Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K m 1 M). Blocking AKT activation with AKTib1/2 also prevented fMLP-but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67 phox and p47 phox , ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47 phox , p67 phox , and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase. Phospholipase D (PLD)2 is a major source of lipid mediators in many cell types and has emerged as a key regulator of various physiological responses, such as cytoskeleton rearrangement, endocytosis, vesicle trafficking, cell migration, as well pathological processes (1). PLD cleaves phosphatidylcholine to produce free choline and a lipid second messenger, phosphatidic acid (PA). This latter is rapidly dephosphorylated into diglycerides (diacylglycerol, alkyl-acyl glycerol) which are potent PKC activators (2). Two distinct families of PLD, PLD1 and PLD2, have been cloned in mammalian cells and share ϳ50% amino acid identity. Both PLDs are expressed ubiquitously and show a different intracellular distribution and are activated differently suggesting they regulate distinct functions (2, 3). PLD1 is located preferentially at the membrane of internal compartments and is activated by protein kinase C (PKC), small G proteins of the Rho and ADP-ribosylation factor families, Rac1, whereas PLD2, located at the plasma membrane, is not directly activated by these regulators (2). The biochemical regulation of PLD2 is poorly documented, although this isoform is apparently tightly regulated upon activation of various types of membrane receptors.In a previous study with retinoic acid-differentiated human promyelocyte leukemia HL-60 cells (dHL-60 cells), we show...
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