ABCMdb

ABCC1 p.Thr249Glu

Predicted by SNAP2:	A: N (82%), C: N (87%), D: N (72%), E: N (82%), F: N (57%), G: N (72%), H: N (82%), I: N (66%), K: N (93%), L: N (72%), M: N (87%), N: N (82%), P: N (72%), Q: N (93%), R: N (93%), S: N (97%), V: N (72%), W: N (53%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: D, G: D, H: D, I: D, K: N, L: N, M: N, N: N, P: D, Q: N, R: N, S: N, V: N, W: D, Y: D,

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[hide] Casein kinase 2alpha regulates multidrug resistanc... Mol Pharmacol. 2012 Sep;82(3):488-99. doi: 10.1124/mol.112.078295. Epub 2012 Jun 13. Stolarczyk EI, Reiling CJ, Pickin KA, Coppage R, Knecht MR, Paumi CM
Casein kinase 2alpha regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.
Mol Pharmacol. 2012 Sep;82(3):488-99. doi: 10.1124/mol.112.078295. Epub 2012 Jun 13., [PMID:22695718]

Abstract [show]
We have shown previously that the function of Ycf1p, yeast ortholog of multidrug resistance-associated protein 1 (MRP1), is regulated by yeast casein kinase 2alpha (Cka1p) via phosphorylation at Ser251. In this study, we explored whether casein kinase 2alpha (CK2alpha), the human homolog of Cka1p, regulates MRP1 by phosphorylation at the semiconserved site Thr249. Knockdown of CK2alpha in MCF7-derived cells expressing MRP1 [MRP1 CK2alpha(-)] resulted in increased doxorubicin sensitivity. MRP1-dependent transport of leukotriene C(4) and estradiol-17beta-d-glucuronide into vesicles derived from MRP1 CK2alpha(-) cells was decreased compared with MRP1 vesicles. Moreover, mutation of Thr249 to alanine (MRP1-T249A) also resulted in decreased MRP1-dependent transport, whereas a phosphomimicking mutation (MRP1-T249E) led to dramatic increase in MRP1-dependent transport. Studies in tissue culture confirmed these findings, showing increased intracellular doxorubicin accumulation in MRP1 CK2alpha(-) and MRP1-T249A cells compared with MRP1 cells. Inhibition of CK2 kinase by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole resulted in increased doxorubicin accumulation in MRP1 cells, but not in MRP1 CK2alpha(-), MRP1-T249A, or MRP1-T249E cells, suggesting that CK2alpha regulates MRP1 function via phosphorylation of Thr249. Indeed, CK2alpha and MRP1 interact physically, and recombinant CK2 phosphorylates MRP1-derived peptide in vitro in a Thr249-dependent manner, whereas knockdown of CK2alpha results in decreased phosphorylation at MRP1-Thr249. The role of CK2 in regulating MRP1 was confirmed in other cancer cell lines where CK2 inhibition decreased MRP1-mediated efflux of doxorubicin and increased doxorubicin cytotoxicity. This study supports a model in which CK2alpha potentiates MRP1 function via direct phosphorylation of Thr249.

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No. Sentence Comment
4 Moreover, mutation of Thr249 to alanine (MRP1-T249A) also resulted in decreased MRP1-dependent transport, whereas a phospho-mimicking mutation (MRP1-T249E) led to dramatic increase in MRP1-dependent transport. Login to comment
6 Inhibition of CK2 kinase by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole resulted in increased doxorubicin accumulation in MRP1 cells, but not in MRP1 CK2␣(-), MRP1-T249A, or MRP1-T249E cells, suggesting that CK2␣ regulates MRP1 function via phosphorylation of Thr249. Login to comment
67 MRP1 was mutated at Thr249 to Ala or Glu using QuikChange XL (Stratagene/Agilent Technologies, Santa Clara, CA). Login to comment
69 Mutagenesis was carried-out according to the manufacturer`s instructions and yielded two new products, pLNCX-mcs-X/S-MRP1-T249A and pLNCX-mcs-X/S-MRP1-T249E. Login to comment
71 Expression vectors pLNCX-mcs-X/S-MRP1-T249A and pLNCX-mcs-X/S-MRP1-T249E were transfected into PA317 packaging cell line by the calcium phosphate precipitation method (Ausubel et al., 1987). Login to comment
180 To determine whether phosphorylation of Thr249 affects MRP1 transporter function, two site-specific mutants were generated: MRP1-T249A, with alanine blocking phosphorylation at Thr249; and MRP1-T249E mutant, mimicking threonine phosphorylation. Login to comment
181 WT cells were retrovirally transduced with each mutant vector, and stable clones of both MRP1-T249A and MRP1-T249E were selected to have expression comparable with MRP1 cells (Fig. 2A, lanes g and h versus lane d). Login to comment
192 h, MRP1-T249E. Login to comment
197 In addition, we performed cycloheximide chase assay to address whether mutation of Thr249 to Ala or Glu affects MRP1 protein stability. Login to comment
198 We have found that the stability of both MRP1-T249A and MRP1-T249E does not differ from that of the wild-type MRP1 protein, suggesting that the mutations at Thr249 do not alter protein folding (Supplemental Fig. 1). Login to comment
199 The resistance of MRP1-, MRP1-T249A-, and MRP1-T249E-expressing cells to doxorubicin and in vitro transport assays were carried out as described above for the CK2␣ knockdowns. Login to comment
201 Although cell survival plots for MRP1-T249A and MRP1-T249E do not seem to differ from those for the MRP1 cell line, a modest increase in sensitivity can be seen for MRP1-T249A cells, and a trend to confer more resistance can be seen for MRP1-T249E cells (Fig. 3, B and D; Table 1). Login to comment
202 In vitro transport assays (Fig. 3, E and F) revealed that MRP1-T249A dependent transport of both LTC4 and E217betaG was decreased by approximately half, and MRP1-T249E dependent transport of LTC4 and E217betaG was roughly double that of MRP1. Login to comment
206 Consistent with cytotoxicity and transport data described earlier, CK2␣ knockdown in MRP1-expressing cells resulted in increased doxorubicin accumulation in these cells compared with MRP1 scrambled control [Fig. 4, com- W T W T Scram bled (-) α W T C K 2 M R P1 M R P1 Scram bled (-) α M R P1 C K 2M R P1-T249A M R P1-T249E 0 10 20 30 40 50 ** * ** ** ** p<0.01 pmoleofLTC4/min/mgofprotein W T W T Scram bled (-) α W T C K 2 M R P1 M R P1 Scram bled (-) α M R P1 C K 2M R P1-T249A M R P1-T249E 0 100 200 300 ** p<0.05 pmoleofE2β17G/min/mgofprotein FE 0 1 2 3 4 0.0 0.5 1.0 MRP1 CK2A(-) WT scramble WT CK2A(-) MRP1 scramble log [doxorubicin] nM FractionofCellsSurviving 0 1 2 3 4 0.0 0.5 1.0 WT MRP1 MRP1-T249E MRP1-T249A log [doxorubicin] nM FractionofCellsSurviving Doxorubicin (nM) FractionofCellsSurviving 0 500 1000 0.0 0.5 1.0 WT scramble WT CK2A(-) MRP1 scramble MRP1 CK2A(-) Doxorubicin (nM) FractionofCellsSurviving 0 1000 2000 3000 4000 5000 0.0 0.5 1.0 WT MRP1 MRP1-T249A MRP1-T249E BA DC Fig. 3. Login to comment
212 B, four-parameter logistic nonlinear regression curve fit for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment
214 D, fraction of cell survival-doxorubicin concentration data plot for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment
217 Compared with wild-type MRP1 cells, we observed increased doxorubicin accumulation in MRP1-T249A mutant cell line (Fig. 4, MRP1 versus MRP1 T249A; p Ͻ 0.001) and no change in MRP1-T249E mutant cells (Fig. 4, MRP1 versus MRP1 T249E). Login to comment
220 Furthermore, if CK2␣ regulates MRP1 function via Thr249, as we have proposed, then the doxorubicin efflux from MRP1-T249A and MRP1-T249E cells should not be affected by the addition of the CK2 inhibitor (Fig. 4, DMAT treatments on MRP1-T249A and MRP1-T249E). Login to comment
222 However treatment of MRP1 and MRP1 scrambled cells with DMAT increased doxorubicin accumulation to levels similar to that of the WT, WT scrambled, WT CK2␣(-), MRP1 CK2␣(-), and MRP1-T249A cells (Fig. 4). Login to comment
234 Cell Line AUC IC50 95% CI IC90 95% CI nM nM nM WT scrambled 162a 11.7 10.90-12.52 2.16 1.81-2.58 WT CK2A(-) 155a 10.6 9.459-11.90 1.46 1.08-1.98 MRP1 scramble 360a 88.6b 79.23-98.97 5.45b 3.99-7.46 MRP1 CK2A(-) 229a 24.9b,c 21.55-28.73 1.31c 0.9036-1.885 WT 564 12.2 10.98-13.47 2.45 1.9-3.16 MRP1 1114 105.3d 97.85-113.3 12.93d 10.68-15.67 MRP1-T249A 961 101.7d 87.53-118.2 5.58d,e 3.76-8.28 MRP1-T249E 1514 107.7d 94.61-122.6 11.67d 0.39-16.22 CI, confidence interval. Login to comment
240 W T W T Scram bled (-) α W T C K 2 M R P1 M R P1 Scram bled (-) α M R P1 C K 2 M R P1-T249AM R P1-T249E 0 50 100 150 control 10 µM DMAT *** * * * *** * ** p<0.05 p<0.01 p<0.001 *** *** *** RelativeDoxorubicinAccumulation (ExpressedasPercentofControl) Fig. 4. Login to comment
294 Figure 3D clearly shows that the survival curve for MRP1-T249E mutant reaches a plateau much ear- HeLa H460 p<0.05* n.s.= not significant C ontrol M K571 TBBz M K571+TBBz FTC PSC 833 0 50 100 150 200 n.s. * *** *** C ontrol M K571 TBBz M K571+TBBz FTC PSC 833 0 50 100 150 200 * *** n.s. *** RelativeDoxorubicinAccumulation (PercentofControl) H460 A549C 1 2 3 4 0.0 0.5 1.0 FractionofCellsSurviving HeLa 1 2 3 4 0.0 0.5 1.0 1 2 3 4 0.0 0.5 1.0 A549 C ontrol M K571 TBBz M K571+TBBz FTC PSC 833 0 50 100 150 200 n.s. *** * *** p<0.001*** Control MK571 TBBz MK571 + TBBz A MRP1 ABCG2 CK2α β-actin ABCB1 ABC B1 H eLa H 460 A549 M C F7 M R P1 B Symbols used: Fig. 6. Login to comment
302 The earlier plateau for MRP1-T249E indicates reduction in lethality beyond 100 to 200 nM and suggests the presence of cell subpopulations biochemically or kinetically resistant to doxorubicin (Moon et al., 1981). Login to comment
303 Taken together, we postulate that there are differences in doxorubicin sensitivity among MRP1, MRP1-T249A, and MRP1-T249E cell lines, although they are difficult to demonstrate with this assay for several reasons (Chambers et al., 1984; Cole, 1986; Campling et al., 1988; Campling et al., 1991). Login to comment
318 It is noteworthy that transport analysis suggests that in MCF7 cells, MRP1 is phosphorylated at Thr249 at approximately 50% of capacity, because T249E mutation increases transport by 2-fold and T249A mutation decreases it by 50%. Login to comment
319 However, tissue culture experiments show similar doxorubicin accumulation for MRP1 and MRP1-T249E, whereas the MRP1-T249A and MRP1 CK2␣(-) cells accumulate 2-fold more doxorubicin compared with MRP1 cells, suggesting that a large fraction of MRP1 cellular pool is phosphorylated (50-100%). Login to comment
334 Pretreatment of MRP1 cells with DMAT increased doxorubicin cellular accumulation by 2-fold, whereas no change was observed for pretreatments of MRP1 CK2␣(-), MRP1-T249A, and MRP1-T249E cells, indicating that Thr249 is the primary site of CK2␣-mediated phosphorylation of MRP1. Login to comment
213 B, four-parameter logistic nonlinear regression curve fit for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment
215 D, fraction of cell survival-doxorubicin concentration data plot for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment
219 Compared with wild-type MRP1 cells, we observed increased doxorubicin accumulation in MRP1-T249A mutant cell line (Fig. 4, MRP1 versus MRP1 T249A; p b0d; 0.001) and no change in MRP1-T249E mutant cells (Fig. 4, MRP1 versus MRP1 T249E). Login to comment
236 Cell Line AUC IC50 95% CI IC90 95% CI nM nM nM WT scrambled 162a 11.7 10.90-12.52 2.16 1.81-2.58 WT CK2A(afa;) 155a 10.6 9.459-11.90 1.46 1.08-1.98 MRP1 scramble 360a 88.6b 79.23-98.97 5.45b 3.99-7.46 MRP1 CK2A(afa;) 229a 24.9b,c 21.55-28.73 1.31c 0.9036-1.885 WT 564 12.2 10.98-13.47 2.45 1.9-3.16 MRP1 1114 105.3d 97.85-113.3 12.93d 10.68-15.67 MRP1-T249A 961 101.7d 87.53-118.2 5.58d,e 3.76-8.28 MRP1-T249E 1514 107.7d 94.61-122.6 11.67d 0.39-16.22 CI, confidence interval. Login to comment
296 Figure 3D clearly shows that the survival curve for MRP1-T249E mutant reaches a plateau much ear- HeLa H460 p<0.05 * n.s.= not significant C ontrol M K571 TBBz M K571+TBBz FTC PSC 833 0 50 100 150 200 n.s. * *** *** C ontrol M K571 TBBz M K571+TBBz FTC PSC 833 0 50 100 150 200 * *** n.s. *** Relative Doxorubicin Accumulation (Percent of Control) H460 A549 C 1 2 3 4 0.0 0.5 1.0 Fraction of Cells Surviving HeLa 1 2 3 4 0.0 0.5 1.0 1 2 3 4 0.0 0.5 1.0 A549 C ontrol M K571 TBBz M K571+TBBz FTC PSC 833 0 50 100 150 200 n.s. *** * *** p<0.001 ** * Control MK571 TBBz MK571 + TBBz A MRP1 ABCG2 CK2b1; b2;-actin ABCB1 ABC B1 H eLa H 460 A549 M C F7 M R P1 B Symbols used: Fig. 6. Login to comment
304 The earlier plateau for MRP1-T249E indicates reduction in lethality beyond 100 to 200 nM and suggests the presence of cell subpopulations biochemically or kinetically resistant to doxorubicin (Moon et al., 1981). Login to comment
305 Taken together, we postulate that there are differences in doxorubicin sensitivity among MRP1, MRP1-T249A, and MRP1-T249E cell lines, although they are difficult to demonstrate with this assay for several reasons (Chambers et al., 1984; Cole, 1986; Campling et al., 1988; Campling et al., 1991). Login to comment
320 However, tissue culture experiments show similar doxorubicin accumulation for MRP1 and MRP1-T249E, whereas the MRP1-T249A and MRP1 CK2ॷ(afa;) cells accumulate 2-fold more doxorubicin compared with MRP1 cells, suggesting that a large fraction of MRP1 cellular pool is phosphorylated (50-100%). Login to comment
335 Pretreatment of MRP1 cells with DMAT increased doxorubicin cellular accumulation by 2-fold, whereas no change was observed for pretreatments of MRP1 CK2ॷ(afa;), MRP1-T249A, and MRP1-T249E cells, indicating that Thr249 is the primary site of CK2ॷ-mediated phosphorylation of MRP1. Login to comment