Abstract
The oncogenic serine/threonine kinase Pim-1 phosphorylates and activates the ATP-binding cassette transporter breast cancer resistance protein (ABCG2). The ABC transporter P-glycoprotein (Pgp; ABCB1) also contains a Pim-1 phosphorylation consensus sequence, and we hypothesized that Pim-1 also regulates Pgp. Pgp is exported from the endoplasmic reticulum (ER) as a 150-kDa species that is glycosylated to 170-kDa Pgp, translocates to the cell surface, and mediates drug efflux; alternatively, 150-kDa Pgp is cleaved to a 130-kDa proteolytic product by ER proteases or undergoes ubiquitination and proteasomal degradation. Pim-1 and Pgp interaction was studied in GST pull-down and phosphorylation in in vitro kinase assays. Pim-1 knockdown and inhibition effects on Pgp expression were studied by immunoblotting and flow cytometry and on Pgp stability by immunoblotting after cycloheximide treatment. Pim-1 directly interacted with and phosphorylated Pgp in intact cells and in vitro. Pim-1 knockdown or inhibition decreased cellular and cell surface 170-kDa Pgp, in association with both transient increase in 130-kDa Pgp and increased Pgp ubiquitination and proteasomal degradation. Pim-1 inhibition also decreased expression of 150-kDa Pgp in the presence of the glycosylation inhibitor 2-deoxy-d-glucose. Finally, Pim-1 inhibition sensitized Pgp-overexpressing cells to doxorubicin. Thus, Pim-1 regulates Pgp expression by protecting 150-kDa Pgp from proteolytic and proteasomal degradation and enabling Pgp glycosylation and cell surface translocation and thus Pgp-mediated drug efflux. Pim-1 inhibitors are entering clinical trials and may provide a novel approach to abrogating drug resistance.
Footnotes
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This work was funded by the Leukemia and Lymphoma Society Translational Research Award; and by the University of Maryland, Baltimore University of Maryland Medical Group Cancer Research [Grant CH 649 CRF].
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Y.X. and M.B. are co-first authors.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.061713.
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ABBREVIATIONS:
- BCRP
- breast cancer resistance protein
- ABC
- ATP-binding cassette
- ER
- endoplasmic reticulum
- Pgp
- P-glycoprotein
- PKC
- protein kinase C
- FCS
- fetal calf serum
- shRNA
- small hairpin RNA
- siRNA
- small interfering RNA
- GST
- glutathione transferase
- 2-DG
- 2-deoxy-d-glucose
- MG-132
- carbobenzoxy-l-leucyl-l-leucyl-l-leucinal
- CHX
- cycloheximide
- DMSO
- dimethyl sulfoxide
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- PSC-833
- valspodar.
- Received October 11, 2009.
- Accepted May 11, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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