Pim-1 kinase protects P-glycoprotein from degradation and enables its glycosylation and cell surface expression.

  • Xie Y
  • Burcu M
  • Linn D
 et al. 
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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.

Author-supplied keywords

  • Cell Membrane
  • Cell Membrane: metabolism
  • Cycloheximide
  • Cycloheximide: pharmacology
  • Flow Cytometry
  • Gene Knockdown Techniques
  • Glycosylation
  • HL-60 Cells
  • Humans
  • Hydrolysis
  • P-Glycoprotein
  • P-Glycoprotein: metabolism
  • Phosphorylation
  • Proteasome Endopeptidase Complex
  • Proteasome Endopeptidase Complex: metabolism
  • Proto-Oncogene Proteins c-pim-1
  • Proto-Oncogene Proteins c-pim-1: genetics
  • Proto-Oncogene Proteins c-pim-1: metabolism
  • Ubiquitination

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Authors

  • Yingqiu Xie

  • Mehmet Burcu

  • Douglas E Linn

  • Yun Qiu

  • Maria R Baer

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