Fig 1.
De novo purine biosynthesis and PDK1-associated signaling pathways.
(A) Biosynthesis of inosine monophosphate (IMP) from phosphoribosyl pyrophosphate (PRPP) is catalyzed by six enzymes and their assemblies. A 3-enzyme core assembly (blue box) catalyzes the first half of the pathway while the purinosome (red box) regulates the entire pathway. (B) 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is recruited to the plasma membrane upon activation of phosphoinositide 3-kinase (PI3K). Subsequently, PI3K and PDK1 activate Akt for various signaling cascades, including the mammalian target of rapamycin (mTOR). Alternatively, cytoplasmic PDK1 phosphorylates several kinases, including protein kinase C (PKC) and p70 ribosomal protein S6 kinase (S6K), in an Akt-independent manner. Pharmacological inhibitors used in this work are also indicated.
Fig 2.
Regulation of cytoplasmic activity of PDK1 with small molecules promotes clustering of FGAMS-mEGFP.
HeLa and Hs578T cells expressing FGAMS-mEGFP were treated with a PDK1 inhibitor GSK2334470 (A-D; NHeLa = 158 and NHs578T = 791) or a PKC inhibitor Gö6983 (E-H; NHeLa = 623 and NHs578T = 146) for 4–5 hours. HeLa cells expressing FGAMS-mEGFP were also treated with a S6K inhibitor PF4708671 (I-J; NHeLa = 623). As a negative control, Hs578T cells expressing purine salvage enzyme HPRT1-mEGFP were treated with GSK2334470 for 5–6 hours. Unlike FGAMS-mEGFP, clustering of HPRT1-mEGFP was not observed (K-L). The representative images were selected from at least three independent imaging sessions. N indicates the number of the cells we have imaged in our study. Scale bar, 10 μm.
Fig 3.
Knock-down of PDK1 induces the clustering of FGAMS-mEGFP.
HeLa cells were co-transfected with shRNAPDK1 (A and C) and FGAMS-mOFP (B and D). As a control, HeLa cells were also co-transfected with scrambled shRNA in the presence of FGAMS-mOFP (E-F). The representative images were selected from at least five independent imaging sessions. The fold changes of FGAMS-mOFP clustering were quantified in the presence of shRNAs relative to no shRNA control (G). At least 270 cells were analyzed for each condition. Statistical analyses were performed using two-sample two-tail t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar, 10 μm.
Fig 4.
Inhibition of PI3K/Akt/mTOR signaling pathway has no effect on FGAMS-mEGFP localization.
HeLa and Hs578T cells expressing FGAMS-mEGFP (A, C, E, G, I and K), were treated with inhibitors against PI3K (B and D, LY294002; NHeLa = 200 and NHs578T = 408), Akt (F, H and L, Akt Inhibitor X; NHeLa = 200 and NHs578T = 752 or VI; NHeLa = 46), and mTOR (J, rapamycin; NHeLa = 200). Treatment with each inhibitor did not change the subcellular localization of FGAMS-mEGFP. Cells were exposed to the small molecules for at least 4–5 hours. The representative images were selected from at least three independent imaging sessions. N indicates the number of the cells we have imaged in our study. Scale bar, 10 μm.
Fig 5.
Pharmacological inhibition of PDK1 results in the formation of the 3-enzyme core assembly for de novo purine biosynthesis.
HeLa cells expressing FGAMS-mOFP with PPAT-mEGFP (A-C) or TrifGART-GFP (G-I) promoted respective co-clusters in the presence of GSK2334470 (D-F and J-L, respectively) (NDually-Transfected-Cells > 80). As a control, HeLa cells expressing FGAMS-mOFP with mEGFP-ATIC (M-O), however, did not induce co-clusters in response to the treatment of GSK2334470 (P-R) (NDually-Transfected-Cells > 50). Cells were exposed to GSK2334470 for at least 4 hours. The representative images were selected from at least five independent imaging sessions. N indicates the number of the co-transfected cells we have imaged in our study. Pearson’s correlation coefficients (ρ) are indicated in merged images (F, L and R). Scale bar, 10 μm.
Fig 6.
Immunocytochemistry of endogenous TrifGART with FGAMS-mEGFP in fixed HeLa cells.
HeLa cells expressing FGAMS-mEGFP were treated with GSK2334470 (11–33 μM) and fixed with 100% methanol. The representative images demonstrate that clustered FGAMS-mEGFP (A) colocalizes with endogenous TrifGART (B) in fixed HeLa cells. Pearson’s correlation coefficient (ρ) is indicated in a merged image (C). A part of the cell is also zoomed in for clarification (D). At least 300 cells were analyzed. Scale bar, 10 μm, unless otherwise indicated.