ABCMdb

ABCC1 p.Thr249Ala

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
5 Studies in tissue culture confirmed these findings, showing increased intracellular doxorubicin accumulation in MRP1 CK2␣(-) and MRP1-T249A cells compared with MRP1 cells. 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
137 The three peptides synthesized were biotin-Ahx-RRRADDSDDDDDK (Control), biotin- Ahx- LNKEDTSEQVV (MRP1-Thr249 peptide, Thr249 in bold), and biotin-Ahx-LNKEDASEQVV (MRP1-T249A peptide, Ala249 in bold). 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
191 g, MRP1-T249A. 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
231 To determine whether CK2␣ kinase can phosphorylate Thr249 on MRP1, we performed in vitro kinase assays with human recombinant CK2␣ and three peptide substrates: a control peptide to measure general CK2 activity and two peptides derived from the CK2 consensus site in MRP1, the first containing Thr249, the second with Thr249 substituted by alanine. Login to comment
233 Human recombinant CK2␣ phosphorylated both the control and the MRP1244-255 peptides, whereas the phosphorylation of the MRP1244-255-T249A was reduced 6-fold TABLE 1 Doxorubicin sensitivity of MCF7-derived cell lines used in this study AUC was derived from fraction cell survival-doxorubicin concentration plots in Fig. 3, C and D. IC50 and IC90 values derived from four-parameter logistic nonlinear regression curve fit presented in Fig. 3, A and B. 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
241 CK2␣ knockdown, inhibition of CK2, and mutation of the putative CK2 phosphorylation site at Thr249 to alanine results in decreased MRP1-mediated doxorubicin efflux. Login to comment
247 Upon purification of the phosphoantibody by negative affinity chromatography, we tested its specificity by using it to immunoprecipitate the antigen from WT, MRP1, and MRP1-T249A membrane lysates (Fig. 5C). Login to comment
248 We reasoned that if MRP1 was phosphorylated at Thr249, as our data suggested, then MRP1 should be immunoprecipitated from MRP1 cells only, not from WT or MRP1-T249A. Login to comment
249 As anticipated, a strong MRP1 signal was detected in the immunoprecipitate from MRP1 cells and no signal or a very weak signal was detected in WT or MRP1-T249A lysates (Fig. 5C). Login to comment
264 W T M R P 1 M R P 120ug W T M R P 1 M R P 130ug 1 2 3 4 5 6 IP: CK2α IP:MRP1 IB:MRP1 IB:CK2α lysate: nmol\5min\mgofprotein ofphosphorylatedpeptide C ontrol 244-254 M R P1 T249A 244-254 M R P1 0 2 4 6 8 p<0.001 0 50 500 1000 µg IP: T249-P IB: MRP1 A B MRP1 C IP: T249-P IB: MRP1 IB: MRP1 1 0.03 E D MRP1 MRP1CK2α(-) IP: T249-P IB: MRP1 W T M R P1 M R P1-T249A Relative intensity 1 2 3 4 1 2 3 Fig. 5. Login to comment
275 C, MRP1-Thr249-P antibody pulls down MRP1 protein from MRP1 cells but not from MRP1-T249A line. Login to comment
276 Custom-made rabbit antibody against synthetic peptide corresponding to MRP1-Thr249-P consensus site [WSLNKEDT(p)SEQVVP] was used to immunoprecipitate (IP) MRP1 protein from membrane fractions prepared from WT, MRP1, and MRP1-T249A cells, followed by IB with MRP1 antibody (QCRL-1). Login to comment
301 The data were normalized to the baseline absorbance, and four-parameter logistic nonlinear regression curves were generated with use of GraphPad Prism 5. Summary statistics derived from these data are listed in Table 2. lier than for either MRP1 or MRP1-T249A, represented by a larger AUC value. 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
316 The regulation of MRP1 via CK2␣ seems to be general, not substrate-specific, because both CK2␣ knockdown and Thr249Ala mutation similarly alter MRP1 function toward different substrates: LTC4, E217betaG, and doxorubicin (Figs. 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
321 Support for our model comes from coIPs showing that MRP1 and CK2␣ physically interact (Fig. 5A), CK2␣ kinase assays showing that CK2 consensus site of MRP1 is phosphorylated in a Thr249-dependent manner (Fig. 5B), and immunoprecipitations with phosphospecific antibody demonstrating absent phosphorylation of Thr249 in MRP1-T249A and reduced in MRP1 CK2␣(-) cells (Fig. 5E). Login to comment
332 This observation is in agreement with our finding that vesicles derived from MRP1 CK2␣ cells have an intermediate ability to transport LTC4 and E217betaG compared with MRP1 and MRP1-T249A. 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
224 However treatment of MRP1 and MRP1 scrambled cells with DMAT increased doxorubicin accumulation to levels similar to that of the WT, WT scrambled, WT CK2ॷ(afa;), MRP1 CK2ॷ(afa;), and MRP1-T249A cells (Fig. 4). Login to comment
235 Human recombinant CK2ॷ phosphorylated both the control and the MRP1244-255 peptides, whereas the phosphorylation of the MRP1244-255-T249A was reduced 6-fold TABLE 1 Doxorubicin sensitivity of MCF7-derived cell lines used in this study AUC was derived from fraction cell survival-doxorubicin concentration plots in Fig. 3, C and D. IC50 and IC90 values derived from four-parameter logistic nonlinear regression curve fit presented in Fig. 3, A and B. 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
243 CK2ॷ knockdown, inhibition of CK2, and mutation of the putative CK2 phosphorylation site at Thr249 to alanine results in decreased MRP1-mediated doxorubicin efflux. Login to comment
250 We reasoned that if MRP1 was phosphorylated at Thr249, as our data suggested, then MRP1 should be immunoprecipitated from MRP1 cells only, not from WT or MRP1-T249A. Login to comment
251 As anticipated, a strong MRP1 signal was detected in the immunoprecipitate from MRP1 cells and no signal or a very weak signal was detected in WT or MRP1-T249A lysates (Fig. 5C). Login to comment
277 C, MRP1-Thr249-P antibody pulls down MRP1 protein from MRP1 cells but not from MRP1-T249A line. Login to comment
278 Custom-made rabbit antibody against synthetic peptide corresponding to MRP1-Thr249-P consensus site [WSLNKEDT(p)SEQVVP] was used to immunoprecipitate (IP) MRP1 protein from membrane fractions prepared from WT, MRP1, and MRP1-T249A cells, followed by IB with MRP1 antibody (QCRL-1). 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
322 Support for our model comes from coIPs showing that MRP1 and CK2ॷ physically interact (Fig. 5A), CK2ॷ kinase assays showing that CK2 consensus site of MRP1 is phosphorylated in a Thr249-dependent manner (Fig. 5B), and immunoprecipitations with phosphospecific antibody demonstrating absent phosphorylation of Thr249 in MRP1-T249A and reduced in MRP1 CK2ॷ(afa;) cells (Fig. 5E). Login to comment
333 This observation is in agreement with our finding that vesicles derived from MRP1 CK2ॷ cells have an intermediate ability to transport LTC4 and E217betaG compared with MRP1 and MRP1-T249A. 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