sq-23377 and phosphatidylinositol-4-phosphate

The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 microM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIalpha or PI4KIIIbeta by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ ionophores dissociated the domains from all membranes, but after Ca2+ chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+ transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIbeta or -IIalpha, but was inhibited by down-regulation of PI4KIIIalpha, or by 10 microM PAO, which also inhibits PI4KIIIalpha. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.

Topics: 1-Phosphatidylinositol 4-Kinase; Acetylcysteine; Adaptor Proteins, Signal Transducing; Androstadienes; Animals; Blotting, Western; Calcium; Carrier Proteins; Cell Membrane; Chelating Agents; Chlorocebus aethiops; COS Cells; Cytoplasm; DNA; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Fluorescence Resonance Energy Transfer; Golgi Apparatus; Green Fluorescent Proteins; Humans; Immunohistochemistry; Ionomycin; Ionophores; Lipid Metabolism; Microscopy, Confocal; Phosphatidylinositol Phosphates; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Receptors, Steroid; RNA, Small Interfering; Subcellular Fractions; Time Factors; Transfection; Wortmannin

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Diglycerides; Ethers; Hydrolysis; Ionomycin; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pituitary Gland; Rats; Thyrotropin-Releasing Hormone; Time Factors