ABCMdb

ABCC7 p.Lys68Arg

ClinVar:	c.204A>T , p.Lys68Asn ? , not provided c.202A>G , p.Lys68Glu ? , not provided
CF databases:	c.202A>G , p.Lys68Glu (CFTR1) ? , This mutation was found in a male Turkish patient (first reported Aug 4, 1997). His sweat chloride was 60 meq/l and showed lung disease at age of 26 months. His other CF mutation was later reported to be 406+3T->C (on March 23, 1998). c.204A>T , p.Lys68Asn (CFTR1) ? , This mutation was detected in a single Turkish CF patient of Syrian origin. The second mutation is yet unknown. The mutation segregates with haplotype C.
Predicted by SNAP2:	A: D (66%), C: D (75%), D: D (91%), E: D (80%), F: D (85%), G: D (85%), H: D (53%), I: D (80%), L: D (66%), M: D (75%), N: D (63%), P: D (85%), Q: D (53%), R: N (66%), S: N (61%), T: D (63%), V: D (80%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: D, I: D, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D,

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Publications
[hide] Interference with ubiquitination in CFTR modifies ... Mol Cell Biol. 2014 Jul;34(14):2554-65. Lee S, Henderson MJ, Schiffhauer E, Despanie J, Henry K, Kang PW, Walker D, McClure ML, Wilson L, Sorscher EJ, Zeitlin PL
Interference with ubiquitination in CFTR modifies stability of core glycosylated and cell surface pools.
Mol Cell Biol. 2014 Jul;34(14):2554-65., [PMID:24777605]

Abstract [show]
It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band CCFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

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No. Sentence Comment
5 N-tail mutants, K14R and K68R, lead to increased mature band C CFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. Login to comment
73 We hypothe- TABLE 1 Primers for site-directed mutagenesis Mutation Directiona Sequence (5=-3=)b K14R F GGCCAGCGTTGTCTCCAGACTTTTTTTCAGCTGGACC R GGTCCAGCTGAAAAAAAGTCTGGAGACAACGCTGGCC K68R F GGCTTCAAAGAAAAATCCTAGACTCATTAATGCCCTTCGGCG R CGCCGAAGGGCATTAATGAGTCTAGGATTTTTCTTTGAAGCC K710R F CCAATCAACTCTATACGAAGATTTTCCATTGTGCAAAAG R CTTTTGCACAATGGAAAATCTTCGTATAGAGTTGATTGG K716R F GAAAATTTTCCATTGTGCAAAGGACTCCCTTACAAATGAATGG R CCATTCATTTGTAAGGGAGTCCTTTGCACAATGGAAAATTTTC K710/716R F CCAATCAACTCTATACGAAGATTTTCCATTGTGCAAAGGACTCCCTTACAAATGAATGG R CCATTCATTTGTAAGGGAGTCCTTTGCACAATGGAAAATCTTCGTATAGAGTTGATTGG K1041R F CCTCACAGCAATTCAGACAACTGGAATCTGAAG R CTTCAGATTCCAGTTGTCTGAGTTGCTGTGAGG K1080R F GAAACTCTGTTCCACAGAGCTCTGAATTTACATAC R GTATGTAAATTCAGAGCTCTGTGGAACAGAGTTTC K1218R F GATCTCACAGCAAGATACACAGAAGG R CCTTCTGTGTATCTTGCTGTGAGATC a F, forward; R, reverse. Login to comment
97 Lysine-to-arginine mutations caused minor changes in CFTR conformation, except for the K68R mutation which caused changes in the orientation of third alpha helix, where 68R is located (Fig. 3A). Login to comment
99 K14R and K68R mutations result in increased levels of immature (A/B band) and mature (C band) forms of CFTR expression (Fig. 2B). Login to comment
117 To test this hypothesis for K14R and K68R, we expressed these N-tail mutants in the presence and absence of proteasomal inhibition. Login to comment
119 4A), but proteasome inhibition during expression of the K14R and K68R mutant CFTR cDNAs led to even higher expression levels. Login to comment
130 This immunoblot demonstrates increased levels of band C CFTR expression from the K14R, K68R, and K1218R cDNA vectors and decreased levels of band C CFTR expression in K710R, K716R, K710/716R, and K1080R vectors. Login to comment
135 However, unexpectedly, expression levels in K14R- and K68R- transfected cells were not increased by lysosomal inhibition, perhaps because they were already slow to recycle. Login to comment
136 Densitometry analysis showed that a proteasomal inhibitor increased C bands of wt, K14R, and K68R compared to control levels, but changes in A/B bands were insignificant. Login to comment
137 Lysosomal inhibitor did not change C bands, but A/B bands of K14R and K68R were decreased compared to control levels. Login to comment
138 These data suggest that K14R and K68R mutations allow CFTR to bypass the lysosome for degradation so that preventing lysosomal activity does not further increase the expression level of the mutant compared to wild-type CFTR. Login to comment
144 A K-to-R mutation does not alter the conformational structure of each domain except for the K68R mutant. Login to comment
163 (A) The results from expression of control vector, wt CFTR, K14R, and K68R with and without proteasomal or lysosomal inhibition are shown in representative blots. Login to comment
164 MG132 increased total A/B/C bands of CFTR in wild-type, K14R, and K68R proteins. Login to comment
200 There were decreased levels of cell surface CFTR detection in the K68R, K710R, K716R, K710/ K716R, and K1041R mutant forms of CFTR, but the wild-type, K14R, K1080R, and K1218R mutants accumulated similar levels of visible cell surface CFTR. Login to comment
201 Analysis of the Z stack of images for the wild type, K68R, K710R, K1080R, and K1218R confirmed that the CFTR immunostaining, shown in red, resided at the apical surface in these nonpermeabilized cells (data not shown). Login to comment
207 Again, there was a reduction in immunodetectable CFTR (K14R, K68R, K710R, K716R, K710/716R, and K1080R forms) in cell surface membranes compared to wild-type levels. Login to comment
218 Control vector, wt CFTR, K14R, K68R, K710R, K716R, K710/716R, K1041R, K1080R, and K1218R were expressed in IB3-1 cells for 48 h. CFTR was immunoprecipitated (IP) with M3A7 as described in Materials and Methods and separated by SDS-PAGE. Login to comment
221 K63-linked ubiquitin on CFTR is reduced in the K68R, K710R, K716R, and K1218R mutants. Login to comment
253 A lighter signal is visible from cells expressing K68R and the R domain mutants. Login to comment
275 Since ZO-1 interacts with PDZ domains, the reduced interactions in K14R, K68R, K710R, K716R, K710/716R, and K1041R and the absence of interactions with K1080R and K1218R raise the issue of alternative trafficking routes that are not dependent on the C-tail PDZ domain. Login to comment
279 K14R and K68R experience some proteasome degradation, but a sizeable fraction is localized near the plasma membrane. Login to comment
296 Interestingly, ZO-1 was highly abundant in complexes formed with wt CFTR; K14R, K68R, K710R, K716R, K710/716R, and K1041R mutants bound less ZO-1, whereas K1080R and K1218R mutant complexes had none. Login to comment