Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells.
Human erythroleukemia (HEL) cells phosphorylate [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate; they also contain all the enzymes to sequentially dephosphorylate [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate to inositol. alpha-Thrombin, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, and sodium fluoride caused the formation of [3H]inositol phosphates in HEL cells that were previously labeled with [3H]inositol. This indicates agonist-induced activation of phospholipase C and hydrolysis of the inositol phospholipids. Pretreatment of the HEL cells with iloprost, a prostacyclin analog that increases cellular cyclic AMP levels, dramatically reduced the formation of inositol phosphates and the increase of [3H]phosphatidylinositol 4,5-bisphosphate. The inhibitory effects of iloprost were associated with the phosphorylation of a 24-kDa protein, which was detected with an antiserum obtained against the rap 1 protein. The catalytic subunit of protein kinase A inhibited formation of polyphosphoinositides during phosphorylation of the rap 1 protein in membranes. This rap 1 protein might have functional relevance in the inhibition of agonist-induced inositide metabolism.
Division of Cell Biology, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
E R
ER
Lazarowski
D A
DA
Winegar
R D
RD
Nolan
E
E
Oberdisse
E G
EG
Lapetina
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D016285:Iloprost; D007294:Inositol / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D007295:Inositol Phosphates; D008565:Membrane Proteins; C081655:inositol-1,3,4,5-tetrakisphosphate; D007294:Inositol; D015544:Inositol 1,4,5-Trisphosphate; D011464:Epoprostenol; D011494:Protein Kinases; D013917:Thrombin; D016285:Iloprost