ABCC7 p.Lys1218Arg
| Predicted by SNAP2: | A: N (57%), C: D (53%), D: D (85%), E: D (66%), F: D (63%), G: D (71%), H: D (53%), I: D (66%), L: D (66%), M: N (66%), N: N (61%), P: D (80%), Q: N (61%), R: N (93%), S: N (82%), T: N (72%), V: D (66%), W: D (63%), Y: D (63%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: N, I: D, L: D, M: D, N: N, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D, |
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[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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None has been submitted yet.
No. Sentence Comment
7 The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition.
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ABCC7 p.Lys1218Arg 24777605:7:4
status: NEW73 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.
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ABCC7 p.Lys1218Arg 24777605:73:760
status: NEW102 The K1218R mutation increases CFTR B and C bands even more significantly than N-tail mutants (Fig. 2B).
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ABCC7 p.Lys1218Arg 24777605:102:4
status: NEW130 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.
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ABCC7 p.Lys1218Arg 24777605:130:97
status: NEW157 K1080R and K1218R mutants displayed both bands B and C after proteasomal inhibition.
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ABCC7 p.Lys1218Arg 24777605:157:11
status: NEW158 This supports the hypothesis that the K1080R and K1218R mutants undergo the same degree of proteasomal degradation as wild-type CFTR and are still trafficked.
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ABCC7 p.Lys1218Arg 24777605:158:49
status: NEW169 (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.
X
ABCC7 p.Lys1218Arg 24777605:169:80
status: NEW171 K1218R shows the most robust rescue by both.
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ABCC7 p.Lys1218Arg 24777605:171:0
status: NEW174 pression of K1080R C band and improved the K1218R mutant, indicating that the K1218R mutant can avoid lysosomal degradation.
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ABCC7 p.Lys1218Arg 24777605:174:43
status: NEWX
ABCC7 p.Lys1218Arg 24777605:174:78
status: NEW191 The K710R, K716R, and K710/716R mutants showed faster degradation of the C band of CFTR than the wild type and K1218R.
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ABCC7 p.Lys1218Arg 24777605:191:111
status: NEW193 The initial expression level of K1218R is higher than that of wt CFTR, and the degradation rate of K1218R is similar to that of the wt CFTR.
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ABCC7 p.Lys1218Arg 24777605:193:32
status: NEWX
ABCC7 p.Lys1218Arg 24777605:193:99
status: NEW194 K14R, K1080R, and K1218R but not K1041R reach the cell surface.
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ABCC7 p.Lys1218Arg 24777605:194:18
status: NEW200 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.
X
ABCC7 p.Lys1218Arg 24777605:200:169
status: NEW201 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).
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ABCC7 p.Lys1218Arg 24777605:201:78
status: NEW208 As expected, increased amounts of K1218R were detected from the cell surface fraction (Fig.
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ABCC7 p.Lys1218Arg 24777605:208:34
status: NEW214 Preventing ubiquitination at K14R, K1080R, and K1218R allows the mutant protein to gain access to the cell surface.
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ABCC7 p.Lys1218Arg 24777605:214:47
status: NEW218 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.
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ABCC7 p.Lys1218Arg 24777605:218:82
status: NEW221 K63-linked ubiquitin on CFTR is reduced in the K68R, K710R, K716R, and K1218R mutants.
X
ABCC7 p.Lys1218Arg 24777605:221:71
status: NEW226 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.
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ABCC7 p.Lys1218Arg 24777605:226:91
status: NEW230 Wild-type and K1218R CFTR maintain the highest stability with little turnover in 24 h.
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ABCC7 p.Lys1218Arg 24777605:230:14
status: NEW237 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.
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ABCC7 p.Lys1218Arg 24777605:237:169
status: NEW241 HDAC6 was abundant in all cases, and ZO-1 was conspicuously absent from the K1080R and K1218R mutants.
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ABCC7 p.Lys1218Arg 24777605:241:87
status: NEW247 K1218R is the most interesting construct since this mutant CFTR does better than the wild type in remaining at the cell surface in large-mass quantities.
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ABCC7 p.Lys1218Arg 24777605:247:0
status: NEW252 The F508del from the surface of cells expressing wt CFTR, K14R, K1080R, and K1218R.
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ABCC7 p.Lys1218Arg 24777605:252:76
status: NEW261 Band C was most reduced in K1041R and highest in the wt, K1080R, and K1218R, consistent with data in panels A and B.
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ABCC7 p.Lys1218Arg 24777605:261:69
status: NEW267 Cell surface band C, prominent for wt CFTR and K1218R located on the PM, was unaffected by MG132 and increased by lysosomal inhibition.
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ABCC7 p.Lys1218Arg 24777605:267:47
status: NEW269 A similar pattern was seen with lysosomal inhibition of cells expressing K1218R, suggesting that there is a ubiquitination step between the cell surface and the lysosome occurring through an alternate site.
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ABCC7 p.Lys1218Arg 24777605:269:73
status: NEW274 We did not detect ZO-1 in the K1080R or K1218R complex.
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ABCC7 p.Lys1218Arg 24777605:274:40
status: NEW275 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.
X
ABCC7 p.Lys1218Arg 24777605:275:163
status: NEW280 K1218R bypasses the proteasome fairly efficiently and further is slow to undergo lysosomal degradation.
X
ABCC7 p.Lys1218Arg 24777605:280:0
status: NEW296 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.
X
ABCC7 p.Lys1218Arg 24777605:296:166
status: NEW