ABCMdb

PMID: 22695718

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., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala MRP1 was mutated at Thr249 to Ala or Glu using QuikChange XL (Stratagene/Agilent Technologies, Santa Clara, CA). Login to comment 69 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala 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 178 ABCC7 p.Glu217Gly Transport of E217G also shows a trend to be reduced; however, it did not reach statistical significance (Fig. 3F). Login to comment 180 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala g, MRP1-T249A. Login to comment 192 ABCC1 p.Thr249Glu h, MRP1-T249E. Login to comment 197 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala B, four-parameter logistic nonlinear regression curve fit for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment 213 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala B, four-parameter logistic nonlinear regression curve fit for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment 214 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala D, fraction of cell survival-doxorubicin concentration data plot for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment 215 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala D, fraction of cell survival-doxorubicin concentration data plot for WT, MRP1, MRP1-T249A, and MRP1-T249E. Login to comment 217 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 219 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 220 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 224 ABCC1 p.Thr249Ala 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 231 ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 235 ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 240 ABCC1 p.Thr249Glu 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 241 ABCC1 p.Thr249Ala 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 243 ABCC1 p.Thr249Ala 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 247 ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 250 ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala 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 257 ABCG2 p.Arg482Thr We considered the expression of other ABC proteins that transport doxorubicin, [e.g., ABCB1 and a mutant form of ABCG2 (R482T/G)]. Login to comment 264 ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala C, MRP1-Thr249-P antibody pulls down MRP1 protein from MRP1 cells but not from MRP1-T249A line. Login to comment 276 ABCC1 p.Thr249Ala 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 277 ABCC1 p.Thr249Ala C, MRP1-Thr249-P antibody pulls down MRP1 protein from MRP1 cells but not from MRP1-T249A line. Login to comment 278 ABCC1 p.Thr249Ala 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 294 ABCC1 p.Thr249Glu 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 296 ABCC1 p.Thr249Glu 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 301 ABCC1 p.Thr249Ala 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 302 ABCC1 p.Thr249Glu 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 304 ABCC1 p.Thr249Glu 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 ABCC1 p.Thr249Glu ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala ABCC1 p.Thr249Ala 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 320 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 321 ABCC1 p.Thr249Ala 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 322 ABCC1 p.Thr249Ala 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 332 ABCC1 p.Thr249Ala 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 333 ABCC1 p.Thr249Ala 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 334 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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 335 ABCC1 p.Thr249Glu ABCC1 p.Thr249Ala 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