ABCG2 p.Thr362Asp
| Predicted by SNAP2: | A: N (82%), C: N (82%), D: N (78%), E: N (82%), F: N (87%), G: N (82%), H: N (93%), I: N (78%), K: N (82%), L: N (82%), M: N (82%), N: N (87%), P: N (87%), Q: N (87%), R: N (78%), S: N (97%), V: N (87%), W: N (66%), Y: N (93%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, V: N, W: D, Y: N, |
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[hide] The 44-kDa Pim-1 kinase phosphorylates BCRP/ABCG2 ... J Biol Chem. 2008 Feb 8;283(6):3349-56. Epub 2007 Dec 5. Xie Y, Xu K, Linn DE, Yang X, Guo Z, Shimelis H, Nakanishi T, Ross DD, Chen H, Fazli L, Gleave ME, Qiu Y
The 44-kDa Pim-1 kinase phosphorylates BCRP/ABCG2 and thereby promotes its multimerization and drug-resistant activity in human prostate cancer cells.
J Biol Chem. 2008 Feb 8;283(6):3349-56. Epub 2007 Dec 5., 2008-02-08 [PMID:18056989]
Abstract [show]
We previously showed that the 44-kDa serine/threonine kinase Pim-1 (Pim-1L) can protect prostate cancer cells from apoptosis induced by chemotherapeutic drugs (Xie, Y., Xu, K., Dai, B., Guo, Z., Jiang, T., Chen, H., and Qiu, Y. (2006) Oncogene 25, 70-78). To further explore the mechanisms of Pim-1L-mediated resistance to chemotherapeutic drugs in prostate cancer cells, we employed a yeast two-hybrid screening to identify cellular proteins that were associated with Pim-1L, and we found the ABC transporter BCRP/ABCG2 as one of the potential interacting partners of Pim-1L. We also showed that the expression level of Pim-1L and BCRP was up-regulated in mitoxantrone and docetaxel-resistant prostate cancer cell lines. Pim-1L was co-localized with BCRP on the plasma membrane and induced phosphorylation of BCRP at threonine 362. Knocking-down Pim-1L expression in the drug-resistant prostate cancer cells abolished multimer formation of endogenous BCRP and resensitized the resistant cells to chemotherapeutic drugs suggesting that BCRP phosphorylation induced by Pim-1L was essential for its functionality. This is further corroborated by our finding that the plasma membrane localization and drug-resistant activity of BCRP were compromised by T362A mutation. Our data suggest that Pim-1L may protect prostate cancer cells from apoptosis, at least in part, through regulation of transmembrane drug efflux pump. These findings may provide a potential therapeutic approach by disrupting Pim-1 signaling to reverse BCRP-mediated multidrug resistance.
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No. Sentence Comment
50 To generate the BCRP T362A or T362D mutant, the threonine residue at position 362 was mutated to alanine or aspartic acid via oligonucleotide-directed mutagenesis with the sense primers 5Ј-GAAGAAGATCGCAGTCTTCAAGG-3Ј or 5Ј-GAAG- AAGATCGACGTCTTCAAGG-3Ј, respectively and their complementary antisense primers by using the QuikChange Mutagenesis kit (Stratagene).
X
ABCG2 p.Thr362Asp 18056989:50:30
status: VERIFIED150 Co-expression of BCRP and Pim-1L in 293T cells induced threonine phosphorylation of BCRP, and the substitution of Thr-362 with alanine (T362A) or the phosphorylation-mimicking aspartate (T362D) completely abolished threonine phosphorylation of BCRP, suggesting that Thr-362 may indeed be phosphorylated by Pim-1L in vivo (Fig. 3C).
X
ABCG2 p.Thr362Asp 18056989:150:187
status: VERIFIED160 In addition, the BCRP drug-resistant activity was significantly reduced when endogenous Pim-1L expression was knocked down by the siRNA, whereas the phosphorylation-mimicking mutant T362D remained active independent of Pim-1L (Fig. 4B).
X
ABCG2 p.Thr362Asp 18056989:160:182
status: VERIFIED166 *, p Ͻ 0.01 compared with the vector control. B, T362D mutant-mediated drug resistance is independent of Pim-1L.
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ABCG2 p.Thr362Asp 18056989:166:55
status: VERIFIED172 Immunofluorescence microscopy revealed that the wild-type BCRP and the phosphorylation- mimmicking mutant T362D were predominantly localized on plasma membrane, whereas the T362A mutant was mainly localized in cytoplasmic compartment (Fig. 5A).
X
ABCG2 p.Thr362Asp 18056989:172:106
status: VERIFIED173 The retention of BCRP in the cytoplasmic compartment caused by the T362A mutation was further confirmed by fractionation experiments (Fig. 5B).
X
ABCG2 p.Thr362Asp 18056989:173:106
status: NEW192 LNCaP cells were treated with the lentivirus encoding the wild-type BCRP, T362A, or T362D mutant.
X
ABCG2 p.Thr362Asp 18056989:192:84
status: VERIFIED151 Co-expression of BCRP and Pim-1L in 293T cells induced threonine phosphorylation of BCRP, and the substitution of Thr-362 with alanine (T362A) or the phosphorylation-mimicking aspartate (T362D) completely abolished threonine phosphorylation of BCRP, suggesting that Thr-362 may indeed be phosphorylated by Pim-1L in vivo (Fig. 3C).
X
ABCG2 p.Thr362Asp 18056989:151:187
status: NEW161 In addition, the BCRP drug-resistant activity was significantly reduced when endogenous Pim-1L expression was knocked down by the siRNA, whereas the phosphorylation-mimicking mutant T362D remained active independent of Pim-1L (Fig. 4B).
X
ABCG2 p.Thr362Asp 18056989:161:182
status: NEW167 *, p Ͻ 0.01 compared with the vector control. B, T362D mutant-mediated drug resistance is independent of Pim-1L.
X
ABCG2 p.Thr362Asp 18056989:167:55
status: NEW193 LNCaP cells were treated with the lentivirus encoding the wild-type BCRP, T362A, or T362D mutant.
X
ABCG2 p.Thr362Asp 18056989:193:84
status: NEW