ABCMdb

ABCB1 p.Lys48Arg

Predicted by SNAP2:	A: N (66%), C: N (82%), D: D (53%), E: D (59%), F: N (72%), G: N (66%), H: N (78%), I: N (82%), L: N (78%), M: N (78%), N: N (57%), P: D (53%), Q: N (61%), R: N (82%), S: N (72%), T: N (78%), V: N (78%), W: N (66%), Y: N (87%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: D, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] Regulation of the stability of P-glycoprotein by u... Mol Pharmacol. 2004 Sep;66(3):395-403. Zhang Z, Wu JY, Hait WN, Yang JM
Regulation of the stability of P-glycoprotein by ubiquitination.
Mol Pharmacol. 2004 Sep;66(3):395-403., [PMID:15322230]

Abstract [show]
Ubiquitination plays a crucial role in regulating protein turnover. Here we show that ubiquitination regulates the stability of the MDR1 gene product, P-glycoprotein, thereby affecting the functions of this membrane transporter that mediates multidrug resistance. We found that P-glycoprotein was constitutively ubiquitinated in drug-resistant cancer cells. Transfection of multidrug-resistant cells with wild-type ubiquitin or treatment with an N-glycosylation inhibitor increased the ubiquitination of P-glycoprotein and increased P-glycoprotein degradation. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), a proteasome inhibitor, induced accumulation of ubiquitinated P-glycoprotein, suggesting the involvement of the proteasome in the turnover of the transporter. Treatment of multidrug-resistant cells with 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester that increases the phosphorylation of P-glycoprotein through activation of protein kinase C, or substituting phosphorylation sites of P-glycoprotein by nonphosphorylatable residues did not affect the ubiquitination of the transporter. Enhanced ubiquitination of P-glycoprotein resulted in a decrease of the function of the transporter, as demonstrated by increased intracellular drug accumulation and increased cellular sensitivity to drugs transported by P-glycoprotein. Our results indicate that the stability and function of P-glycoprotein can be regulated by the ubiquitin-proteasome pathway and suggest that modulating the ubiquitination of P-glycoprotein might be a novel approach to the reversal of drug resistance.

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No. Sentence Comment
66 Plasmids PCW7 (wild-type ubiquitin) and PCW8 (ubiquitin K48R mutant) were kindly provided by Dr. Ron Kopito (Stanford University, Stanford, CA). Login to comment
85 To determine the effect of ubiquitination on P-gp stability, we transiently transfected the MDR MCF-7 cells with a wild-type ubiquitin plasmid, PCW7, or a dominant-negative ubiquitin, PCW8 (K48R mutant), in which the invariant lysine at position 48 was replaced by arginine (Finley et al., 1994). Login to comment
87 The K48R ubiquitin mutant (PCW8) produces ubiquitin chain termination and accumulation of incompletely ubiquitinated proteins that are not targeted for proteasomal degradation (Ward et al., 1995; Yu and Kopito, 1999). Login to comment
128 MDR MCF-7 cells were transfected with wild-type ubiquitin (A and B) or mutant ubiquitin K48R (C). Login to comment
198 Acknowledgments We are grateful to Dr. Ron Kopito (Stanford University) for donating the plasmids PCW7 (wild-type ubiquitin) and PCW8 (ubiquitin K48R mutant), and to Dr. Michael Gottesman (National Cancer Institute) for providing the NIH3T3, N3V2400, N4V600, and N5V2400 cell lines. Login to comment

[hide] Discrete mutations introduced in the predicted nuc... Mol Cell Biol. 1989 Dec;9(12):5289-97. Azzaria M, Schurr E, Gros P
Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.
Mol Cell Biol. 1989 Dec;9(12):5289-97., [PMID:2573836]

Abstract [show]
In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

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No. Sentence Comment
410 Mutation of lysine48 to arginine in the yeast RAD3 protein abolishes its ATPase and DNA helicase activities but not the ability to bind ATP. Login to comment