide (boxed inset) covering part of the ER exit motif (blue). The abundance of mass shifts detected by Blind-PTM is shown, and major modifications are indicated. The inset further shows N- and C-terminal overlapping peptides, with their respective modifications shown in red. ∆F508 CFTR PTMs are above the sequence, and wild-type PTMs are below. (C) PTM spectrum of peptide covering the N-terminal NBD1 sequence (RI element). N- or C-terminal overlapping peptides containing additional modifications are indicated in the inset, where ∆F508 CFTR PTMs are above and wild-type PTMs are below the sequence. Ox, oxidation; Me, mono-methylation; di-Me, di-methylation; +Lys, mis-cleavage upstream of lysine; Ub, ubiquitination. Data represent n = 14 independent biological replicates.

  • Fig. 2 Misregulation of domain-specific phosphorylation in CF.

    (A) SILAC ratios and differential PTMs of peptides across the CFTR sequence from 16HBE41o and CFBE41o cells. Data are representative of n = 4 experiments. Non-mod, not modified; Phospho, phosphorylated; Me, methylated. (B) TMT-based quantification of phosphorylation of sites in ∆F508 CFTR relative to wild type (wt) in 16HBE41o and CFBE41o cells. Values are log2(ratio). Data are representative of n = 7 experiments. (C) Differential phosphorylation of Thr421, Ser422, Ser427 (RI element) and Ser660, Ser700, Ser813, and Thr816 (R region) was confirmed by simultaneous detection of spiked-in synthetic heavy (13C615N2 Lys; 13C615N4 Arg)–labeled peptides in 16HBE41o and CFBE41o cells. Data are means ± SD from n = 5 independent biological replicates. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001 by unpaired, two-tailed t tests, analyzed and corrected by the Bonferroni-Sidak method.

  • Fig. 3 CSNK2A1-mediated phosphorylation of Thr421 to Ser427 is essential for CFTR maturation.

    (A) CX-4945–mediated inhibition of CK2α prevents phosphorylation of wild-type CFTR at Thr421, Ser422, and Ser427 as quantified with spiked-in synthetic, heavy isotope–labeled peptides. Data are means ± SD of n = 3 experiments; statistical analysis noted below. (B) Experimental outline for AHA labeling and enrichment of newly synthesized CFTR by click chemistry. (C) Western blot of input lysate for CFTR-IP before click reaction. (D) Western blot showing that wild-type CFTR maturation is dependent on CK2α phosphorylation in the RI element. Blots are representative of n = 3 experiments. (E) Quantification of newly synthesized immature (band B) and mature (band C) CFTR relative to time point 0 (h, hours). (F) Western blot of mature CFTR (band C) in FRT cells expressing various CFTR mutants. (G) Phosphorylation at Thr421, Ser422, and Ser427 in the various CFTR mutants described in (F) was quantified with spiked-in synthetic, heavy isotope–labeled peptides relative to the amount of mature CFTR present. Data are means ± SD of n = 4 independent measurements per site; statistical analysis noted below. (H to K) AHA pulse-chase assays in NHBE cells (H) and FRT cells expressing G551D or wild-type CFTR (K) treated with CX-4945 or DMSO. Subsequent quantification of newly synthesized immature CFTR (band B) and mature CFTR (band C) at the time points indicated relative to time 0 (J and L). Top panels show input and loading control. SA-HRP, streptavidin-conjugated horseradish peroxidase. Time of AHA labeling before CFTR-IP is indicated. Blots are representative, and data are means ± SD of n = 3 independent biological replicates. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001 by unpaired, two-tailed t tests with Bonferroni-Sidak correction (A) or one-way ANOVA with Tukey post-test (G, J, and L).

  • Fig. 4 CK2α inhibition induces wild-type CFTR degradation.

    Degradation network identified for wild-type CFTR, wild-type CFTR treated with CX-4945, or ∆F508 CFTR in 16HBE41o and CFBE41o cells shows recruitment of ∆F508-specific interactions in wild-type CFTR–expressing cells treated with CX-4945 (red nodes). Data represent independent biological replicates (wt, n = 8; ∆F508, n = 8; wt + CX-4945, n = 3).

  • Fig. 5 Rescue of ∆F508 CFTR function at permissive temperature depends on CSNK2A1 phosphorylation of Thr421 to Ser427.

    (A) Quantification of phosphorylated sites Ser422 and Ser427, methylation site Lys442, and ubiquitination site Lys420 at 37°C (red bars) or at permissive temperature of 30°C (blue bars). (B) Quantification of ∆F508 CFTR phosphorylation at Thr421, Ser422, and Ser427 by synthetic heavy-labeled peptides shows reduced phosphorylation upon shRNA-mediated knockdown of CSNK2A1. (C) Western blot showing that shRNA-mediated knockdown of CSNK2A1 impairs ∆F508 CFTR maturation at permissive temperature. Detection of β-actin was used as loading control. (D) Representative traces of forskolin (F)– and genistein (G)–stimulated ∆F508 CFTR short-circuit currents (Isc), showing that knockdown of CSNK2A1 (right) or treatment with 10 μM CX-4945 (left) prevents temperature shift (28°C)–induced rescue of ∆F508 CFTR channel activity. Specificity of the current is established by treatment with CFTR inhibitor 172 (I). (E) Quantification of ∆Isc currents as log2 fold change relative to control cells. All data are representative or means ± SD of n = 3 independent biological replicates. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001 by one-way ANOVA with Bonferroni post-test.

  • Fig. 6 A minimal PTM code for CFTR maturation.

    (A) Heatmap of CFTR PTMs at permissive temperature or upon treatment with different CFTR modifier drugs. (B) Heatmap of signature PTMs organized by CFTR domains. Modified residues are indicated either below (A) or next to the heatmap (B). P, phosphorylation; U, ubiquitination; M, methylation; A, acetylation. Gray color in the heatmap represents values not measured (NaN), whereas all additional values are normalized to ∆F508 CFTR whenever possible. The increase in ∆F508 CFTR anion channel activity is indicated for each of the different conditions, and the Pearson correlation coefficient between the different PTM signatures and the wild-type CFTR PTM signature is shown. Data represent two or more independent biological replicates per condition. nd, not determined. (C) Schematic representation of the proposed PTM code for CFTR maturation. Correctly folded CFTR displays a methylation-phosphorylation pattern in the RI element PTM hotspot that allows maturation of the protein and functions as a combinatorial PTM code. Upon misfolding, this code is quantitatively and qualitatively altered, which leads to degradation of the CFTR protein. At a permissive temperature, the wild-type code is reinstated and enables maturation of ∆F508 CFTR. However, if phosphorylation in the RI element is inhibited, then maturation at permissive temperature is blocked and the code resembles that of ∆F508 CFTR at 37°C.

  • Table 1 Clinical significance of naturally occurring point mutants of CFTR PTM sites.

    Given are the residue, observed point mutation, and clinical significance of point mutations as reported in the CFTR mutation databases (30).

    Modified peptide sequenceSiteA score [−10× log(P)]Mutation in CF patientsClinical significance
    T*SNGDDSLFFSNFSLLGTPVLKT42119.21T421ACBAVD
    GQLLAVAGSTGAGK(me)TK464K464 NCF, severe phenotype at
    early age with pancreatic
    insufficiency, chronic
    cough and bronchial
    infection, 3659delC
    mutation on the other
    chromosome (expected to
    lead to pancreatic
    insufficiency)
    FAEK(ub)DNIVLGEGGITLSGGQRK536K536EParent of a child with a
    positive newborn
    screening test
    DNIVLGEGGITLS*GGQRS54964.79S549 N, S549I, S549F, S549RCF, severe clinical phenotype
    AVYK(ub)DADLYLLDSPFGYLDVLTEKK564K564ECBAVD
    NS*ILTETLHRS66056.02S660 TAsymptomatic
    NSILTETHR(me)R668R668CDoes not cause CF
    LS*LVPDSEQGEAILPRS73794.12S737FElevated sweat chloride
    LSLVPDSEQGEAILPR(me)IR751R751P, R751C, R751LLung disease, carrier testing
    for R751C
    VSLAPQANLTELDIYSR(me)RR810R810GCBAVD (∆F508 on other
    allele)
    LS*QETGLEISEEINEEDLKS81369.89S813PVery mild CF
    AYFLQTSQQLK(ub)QLESEGRK1041K1081RReduction of band C
    (McClure 2014)
    TGSGK(ub)STLLSAFLRK1250K1250A mutation
    dramatically prolonged
    burst duration (abolishes
    adenosine triphosphate
    hydrolysis)

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/562/eaan7984/DC1

    Fig. S1. Identification of CFTR single-nucleotide polymorphisms in CFBE41o and HBE41o cell lines.

    Fig. S2. Balanced SILAC approach.

    Fig. S3. Kinase families in the CFTR interactome.

    Fig. S4. CK2a knockdown.

    Table S1. Identified phosphopeptides.

    Table S2. CFTR ubiquitination and methylation sites identified in the forward search.

    Data file S1. Interactome of wild-type CFTR in the presence of CX-4945.

  • The PDF file includes:

    • Fig. S1. Identification of CFTR single-nucleotide polymorphisms in CFBE41o and HBE41o cell lines.
    • Fig. S2. Balanced SILAC approach.
    • Fig. S3. Kinase families in the CFTR interactome.
    • Fig. S4. CK2a knockdown.
    • Table S1. Identified phosphopeptides.
    • Table S2. CFTR ubiquitination and methylation sites identified in the forward search.
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Interactome of wild-type CFTR in the presence of CX-4945.

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