ABCMdb

ABCC7 p.Lys1041Arg

Predicted by SNAP2:	A: D (63%), C: D (53%), D: D (80%), E: D (71%), F: D (59%), G: D (71%), H: D (63%), I: D (59%), L: D (59%), M: N (53%), N: D (63%), P: D (80%), Q: D (53%), R: D (53%), S: D (59%), T: D (63%), V: D (59%), W: D (71%), Y: D (59%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: N, R: N, S: D, T: D, 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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Sentences [show]
No. Sentence Comment
6 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. 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
86 We generated K1041R and K1080R mutants and expressed them in IB3-1. Login to comment
89 Interference with residue 1080 drastically reduced CFTR but, in contrast to the K1041R mutation, significant band C continued to appear, similar to results with the R domain mutants. Login to comment
90 C-band expression was quantified by densitometry, as shown in Fig. 2B, and K1041R was the lowest although band B expression was higher than in the wt, further supporting a maturational block after band B and before C. Login to comment
104 The observed reduction in expression of K710R, K716R, K710/ 716R, K1041R, and K1080R CFTRs might be the consequence of disordered conformational structure or folding/assembly. Login to comment
106 The data in Fig. 3B show similar patterns of proteolytic digestion for wt and mutant CFTR; K1041R is the only mutant which shows significantly less CFTR at the final trypsin concentration. Login to comment
131 At steady state, the K1041R mutation in TM10 is almost completely lacking band C CFTR expression. Login to comment
151 The K1041R mutant was included to highlight the differences that will be explored further. Login to comment
156 Interestingly, MG132 treatment rescued only B band expression in the K1041R mutant form of CFTR, but this was not sufficient for trafficking beyond the ER. Login to comment
159 Prevention of proteolysis at the lysosome accumulates more C band K1041R and K1080R. Login to comment
169 (C) The results from expression of control vector, wt CFTR, K1041R, K1080R, and K1218R with and without proteasomal or lysosomal inhibition are shown in representative blots similar to the results show in panel A. MG132 rescues band B from K1041R to some extent, as well as a small portion of band C. Login to comment
175 Therefore, these data suggest that K1041R and K1080R mutants are degraded at the proteasome and lysosome, respectively. Login to comment
185 For K1041R the reduced Ub-K63 immunoblot signal likely derives from the reduced amount of immunoprecipitated product. Login to comment
194 K14R, K1080R, and K1218R but not K1041R reach the cell surface. 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
215 On the other hand, prevention of ubiquitination at 1041 by making the K1041R construct promotes degradation primarily by the proteasome. 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
226 FIG 6 K-to-R mutation alters the stability of CFTR protein, and wt CFTR, K14R, K1080R, and K1218R, but not K1041R, are detectable at the cell surface. Login to comment
236 As expected, CFTR is virtually undetectable in cells expressing control vector or K1041R. Login to comment
237 CFTR is easily detected HDAC6 must interact with CFTR to promote trafficking beyond the ER, then we predict that K1041R would interact preferentially with VCP and that K1218R would interact preferentially with HDAC6. Login to comment
261 Band C was most reduced in K1041R and highest in the wt, K1080R, and K1218R, consistent with data in panels A and B. Login to comment
268 Interestingly, higher-molecular-weight forms of K1041R were pulled down from the biotinylated surfaces of cells treated with proteasomal or lysosomal inhibition. 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
278 Poly- or monoubiquitination in K14, K68, K710, K716, 1041, K1080, and K1218 sites allows CFTR to pass through its regular quality control process, producing adequate band C. K710R, K716R, K710/K716R, and K1080R mutants undergo some proteasome- and some lysosome-dependent degradation, whereas K1041R is primarily degraded by the proteasome. 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