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. 2015 Jul 16;6(7):e1825.
doi: 10.1038/cddis.2015.64.

Intracellular oxygen determined by respiration regulates localization of Ras and prenylated proteins

A Kim  1 , R Davis  2 , M Higuchi  1
Affiliations

Intracellular oxygen determined by respiration regulates localization of Ras and prenylated proteins

A Kim et al. Cell Death Dis. .

Abstract

Reduction of mitochondrial DNA (mtDNA) content induces the reduction of oxidative phosphorylation and dependence on fermentative glycolysis, that is, the Warburg effect. In aggressive prostate cancer (PCa), the reduction of mtDNA reduces oxygen consumption, increases intracellular oxygen concentration, and induces constitutive activation of Ras. Many essential proteins for cell death, growth, differentiation, and development, such as Ras, require prenylation for subcellular localization and activation. Prenylation of a protein is defined as the attachment of isoprenoids to a cysteine residue at or near the C-terminus. 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) produces isoprenoids, and is posttranslationally regulated by oxygen. We investigated a critical role of intracellular oxygen in membrane localization of prenylated proteins. Localization of prenylated proteins (H-Ras, prelamin A/C, and Rab5a) was observed in poorly differentiated PCa (PC-3) and well-differentiated PCa (LNCaP) cells. PC-3 cells exhibited high intracellular oxygen concentration, and H-Ras, prelamin A/C, and Rab5a were localized to various membranes (Golgi and plasma membrane, nuclear membrane, and early endosomes, respectively). Remarkably, exogenous hypoxia (0.2% O2) in PC-3 cells induced intracellular hypoxia and changed the localization of the prenylated proteins. H-Ras and Rab5a were translocated to cytosol, and prelamin A/C was in the nucleus forming an abnormal nuclear envelope. The localization was reversed by mevalonate indicating the involvement of mevalonate pathway. In contrast, in LNCaP cells, exhibiting low intracellular oxygen concentration, H-Ras and Rab5a were localized in the cytosol, and prelamin A/C was inside the nucleus forming an inadequate nuclear envelope. Exogenous hyperoxia (40% O2) increased the intracellular oxygen concentration and induced Ras translocation from cytosol to the membrane. Prelamin A/C was translocated to the nuclear membrane and formed a proper nuclear envelope. Rab5a was translocated to the early endosomes. The specific localizations of the prenylated proteins were dependent on intracellular oxygen concentration. These results demonstrate that intracellular oxygen concentration regulates the localization and activation of prenylated proteins.

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Figures

Figure 1
Figure 1
Ras localization is controlled by intracellular oxygen concentration. (a) Immunofluorescence analysis of localization of endogenous Ras in LNCaP and PC-3. (* indicates overexposed image of LNCaP). (b) Confocal images of EGFP-H-Ras-transfected LNCaP and PC-3 cells. Green fluorescent images show EGFP-H-Ras. Scale bar, 10 μm. EGFP-H-Ras-transfected PC-3 cells were treated with 5 μM lovastatin and/or 2 mM mevalonate. (c) and (d) oxygen concentration in the medium surrounding the cells. (mean±standard error, n=3), c is the change in oxygen concentration over time and d is the oxygen concentration surrounding the cells at the 3-h time point. (e) BTP phosphorescent imaging of the indicated cell lines. LNCaP and PC-3 were cultured at 20% O2/5% CO2. BTP phosphorescence is indicated by the red images and DIC images show the whole cells. Scale bar, 10 μm
Figure 2
Figure 2
Oxygen concentration determines localization of H-Ras. (a) Confocal images of EGFP-H-Ras-transfected PC-3 cells. The cells were treated with indicated oxygen conditions with 5% CO2. Scale bar, 10 μm. (b and c) Each graph illustrates the relative fluorescence values of EGFP-HRas in PC-3 cells throughout a cell. The detailed method to measure the fluorescence values is in Supplementary Figure S1B and S1C. (d) Confocal images of EGFP-H-Ras-transfected LNCaP cells. The cells were treated with indicated oxygen conditions with 5% CO2. Scale bar, 10 μm. (e and f) Each graph illustrates the relative fluorescence values of EGFP-HRas in PC-3 cells throughout a cell. The detailed method to measure the fluorescence values is in Supplementary Figure S1B and S1C
Figure 3
Figure 3
(a) The graph illustrates the ratio of localization of H-Ras in membrane to cytosol per cell in the EGFP-H-Ras-transfected LNCaP and PC-3 in the various oxygen conditions, (n=7, *P<0.01). The detailed computation is in Supplementary Figure S1B and S1C. (b) Western blot for ERK activation and HMGR expression. LNCaP and PC-3 lysates were subjected to SDS-PAGE and analyzed by western blotting. (c) Time-dependent changes in localization of H-Ras in LNCaP in exogenous hyperoxic concentration (40% O2/5% CO2)
Figure 4
Figure 4
Localization of prelamin is regulated by intracellular oxygen concentration. (a) Confocal images of DsRed-prelamin A/C-transfected PC-3 cells. The cells were treated with indicated oxygen conditions with 5% CO2. Red fluorescent images show DsRed-prelamin A/C. Scale bar, 10 μm. (b and c) Each graph illustrates the relative fluorescence values of DsRed-prelamin A/C in PC-3 cells throughout a cell. (d) Confocal images of DsRed-prelamin A/C transfected LNCaP cells. The cells were treated with indicated oxygen conditions with 5% CO2. Scale bar, 10 μm. (e and f) Each graph illustrates the relative fluorescence values of DsRed-prelamin A/C in LNCaP cells throughout a cell. (g) Time-dependent changes in localization of prelamin in LNCaP at exogenous hyperoxic concentration (40% O2/5% CO2)
Figure 5
Figure 5
Localization of Rab5a is regulated by intracellular oxygen concentration. (a) Confocal images of EGFP-Rab5a transfected PC-3 cells. The cells were treated with indicated oxygen conditions with 5% CO2. Green fluorescent images show EGFP-Rab5a. Scale bar, 10 μm. (b and c) Each graph illustrates the relative fluorescence values of EGFP-Rab5a in PC-3 cells throughout a cell. (d) Confocal images of EGFP-Rab5a-transfected LNCaP cells. The cells were treated with indicated oxygen conditions with 5% CO2. Scale bar, 10 μm. (e and f) Each graph illustrates the relative fluorescence values of EGFP-Rab5a in LNCaP cells throughout a cell. (g) Time-dependent changes in the localization of EGFP-Rab5a in LNCaP at exogenous hyperoxic concentration (40% O2/5% CO2)
Figure 6
Figure 6
Intracellular oxygen concentration is a key molecule regulating the localization of the prenylated proteins. (a) Confocal images of EGFP-H-Ras-transfected LNCaP cells. The cells were treated with or without 1 μM of Rotenone for 6 h. Scale bar, 10 μm. (b and c) Confocal images of EGFP-H-Ras- and EGFP-Rab5a-transfected PC-3 cells, respectively. Scale bar, 10 μm. EGFP-H-Ras- or EGFP-Rab5a-transfected PC-3 cells were treated with 2 mM mevalonate and/or 0.2% O2/5%CO2
Figure 7
Figure 7
Schematic diagram illustrating that intracellular oxygen concentration, determined by oxidative phosphorylation, regulates prenylation. Green color represents H-Ras and Rab5a and red color represents prelamin A/C
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