ABCMdb

PMID: 23067305

Tosoni K, Stobbart M, Cassidy DM, Venerando A, Pagano MA, Luz S, Amaral MD, Kunzelmann K, Pinna LA, Farinha CM, Mehta A
CFTR mutations altering CFTR fragmentation.
Biochem J. 2013 Jan 1;449(1):295-305. doi: 10.1042/BJ20121240., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Ser511Ala 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). Login to comment 6 ABCC7 p.Ser422Ala ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp ABCC7 p.Thr1471Ala 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). Login to comment 44 ABCC7 p.Ser422Ala ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp ABCC7 p.Ser422Asp ABCC7 p.Thr1471Ala ABCC7 p.Thr1471Asp Cell culture, lysis, protein solubilization and Western blotting The cell culture methods to create the stable CFTR-expressing cell lines (WT, F, WT S422A, WT S422D, WT S511A, WT S511D, WT T1471A and WT T1471D) and their culture protocols have been described recently [25]. Login to comment 187 ABCC7 p.Thr1471Asp The most dramatic effect on synthesis was seen after the T1471D mutation (augmenting the negative charge at the regulatory acid stretch, nine amino acids from the C-terminus of wt-CFTR), which abolished the mature 'band C` form of CFTR reminiscent of the findings when F508delCFTR fails to mature in the Golgi from band B (in the ER) to band C (in the plasma membrane). Login to comment 191 ABCC7 p.Ser422Ala ABCC7 p.Ser422Asp Compared with each other S422D- and S422A-CFTR revealed no gross differences in fragmentation (Figures 7A-7D, compare lanes 3 and 4). Login to comment 197 ABCC7 p.Ser511Ala S511A-CFTR and its cognate NBD2 The diffuse 80 kDa NBD2 and the C-terminal-bearing fragment so characteristic of the wt-CFTR fingerprint is now grossly attenuated (Figure 7C, compare lanes 2 and 5). Login to comment 199 ABCC7 p.Ser511Ala First, the fragment bearing NBD2 and the C-terminus of CFTR may be more easily degraded when the N-terminal 110 kDa (NDB1/N-terminal) fragment (Figures 7A and 7B) is generated by the S511A mutation. Login to comment 200 ABCC7 p.Ser511Ala Secondly, the epitope of NBD2 recognized by this monoclonal antibody (WPSGGQMT) may be masked after S511A mutation by some post-translational event. Login to comment 201 ABCC7 p.Ser511Ala Thirdly, the CFTR fracture point induced by this S511A mutant lies near this region as opposed to the R-domain region seen with wt-CFTR in the Figures above. Login to comment 202 ABCC7 p.Ser511Ala ABCC7 p.Ser511Ala S511A-CFTR and its cognate NBD1 Consistent with the idea of an altered fracture point when S511A-CFTR is present, a new faint ~100-110 kDa band appears. Login to comment 204 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp Note the N-terminal- and NBD1- (but not NBD2) bearing ~120 kDa fragment seen after S511A (but not S511D) mutation in lanes 5 and 6 in (A) and (B), but not in (C) and (D), the latter probing the C-terminus. Login to comment 205 ABCC7 p.Thr1471Asp Compare similarities and differences in fragmentation between lanes 8 and 9 which both reduce the amount of full-length CFTR (T1471D or F508del). Login to comment 211 ABCC7 p.Ser511Ala Thus it is probable that the S511A mutation creates a band at 100-110 kDa-bearing NBD1 and the N-terminus, but we cannot be sure about the relationship between this band and the relative amounts of the Q1-Q4 NBD1-bearing fragments. Login to comment 212 ABCC7 p.Ser511Asp S511D-CFTR and NBD1 and NBD2 This phosphomimic fingerprint is similar to the wt in fragmentation pattern and no 110 kDa band appears, even after overexposure (Figure 7, lanes 6 and results not shown). Login to comment 214 ABCC7 p.Ser511Ala The combined S511 site mutant data suggest the existence of a new S511A-dependent CFTR cleavage whose generation is either dependent on the non-availability of the expected wt hydroxy group on S511 or is induced by a structural change after substitution with alanine (but not the negatively charged aspartate). Login to comment 215 ABCC7 p.Thr1471Ala wt-CFTR with mutated T1471 T1471A fragmentation is very similar to the wt. Login to comment 216 ABCC7 p.Thr1471Asp In contrast T1471D displays gross attenuation of all fragments (only Q4 remains visible with the NBD1 and N-terminal antibodies). Login to comment 217 ABCC7 p.Thr1471Asp The absence of other bands may be a false-negative artefact of low fragment abundance because we find that the full-length wt-CFTR with the T1471D mutation is also attenuated and thus (from an abundance perspective) is indistinguishable from the pattern seen with F508delCFTR, despite the wt background. Login to comment 218 ABCC7 p.Thr1471Asp This confirms recent data [25] showing that T1471D abolished the formation of any fully glycosylated band C CFTR in the same cell line and is consistent with the importance of the acid cluster surrounding T1471 towards the relative rates of opening and closing of CFTR [26]. Login to comment 220 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp ABCC7 p.Thr1471Ala ABCC7 p.Thr1471Asp In summary, the two S511A/S511D and T1471A/T1471D pairs manifested very different CFTR cleavage patterns and abundance of full-length CFTR when present on a wt background. Login to comment 223 ABCC7 p.Ser511Ala Only F508delCFTR and S511A CFTR generated a new CFTR band at about 110 kDa. Login to comment 233 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp It is also unlikely that CK2-relevant CFTR mutants such as S511A- and S511D-CFTR generate an ER-trapped unfolded CFTR, as they have wt patterns of maturation and turnover during pulse-chase experiments in the same cell line [25]; yet, they manifest very different fragmentation. Login to comment