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. 2015 Jul 10;6(19):17237-50.
doi: 10.18632/oncotarget.4044.

Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis

Affiliations

Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis

Agnieszka Gizak et al. Oncotarget. .

Abstract

Phosphoglycerate mutase (PGAM), a conserved, glycolytic enzyme has been found in nucleoli of cancer cells. Here, we present evidence that accumulation of PGAM in the nucleolus is a universal phenomenon concerning not only neoplastically transformed but also non-malignant cells. Nucleolar localization of the enzyme is dependent on the presence of the PGAM2 (muscle) subunit and is regulated by insulin/IGF-1-PI3K signaling pathway as well as drugs influencing ribosomal biogenesis. We document that PGAM interacts with several 40S and 60S ribosomal proteins and that silencing of PGAM2 expression results in disturbance of nucleolar structure, inhibition of RNA synthesis and decrease of the mitotic index of squamous cell carcinoma cells. We conclude that presence of PGAM in the nucleolus is a prerequisite for synthesis and initial assembly of new pre-ribosome subunits.

Keywords: PGAM2; multifunctional enzyme; rRNA; ribosome assembly; squamous cell carcinoma.

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Conflict of interest statement

CONFLICT OF INTERESTS

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Detection of PGAM in KLN-205 cells with antibodies directed to whole PGAM protein or to C-terminal peptide of PGAM
A. control conditions (scan parameters in red channel were set to emphasize nucleolar staining with propidium iodide – PI) B. RNase-treated cells. Bar=15 μm.
Figure 2
Figure 2. Subcellular localization of PGAM with the use of antibody directed to C-terminal peptide of the protein
The localization was examined in cultures of non-small cell lung carcinoma (NSCLC) HL-1 cardiomyocytes, astrocytes and in breast cancer tissue section (BC). Arrows point nucleoli. Bar=5 μm.
Figure 3
Figure 3. Localization of PGAM with the use of antibody against the C-terminal peptide in neoplastically transformed (NSCLC, KLN-205) and non-malignant (HL-1 cardiomiocytes, astrocytes) cells cultured in serum-free media
Bar=10 μm.
Figure 4
Figure 4. Localization of PGAM in KLN-205 cells in different culture conditions
PGAM was localized with the use of antibody against the C-terminal peptide of the protein in the cells cultured in serum-free medium re-supplemented with IGF-1, insulin or insulin and inhibitor of PI3K (wortmannin). Bar=15 μm.
Figure 5
Figure 5. Localization of PGAM with antibody directed to the C-terminal peptide of the protein in nuclei of KLN-205 cells treated with drugs disturbing ribosomal biogenesis
ActD – actinomycin D; PI – propidium iodide. Bar=5 μm.
Figure 6
Figure 6. Correlation of ActD-stimulated changes of PGAM localization in NSCLC cells with blockade of the cell cycle cell in G2 phase
Upper panel: the PGAM2 localization in the cells incubated with 0.04 μg/ml (low) or 2.5 μg/ml (high) actinomycin D (ActD). Lower panel: the cells in G1 (red) and G1/S (yellow) phase in control conditions and blockade of the ActD-treated cells in G2 phase (green fluorescence) as determined by FUCCI Cell Cycle Sensor. Bar=10 μm.
Figure 7
Figure 7. The effects of KLN-205 cells transfection with PGAM2
The cells were transfected with PGAM2-FITC in normal conditions and in the presence of RNase. As a control, cells were transfected with BSA-FITC. Bar=10 μm.
Figure 8
Figure 8. The effects of PGAM2 silencing on nucleolar morphology and new RNA production in KLN-205 cells
A. in PGAM2-silenced cells, unlike control cells, stained with propidium iodide (red), only one centrally located nucleolus can be found and nucleolar PGAM staining (green) is undetectable; B. the level of new RNA (red) synthesis in PGAM2-silenced cells is reduced as compared to control cells. To visualize DNA the cells were counterstained with Hoechst 33342 (blue). Bar=10 μm.

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