Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha.
Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca(2+) 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 (alpha or beta) is responsible for supplying phosphoinositides during agonist-induced Ca(2+) signaling. Using inhibitors that discriminate between the alpha- and beta-isoforms of type III PI4Ks, PI4KIIIalpha was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], and Ca(2+) 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)P(2) levels in (32)P-labeled cells, but only PI4KIIIalpha down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P(2) resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca(2+) 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 PI4KIIIalpha enzyme in maintaining plasma membrane phosphoinositides.
Balla
Andras
A
Section on Molecular Signal Transduction, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Kim
Yeun Ju
YJ
Varnai
Peter
P
Szentpetery
Zsofia
Z
Knight
Zachary
Z
Shokat
Kevan M
KM
Balla
Tamas
T
D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology*; D006728:Hormones / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D054803:Phospholipase C delta / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D034622:RNA Interference / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D002352:Carrier Proteins; D006728:Hormones; C432935:OSH2 protein, S cerevisiae; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C037178:phosphatidylinositol 4-phosphate; D000804:Angiotensin II; D015544:Inositol 1,4,5-Trisphosphate; D019870:1-Phosphatidylinositol 4-Kinase; D054803:Phospholipase C delta; D000077191:Wortmannin