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. 2012 Aug 15;303(4):C436-46.
doi: 10.1152/ajpcell.00105.2012. Epub 2012 May 23.

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

Diego Sbrissa  1 , Ognian C Ikonomov, Catherine Filios, Khortnal Delvecchio, Assia Shisheva
Affiliations

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

Diego Sbrissa et al. Am J Physiol Cell Physiol. .

Abstract

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.

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Figures

Fig. 1.
Fig. 1.
YM201636 preferentially inhibits PIKfyve-catalyzed PtdIns5P synthesis vs. PtdIns(3,5)P2 production in vitro. A: cleared cell lysates derived from 3T3L1 adipocytes were immunoprecipitated (IP) with anti-PIKfyve antibodies and then washed, preincubated with YM201636 (100 nM) or with vehicle for 15 min, and subjected to lipid kinase assay in the presence [γ-32P]ATP (15 min). Extracted lipids were resolved by thin-layer chromatography (TLC) developed with chloroform/methanol/water/ammonia solvent system, as described in materials and methods. Shown is a representative autoradiogram of a TLC plate of 3 independent experiments with similar results. B: anti-PIKfyve immunoprecipitates were incubated with the indicated concentrations of YM201636 and subjected to lipid kinase assay as in A. Extracted lipids were resolved by TLC developed with the n-propanol/2M acetic acid solvent system, as described in materials and methods. Shown is a representative autoradiogram of a TLC plate (top) and quantitation from 4 independent experiments, calculated as a percentage of the corresponding control value (only vehicle) and presented as means ± SE (bottom). *Statistically significant difference (P < 0.05) of the inhibition of PtdIns(3,5)P2 vs. PtdIns5P synthesis.
Fig. 2.
Fig. 2.
In 3T3L1 adipocytes, YM201636 inhibits more powerfully PtdIns5P than PtdIns(3,5)P2 synthesis. 3T3L1 fibroblasts, differentiated to adipocytes on 60-mm dishes, were subjected to inositol and glucose starvation (22 h). Cells were then labeled with myo-[2-3H]inositol (40 h) in the presence of 10% dialyzed FBS and treated with YM201636 (160 nM) or vehicle (DMSO) in DMEM for 40 min. Lipids were extracted, deacylated, and fractionated by HPLC as described in materials and methods. Shown are the radioactivity from the HPLC profiles of a representative experiment (A) and the quantitation from 3 independent experiments, calculated as a percentage of the total PI radioactivity and presented as means ± SE (B). The total PI radioactivity was obtained by summing the counts within the elution times corresponding to the indicated [3H]GroPIns peaks. *P < 0.001, differences in PtdIns(3,5)P2, PtdIns5P, and PtdIns3P in treated vs. untreated 3T3L1 adipocytes.
Fig. 3.
Fig. 3.
In HEK293 cells, YM201636 inhibits preferentially PtdIns5P vs. PtdIns(3,5)P2 synthesis. A HEK293 cell line (DB4), stably expressing PIKfyve at ∼2-fold above the endogenous levels, seeded on 35-mm dishes was subjected to inositol and glucose starvation (22 h). Cells were then labeled with myo-[2-3H]inositol (26 h) in the presence of 10% dialyzed FBS and treated with YM201636 (160 nM) or vehicle in DMEM for 40 min. Lipids were extracted, deacylated, and fractionated by HPLC as described in materials and methods. Presented are the radioactivity from the HPLC profiles of a representative experiment (A) and the quantitation from 3 independent experiments, calculated as a percentage of the total PI radioactivity and presented as means ± SE (B). *P < 0.001, differences in PtdIns(3,5)P2, PtdIns5P, and PtdIns3P in treated vs. untreated cells.
Fig. 4.
Fig. 4.
In Chinese hamster ovary (CHO-T) cells, YM201636 inhibits preferentially PtdIns5P vs. PtdIns(3,5)P2 synthesis. CHO-T cells, stably expressing the human insulin receptor, seeded on 35-mm dishes were subjected to inositol and glucose starvation (12 h) as indicated in materials and methods. Cells were then labeled with myo-[2-3H]inositol (26 h) in the presence of 0.5% BSA and 2.5 mM glucose and treated with YM201636 (160 nM) or vehicle in DMEM for 40 min in the absence (a and b) or presence of 100 nM insulin for the last 10 min of incubation (c). Lipids were extracted, deacylated, and fractionated by HPLC. Presented are the radioactivity from the HPLC profiles of a representative experiment (A) and the quantitation from 3 independent experiments, calculated as a percentage of the total PI radioactivity and presented as means ± SE (B). *P < 0.001, differences in PtdIns(3,5)P2 and PtdIns5P in treated vs. untreated cells.
Fig. 5.
Fig. 5.
YM201636 (YM) at 800 nM, but not at 160 nM, triggers aberrant vacuolation phenotype. CHO-T (a and b), 3T3L1 fibroblasts (c and d), differentiated 3T3L1 adipocytes (e and f), and HEK293 DB4 cells (g and h) were treated with YM201636 at the indicated concentrations for 40 min and fixed. Images were obtained by Hoffman modulation. Pronounced vacuoles are seen only at 800 nM but not at 160 nM of YM201636. In 3T3L1 adipocytes aberrant vacuolation morphology is not apparent at either concentration. Scale bar, 10 μm.
Fig. 6.
Fig. 6.
Insulin-induced disassembly of actin stress fibers is arrested by YM201636 but not by wortmannin (Wort). A: CHO-T cells were serum-starved for 12 h and incubated for 30 min with vehicle (a and b), YM201636 (160 nM; c and d), and wortmannin (100 nM; e and f). Cells were stimulated with 100 nM of insulin for 10 min (b, d, and f) and then stained for F-actin as described in material and methods. Note the disappearance of thick actin filaments crossing the middle of the cell upon insulin stimulation of control and wortmannin-treated (b and f) but not in YM201636-treated (d) cells. B: quantitation of positive cells for F-actin stress fibers, presented as percentage of the total number of inspected cells (counted 150 cells per condition per experiment in 2 separate experiments). Cells displaying at least 2 parallel thick actin fibers crossing nucleus were scored positive. Data are presented as means ± SE. *Statistically significant difference in comparison with the vehicle-treated cells (P < 0.001). Scale bar, 10 μm.
Fig. 7.
Fig. 7.
YM201636 does not alter tyrosine phosphorylation of Dok1. CHO-T cells were serum-starved for 12 h and incubated for 30 min with vehicle or YM201636 at 160 nM. Cells were then stimulated with insulin (100 nM) for 10 min. Cell lysates were collected in the presence of protease and phosphatase inhibitors. Equal amounts of protein (75 μg) were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine (anti-PY), anti-phosphoThr308-Akt (pAkt), anti-Dok, and anti-GDI1 antibodies (GDI1 is used as a loading control) as indicated, with a stripping step between the antibodies. Shown are chemiluminescence detections of a representative blot out of 3 independent experiments with similar results. Note that the insulin-dependent tyrosine phosphorylation of all phospho-proteins, including Dok1, remains unaltered under these conditions. IRS, insulin receptor substrate.
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References

    1. Astle MV, Seaton G, Davies EM, Fedele CG, Rahman P, Arsala L, Mitchell CA. Regulation of phosphoinositide signaling by the inositol polyphosphate 5-phosphatases. IUBMB Life 58: 451–456, 2006 - PubMed
    1. Backer JM. New methods for capturing the mystery lipid, PtdIns5P. Biochem J 428: e1–e2, 2010 - PMC - PubMed
    1. Balla T, Szentpetery Z, Kim YJ. Phosphoinositide signaling: new tools and insights. Physiology (Bethesda) 24: 231–244, 2009 - PMC - PubMed
    1. Carpenter CL. Cantley Phosphoinositide kinases LC Curr Opin Cell Biol 8: 153–158, 1996 - PubMed
    1. Coronas S, Lagarrigue F, Ramel D, Chicanne G, Delsol G, Payrastre B, Tronchere H. Elevated levels of PtdIns5P in NPM-ALK transformed cells: implication of PIKfyve. Biochem Biophys Res Commun 372: 351–355, 2008 - PubMed

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