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Vol. 19, Issue 2, 711-721, February 2008

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Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase III

Andras Balla^*,, Yeun Ju Kim^*, Peter Varnai^*,, Zsofia Szentpetery^*, Zachary Knight, Kevan M. Shokat, and Tamas Balla^*

*Section on Molecular Signal Transduction, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; Department of Physiology, Semmelweis University, School of Medicine, Budapest, Hungary H-1086; Program in Chemistry and Chemical Biology, University of California, San Francisco, CA 94158; and Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158

Submitted July 28, 2007; Revised October 25, 2007; Accepted November 27, 2007
Monitoring Editor: John York

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks ( or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the - and β-isoforms of type III PI4Ks, PI4KIII was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIII down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIII enzyme in maintaining plasma membrane phosphoinositides.

¹ Because AngII treatment increases the labeling of PtdIns/PtdA, and the knockdown of the individual enzymes could also affect this response, the correction for cell loss due to knockdown of the enzymes was based on the PtdIns/PtdA values obtained in the unstimulated cells also for the in AngII-stimulated samples. We assumed that AngII treatment does not cause cell loss.

Address correspondence to: Tamas Balla (ballat{at}mail.nih.gov)

Abbreviations used: 5-ptase, type-IV phosphoinositide 5-phosphatase; AngII, angiotensin II; DTT, dithiothreitol; ER, endoplasmic reticulum; FAPP, four phosphate adaptor protein; FRET, fluorescence resonance energy transfer; GFP, enhanced green fluorescent protein; InsP₃, inositol trisphosphate; Mer, β-mercaptoethanol; mRFP, monomeric red fluorescent protein; OSBP, oxysterol binding protein; PAO, phenylarsine oxide; PI4K, phosphatidylinositol 4-kinase; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PtdA, phosphatidic acid; PLC, phospholipase C; PH, pleckstrin homology; siRNA, small interfering RNA; Wm, wortmannin.

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