ABCMdb

PMID: 18056989

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 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCG2 p.Thr362Ala 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 byPim-1Lwasessentialforitsfunctionality.Thisisfurthercorrob- orated by our finding that the plasma membrane localization and drug-resistant activity of BCRP were compromised by T362A mutation. Login to comment 50 ABCG2 p.Thr362Ala ABCG2 p.Thr362Asp 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). Login to comment 99 ABCG2 p.Thr362Ala The stained slides of BCRP mutant T362A were examined by using an inverted fluorescence microscope under a ϫ60 oil-immersion objective, and the co-localization of BCRP with Pim-1L was scanned with a laser confocal system. Login to comment 100 ABCG2 p.Thr362Ala The stained slides of BCRP mutant T362A were examined by using an inverted fluorescence microscope under a ϫ60 oil-immersion objective, and the co-localization of BCRP with Pim-1L was scanned with a laser confocal system. Login to comment 150 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala ABCG2 p.Thr362Asp 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). Login to comment 151 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala ABCG2 p.Thr362Asp 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). Login to comment 152 ABCG2 p.Thr362Ala As shown in Fig. 3D, this antibody recognized the wild-type BCRP phosphorylated by Pim-1L but not the unphosphorylytable T362A mutant. Login to comment 153 ABCG2 p.Thr362Ala As shown in Fig. 3D, this antibody recognized the wild-type BCRP phosphorylated by Pim-1L but not the unphosphorylytable T362A mutant. Login to comment 157 ABCG2 p.Thr362Ala To test whether phosphorylation of Thr-362 of BCRP is important for BCRP-mediated drug resistance, we infected LNCaP cells with the lentivirus encoding the HA-tagged BCRP or the T362A mutant and then examined whether LNCaP cells were protected by the overexpression of BCRP or its mutant from apoptosis induced by chemotherapeutic drugs MX, TPT, and DTX. Login to comment 158 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala As shown in Fig. 4A, the wild-type BCRP significantly increased the survival of LNCaP cells in response to these drugs, but the T362A mutant failed to do so, suggesting that the integrity of Thr-362 is essential for the functionality of BCRP. Login to comment 159 ABCG2 p.Thr362Ala As shown in Fig. 4A, the wild-type BCRP significantly increased the survival of LNCaP cells in response to these drugs, but the T362A mutant failed to do so, suggesting that the integrity of Thr-362 is essential for the functionality of BCRP. Login to comment 160 ABCG2 p.Thr362Asp 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). Login to comment 161 ABCG2 p.Thr362Asp 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). Login to comment 163 ABCG2 p.Thr362Ala A, the effects of BCRP phosphorylation site mutant T362A on drug-resistant activity. Login to comment 164 ABCG2 p.Thr362Ala A, the effects of BCRP phosphorylation site mutant T362A on drug-resistant activity. Login to comment 166 ABCG2 p.Thr362Asp *, p Ͻ 0.01 compared with the vector control. B, T362D mutant-mediated drug resistance is independent of Pim-1L. Login to comment 167 ABCG2 p.Thr362Asp *, p Ͻ 0.01 compared with the vector control. B, T362D mutant-mediated drug resistance is independent of Pim-1L. Login to comment 171 ABCG2 p.Thr362Ala To test whether Thr-362 phosphorylation plays a role for BCRP membrane localization, we infected LNCaP cells by the lentivirus encoding the wild-type BCRP or the T362A mutant. Login to comment 172 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala ABCG2 p.Thr362Asp 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). Login to comment 173 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala ABCG2 p.Thr362Asp The retention of BCRP in the cytoplasmic compartment caused by the T362A mutation was further confirmed by fractionation experiments (Fig. 5B). Login to comment 174 ABCG2 p.Thr362Ala The retention of BCRP in the cytoplasmic compartment caused by the T362A mutation was further confirmed by fractionation experiments (Fig. 5B). Login to comment 175 ABCG2 p.Thr362Ala We therefore examined the effects of T362A mutation on BCRP dimerization. Login to comment 176 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala We co-transfected the wild-type HA-tagged BCRP with the Myc-tagged BCRP or its T362A mutant into 293T cells. Login to comment 177 ABCG2 p.Thr362Ala We co-transfected the wild-type HA-tagged BCRP with the Myc-tagged BCRP or its T362A mutant into 293T cells. Login to comment 178 ABCG2 p.Thr362Ala As shown in Fig. 5C, the wild-type Myc-BCRP was efficiently associated with the wild-type HA-BCRP but such interaction was disrupted by the T362A mutation. Login to comment 179 ABCG2 p.Thr362Ala As shown in Fig. 5C, the wild-type Myc-BCRP was efficiently associated with the wild-type HA-BCRP but such interaction was disrupted by the T362A mutation. Login to comment 190 ABCG2 p.Thr362Ala Effects of T362A mutation on the subcellular distribution and multimerization of BCRP in prostate cancer cells. Login to comment 191 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala A, T362A mutant predominately localizes in the intracellular compartment. Login to comment 192 ABCG2 p.Thr362Ala ABCG2 p.Thr362Ala ABCG2 p.Thr362Asp LNCaP cells were treated with the lentivirus encoding the wild-type BCRP, T362A, or T362D mutant. Login to comment 193 ABCG2 p.Thr362Ala ABCG2 p.Thr362Asp LNCaP cells were treated with the lentivirus encoding the wild-type BCRP, T362A, or T362D mutant. Login to comment 197 ABCG2 p.Thr362Ala LNCaP cells were treated with the lentivirus encoding the wild-type BCRP or T362A mutant, or the vector control. Login to comment 198 ABCG2 p.Thr362Ala LNCaP cells were treated with the lentivirus encoding the wild-type BCRP or T362A mutant, or the vector control. Login to comment 201 ABCG2 p.Thr362Ala C, T362A mutation disrupts the dimerization of BCRP. Login to comment 202 ABCG2 p.Thr362Ala C, T362A mutation disrupts the dimerization of BCRP. Login to comment