ABCB1 p.Ser661Ala
| Predicted by SNAP2: | A: N (72%), C: N (66%), D: N (72%), E: N (78%), F: N (66%), G: N (72%), H: N (93%), I: N (66%), K: N (82%), L: N (72%), M: N (61%), N: N (87%), P: N (72%), Q: N (82%), R: N (82%), T: N (87%), V: N (72%), W: N (53%), Y: N (72%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: D, W: D, Y: N, |
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[hide] Phosphorylation site mutations in the human multid... J Biol Chem. 1997 Sep 12;272(37):23165-71. Szabo K, Bakos E, Welker E, Muller M, Goodfellow HR, Higgins CF, Varadi A, Sarkadi B
Phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated ATPase activity.
J Biol Chem. 1997 Sep 12;272(37):23165-71., 1997-09-12 [PMID:9287320]
Abstract [show]
In the human multidrug transporter (MDR1), three serine residues located in the "linker" region of the protein are targets of in vivo phosphorylation. These three serines, or all eight serines and threonines in the linker, were substituted by alanines (mutants 3A and 8A) or with glutamic acids (mutants 3E and 8E). The wild-type and mutant proteins were expressed in baculovirus-infected Spodoptera frugiperda (Sf9) ovarian insect cells, and the vanadate-sensitive, drug-stimulated ATPase activity was measured in isolated membrane preparations. The maximum drug-stimulated MDR1-ATPase activity was similar for the wild-type and the mutant proteins. However, wild-type MDR1, which is known to be phosphorylated in Sf9 membranes, and the 3E and 8E mutants, which mimic the charge of phosphorylation, achieved half-maximum activation of MDR1-ATPase activity at lower verapamil, vinblastine, or rhodamine 123 concentrations than the nonphosphorylatable 3A and 8A variants. For some other drugs (e.g. valinomycin or calcein acetoxymethylester) activation of the MDR1-ATPase for any of the mutants was indistinguishable from that of the wild-type protein. Kinetic analysis of the data obtained for the 3A and 8A MDR1 variants indicated the presence of more than one drug interaction site, exhibiting an apparent negative cooperativity. This phenomenon was not observed for the wild-type or the 3E and 8E MDR1 proteins. The dependence of the MDR1-ATPase activity on ATP concentration was identical in the wild-type and the mutant proteins, and Hill plots indicated the presence of more than one functional ATP-binding site. These results suggest that phosphorylation of the linker region modulates the interaction of certain drugs with MDR1, especially at low concentrations, although phosphorylation does not alter the maximum level of MDR1-ATPase activity or its dependence on ATP concentration.
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No. Sentence Comment
30 1 The abbreviations used are: MDR1, human multidrug resistance protein; 3A, mutant MDR1 with mutations S661A, S667A, and S671A; 3E, mutant MDR1 with mutations S661E, S667E, and S671E; 8A, mutant MDR1 with mutations S660A, S661A, S667A, T668A, S671A, S675A, S683A, and T684A; 8E, mutant MDR1 with mutations S660E, S661E, S667E, T668E, S671E, S675E, S683E, and T684E; AM, acetoxymethylester; Sf9, Spodoptera frugiperda ovarian cells.
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ABCB1 p.Ser661Ala 9287320:30:103
status: NEWX
ABCB1 p.Ser661Ala 9287320:30:222
status: NEW40 Baculovirus transfer vectors were constructed by using the human MDR1 cDNA encoding a protein with the following mutants: S661A, S667A, and S671A (3A); S661E, S667E, and S671E (3E); S660A, S661A, S667A, T668A, S671A, S675A, S683A, and T684A (8A); and S660E, S661E, S667E, T668E, S671E, S675E, S683E, and T684E (8E), as described earlier (22, 32).
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ABCB1 p.Ser661Ala 9287320:40:122
status: NEWX
ABCB1 p.Ser661Ala 9287320:40:189
status: NEW[hide] Mechanism of inhibition of P-glycoprotein-mediated... Biochem Pharmacol. 1999 Dec 1;58(11):1723-33. Castro AF, Horton JK, Vanoye CG, Altenberg GA
Mechanism of inhibition of P-glycoprotein-mediated drug transport by protein kinase C blockers.
Biochem Pharmacol. 1999 Dec 1;58(11):1723-33., [PMID:10571246]
Abstract [show]
P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport hy mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.
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No. Sentence Comment
50 The primary screening of the Ser661 to Ala and Ser667 to Ala mutants was done with HinP1 I (site added) and DpnI (site removed), respectively.
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ABCB1 p.Ser661Ala 10571246:50:29
status: NEW46 The primary screening of the Ser661 to Ala and Ser667 to Ala mutants was done with HinP1 I (site added) and DpnI (site removed), respectively.
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ABCB1 p.Ser661Ala 10571246:46:29
status: NEW