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

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.

cdc-Like/Dual-Specificity Tyrosine Phosphorylation–Regulated Kinases Inhibitor Leucettine L41 Induces mTOR-Dependent Autophagy: Implication for Alzheimer’s Disease

“…The effect of L41 on phosphoinositide steady-state levels was assessed in 3T3 L1 adipocytes labeled with myo-[2-3 H]inositol (PerkinElmer) following our protocols for adipocytes in vivo labeling (Sbrissa et al, 2012) with a slight modification. Briefly, cells (35-mm dishes) maintained for 18 hours in glucose-and inositol-free Dulbecco's modified Eagle's medium (DMEM) containing 5% dialyzed fetal bovine serum, 0.5% bovine serum albumin, 5 mg/ml insulin, 5 mg/ml transferrin, 2 mM pyruvate, 25 mM HEPES, pH 7.4, 100 IU/ml penicillin, and 100 mg/ml streptomycin were labeled for 40 hours with 22.5 mCi/ml myo-[2-3 H]inositol in glucose-and inositol-free DMEM containing 5 mg/ ml transferrin, 5 mg/ml insulin, 2 mM pyruvate, and 25 mM HEPES, pH 7.4, at 37°C in a 5% CO 2 /95% air humidified incubator.…”

“…Treatment with 0 [vehicle, final dimethylsulfoxide (DMSO), 0.2% v/v], 1, 5, or 20 mM L41 inhibitor was for the last 75 minutes of the labeling period. Lipids were extracted in the presence of EDTA and tetrabutylammonium sulphate (5 mM each), deacylated, and analyzed by high-performance liquid chromatography (HPLC) (Waters Corporation, Milford, MA) on a 5-mm Partisphere SAX column (4.6 mm  250 mm; Whatman/GE Healthcare Life Sciences, Pittsburgh, PA) as detailed previously (Sbrissa et al, 2012 (Sbrissa et al, 1999) were coinjected as internal HPLC standards. Fractions were collected every 0.25 minutes and analyzed for 3 H and 32 P radioactivity after addition of the scintillation mixture.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…Other recent studies indicated that PIKfyve, together with some of the myotubularin phosphatases that remove the 3-phosphate from PtdIns(3,5)P 2 (see sect. VI), are important for PtdIns5P generation in mammalian cells (1173,1352,1827). One of these studies also found that PtdIns5P generated via this pathway is a regulator of cell migration (1173).…”

Activity-dependent PI(3,5)P 2 synthesis controls AMPA receptor trafficking during synaptic depression

“…S1). PIKfyve provides the sole source of PI(3,5)P 2 (10,15,17,(23)(24)(25)(26)(27)(28). The pools of PI3P that are converted to PI(3,5)P 2 may derive from the class III PI 3-kinase VPS34 (29) and/or the class II PI 3-kinase C2α (30).…”