ABCMdb

ABCB1 p.Ser671Ala

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

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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. Login to comment
40 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). Login to comment

[hide] Phosphorylation of P-glycoprotein by PKA and PKC m... Am J Physiol. 1999 Feb;276(2 Pt 1):C370-8. Vanoye CG, Castro AF, Pourcher T, Reuss L, Altenberg GA
Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl- currents.
Am J Physiol. 1999 Feb;276(2 Pt 1):C370-8., [PMID:9950764]

Abstract [show]
Several proteins belonging to the ATP-binding cassette superfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1). We measured whole cell swelling-activated Cl- currents (ICl,swell) in parental cells and cells expressing wild-type MDR1 or a phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671 replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbol ester reduced the rate of increase in ICl,swell only in cells that express MDR1. PKC stimulation had no effect on steady-state ICl,swell. Stimulation of protein kinase A (PKA) with 8-bromoadenosine 3',5'-cyclic monophosphate reduced steady-state ICl, swell only in MDR1-expressing cells. PKA stimulation had no effect on the rate of ICl,swell activation. The effects of stimulation of PKA and PKC on ICl,swell were additive (i.e., decrease in the rate of activation and reduction in steady-state ICl,swell). The effects of PKA and PKC stimulation were absent in cells expressing the phosphorylation-defective mutant. In summary, it is likely that phosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl- channels by independent mechanisms and that Ser-661, Ser-667, and Ser-671 are involved in the responses of ICl,swell to stimulation of PKA and PKC. These results support the notion that MDR1 phosphorylation affects ICl,swell.

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No. Sentence Comment
12 We measured whole cell swelling-activated Cl-currents (ICl,swell) in parental cells and cells expressing wild-type MDR1 or a phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671 replaced by Ala). Login to comment
50 Site-directed mutagenesis was used to substitute Ser-661, Ser-667, and Ser-671 with Ala, and both MDR1 and MDR1-3SA contain six histidine residues at the COOH-terminal end. Login to comment
143 In summary, PKA and PKC stimulation produced distinct effects on ICl,swell, and these effects were abolished by replacing Ser-661, Ser-667, and Ser-671 with Ala residues. Login to comment

[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
47 The mutagenic oligonucleotides were: 5Ј-TTCAAGATCCGCGCTAATAAGAA-3Ј (Ser [661] to Ala), 5Ј-AAGAAAAAGAGCCACTCGT- AGG-3Ј (Ser667 to Ala), and 5Ј-ACTCGTAGAGCGG- TCCGTGGATC-3Ј (Ser671 to Ala). Login to comment
49 The Ser671 to Ala mutagenic oligonucleotide includes one additional base mutation (silent) that adds a BsrBI site for easy screening. Login to comment
43 The mutagenic oligonucleotides were: 5b18;-TTCAAGATCCGCGCTAATAAGAA-3b18; (Ser [661] to Ala), 5b18;-AAGAAAAAGAGCCACTCGT- AGG-3b18; (Ser667 to Ala), and 5b18;-ACTCGTAGAGCGG- TCCGTGGATC-3b18; (Ser671 to Ala). Login to comment
45 The Ser671 to Ala mutagenic oligonucleotide includes one additional base mutation (silent) that adds a BsrBI site for easy screening. Login to comment