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Comparative Study
. 2013 Feb 12;110(7):2546-51.
doi: 10.1073/pnas.1216182110. Epub 2013 Jan 28.

Arachidonoyl-phosphatidylcholine oscillates during the cell cycle and counteracts proliferation by suppressing Akt membrane binding

Andreas Koeberle  1 , Hideo Shindou, Solveigh C Koeberle, Stefan A Laufer, Takao Shimizu, Oliver Werz
Affiliations
Comparative Study

Arachidonoyl-phosphatidylcholine oscillates during the cell cycle and counteracts proliferation by suppressing Akt membrane binding

Andreas Koeberle et al. Proc Natl Acad Sci U S A. .

Abstract

The activity of protein kinase B (Akt)--a major kinase promoting cell proliferation and survival--oscillates during the cell cycle. To investigate whether membrane phospholipids may regulate Akt phosphorylation and thus activity, we monitored the lipid profile of nocodazole-synchronized mouse NIH 3T3 fibroblasts during the cell cycle by liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The proportion of sn-2-arachidonoyl-phosphatidylcholine (20:4-PC) inversely correlated with Akt activity. Increasing the cellular ratio of 20:4-PC by supplementation of 20:4-PC to the cell culture medium diminished Akt [serine (Ser)473] phosphorylation. Saturated and monounsaturated phosphatidylcholines, used as control had no effect; 20:4-PC reduced cell proliferation relative to controls, interfered with S-phase transition, and suppressed Akt downstream signaling and cyclin expression like LY294002, which is a specific inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. Additive effects of 20:4-PC and LY294002 were not observed, underlining the critical role of Akt for 20:4-PC signaling; 20:4-PC suppressed Akt membrane translocation as shown by immunofluorescence microscopy but left the concentration of the anchor lipid phosphatidylinositol-3,4,5-trisphosphate unchanged. An in vitro binding assay suggests that 20:4-PC attenuates the interaction of Akt with its membrane binding site. We conclude that 20:4-PC oscillates during the cell cycle and delays cell cycle progression by inhibiting Akt membrane binding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
20:4-PC accumulates during the G1-phase of the cell cycle. NIH 3T3 cells (4 × 106 cells/10-cm dish) were synchronized by nocodazole (0 h) and released into a new cell cycle. Relative intensities of PL and LPC species were determined by LC-MS/MS. (A) Signal intensities of 20:4-containing PC and LPC species relative to total PC intensity. (B) Change of 20:4-containing PLs in the S-phase relative to the G2/M-phase (14 vs. 0 h after removal of nocodazole); 100% corresponds to relative intensities of 16.4 ± 1.0%, 31.9 ± 0.6%, 8.0 ± 0.5%, 21.3 ± 1.6%, and 7.14 ± 0.5% for 20:4-PC, -PE, -PS, -PI, and -PG, respectively. Sphingomyelins (SMs) containing 20:4 were not detectable (n.d.). Data are given as means ± SEM; n = 3–6. *P < 0.05, **P < 0.01, ***P < 0.001 vs. G2/M-phase (0 h); ANOVA + Tukey HSD post hoc tests.
Fig. 2.
Fig. 2.
20:4-PC interferes with cell cycle progression. NIH 3T3 cells (1.2 × 106/75-cm2 flask) were treated for 48 h with or without PC(17:0/17:0) (17:0-PC) or PC(16:0/20:4) (20:4-PC) (120 µM, each, or as otherwise indicated) in DMEM supplemented with 10% charcoal-stripped FCS. (A) Cell numbers were determined after trypan blue staining using a Vi-CELL Series Cell Counter (Beckman Coulter). (B) Cell cycle distribution was analyzed by propidium iodide staining and flow cytometry. See Fig. S3F for histogram plots. (C) Expression of cyclin B1, D1, and E was analyzed at the protein level by Western blot. β-Actin expression was determined as control. (Right) Data from densitometric analysis. Data are given as means ± SEM; n = 3–10. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the untreated control (A and B) or 17:0-PC–treated control (C); ANOVA + Tukey HSD post hoc tests (A and B) and Student t test (C). Western blots are representative of three independent experiments (B).
Fig. 3.
Fig. 3.
20:4-PC inhibits Akt signaling. Protein levels of p-Akt (Ser473), Akt, p-p70S6 kinase (p-p70S6K, Thr389; lower band), p-p85S6K (upper band), cyclin B1 and D1, and β-actin were determined by Western blot. (A and B) NIH 3T3 cells were treated as described in Fig. 2. Concentration- (A) and time-dependent effects (B) of PC(17:0/17:0) (17:0-PC) and PC(16:0/20:4) (20:4-PC) (120 µM each or as otherwise indicated). (C) Confluent cells were treated with or without 20:4-PC (120 µM) in DMEM plus 0.2% BSA for 24 h and then stimulated with insulin (2 µM) for 15 min. (D and E) NIH 3T3 cells (5 × 105/25-cm2 flask) were treated with 20:4-PC (120 µM) and/or vehicle (DMSO), LY294002 (10 µM), pyrrolidine-1 (pyrr-1; 2.5 µM) or bromoenol lactone (BEL; 10 µM) for 48 h in DMEM plus 10% charcoal-stripped FCS. (C–E) Blots were densitometrically analyzed. Data are given as means ± SEM; n = 3. **P < 0.01, ***P < 0.001 vs. the untreated control; ANOVA + Tukey HSD post hoc tests. Western blots are representative of two to three independent experiments.
Fig. 4.
Fig. 4.
20:4-PC inhibits Akt membrane translocation. (A and B) Confluent NIH 3T3 cells were treated with or without PC(16:0/20:4) (20:4-PC; 120 µM) in DMEM plus 0.2% BSA for 24 h. After preincubation with or without LY294002 (10 µM) for 10 min, cells were stimulated with insulin (2 µM) for 15 min. (A) Granule structures of Akt are formed at membranes on stimulation with insulin. Their formation is disturbed by LY294002 or 20:4-PC. Akt was detected by rabbit anti-Akt and visualized using IRDye 800CW goat anti-rabbit. (Scale bar, 10 µm.) The stains of Akt (green) are representative for three independent experiments and merged with DNA-stains (DAPI, blue). (B) Cellular PIP3 levels were determined by ELISA; 100% corresponds to 0.3 ± 0.1 nmol PIP3/mg total protein. (C) Effect of 20:4-PC and PC(17:0/17:0) (17:0-PC) on the binding affinity of Akt to PIP3. Human recombinant Akt (1 µg) was spotted onto nitrocellulose and incubated with liposomes of different 20:4-PC or 17:0-PC ratios (PC). Liposomes contained PIP3 for binding Akt and biotinylated PE for labeling with IRDye 800CW streptavidin. (Lower) Stains are representative of three to four independent experiments. (Upper) Results from the densitometric analysis are presented for the 20:4-PC–labeled stains. Data are given as means ± SEM; n = 3–4. *P < 0.05, **P < 0.01 vs. the insulin-stimulated (B) or PIP3-containing but 20:4-PC-free control (C); ANOVA + Tukey HSD post hoc tests.
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