CFTR mutations altering CFTR fragmentation

Abstract

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

“…While the PKA sites, whose phosphorylation has been validated in vivo are concentrated in the R domain (where they play a prominent role in the activation of the channel function) the potential CK2 sites are also localized outside the R domain and, by analogy with other CK2 targets whose phosphorylation commits them to degradation (see e.g. [10]–[12]), they may be implicated in the premature proteolysis of CFTR, also consistent with the outcome of recent mutational studies [13], [14]. It should be noted in fact that, although wild type CFTR is much more stable than its Phe508del counterpart, it nevertheless undergoes a very stringent quality control as well, resulting in perhaps 50% of the protein being discarded and proteolytically degraded [15].…”

“…Accordingly a hCFTR mutant no longer susceptible to such a phosphorylation (T1471A) was overexpressed in BHK cells as compared to hCFTR wild type. This mutant was previously shown to fragment in a manner similar to the wild type protein [14].…”

“…Western blot analysis was performed as described in [14] using a modified Laemmli buffer containing 105 mM dithiothreitol (final concentration) to enhance the solubilization of CFTR. 20–30 µg of total proteins were separated on 4–12% precast NuPage® Bis-Tris gradient gels (Invitrogen) according to the manufacturer’s instructions and transferred on to Optitran BA-S 83 nitrocellulose membranes (Whatman) with a semi-dry blotter (Biometra) for 1 h at 150 mA.…”

NM23 proteins: innocent bystanders or local energy boosters for CFTR?

“…For example, a phosphomimic such as T1471D drastically attenuates the expected wild type CFTR fracture pattern into N and C terminal halves. 28,64 Thus at the time of writing, the molecular mechanisms by which the conformational changes due to the NBDs dimerization lead to the CFTR's channel opening remain clouded (see reference 7 for example) and in particular, how a supposedly remote tail domain of CFTR might 'talk' to the pore is a puzzle that will need a better picture of the full length of the CFTR structure that is currently lacking at atomic resolution. The combined data predict that the negatively charged region of the tail when augmented further by the addition of (CK2-transferred) negative phosphate charge will manifest an interaction with the pore.…”

“…CFTR is driven by one ATP-binding site acting as a non-hydrolyzing 'degenerate' ATP holding 'bridgelike' domain with the other site permitting free hydrolysis that enables domain shifts to facilitate pore access. The relationship between ATP binding and channel gating is a very complex process as reviewed recently by Zwick et al 7 However, these models take no account of the binding of CFTR-associated proteins that drive a cAMP or calciumdependent CFTR activation 12,18 or to the possibility that CFTR might also become a (protease) split molecule that could recombine its N-and C-terminal fragments 28 in the membrane to create an array of different CFTR halves that add to the diversity of CFTR molecules in the membrane. It is accepted that hydrolysis of clamped ATP drives conformational changes in the translocator 'blades' structure, fueling the transport of biological substrates across the cellular membranes.…”