Activation of the Leukocyte NADPH Oxidase by Protein Kinase C in a Partially Recombinant Cell-free System

Lopes, Lucia Rossetti; Hoyal, Carolyn R.; Knaus, Ulla G.; Babior, Bernard M.

doi:10.1074/jbc.274.22.15533

Cited by 46 publications

(39 citation statements)

References 36 publications

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“…PKC is at least one of the enzymes able to convert p47 phox to a functional molecule. 29,49,50 The respective contribution of each PKC isoform has not been completely elucidated. Although an antisense approach has shown that PKC␤ modulates the respiratory burst in differentiated HL-60 cells, 51 another study has shown that PKC phosphorylates specific residues in p47 phox and participates in the signaling cascade between the N-formyl peptide receptor and NADPH oxidase activation.…”

Section: Discussionmentioning

confidence: 99%

Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells

Rabiet

Tardif

Braun

et al. 2002

Blood

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A tetracycline-controlled expression system was adapted to the human promyelocytic HL-60 cell line by placement of the transactivator (tTA-off) sequence under the control of the human EF-1␣ promoter region. Constitutively active and dominant-inhibitory forms of Cdc42 (Cdc42V12 and Cdc42N17, respectively) were conditionally expressed in this system. The expression of Cdc42V12 had no marked effect on chemoattractant-mediated superoxide production, corroborating previous results indicating that the guanosine 5-triphosphate (GTP)-bound form of Cdc42 is ineffective in directly activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in a cell-free system. However, the N17 mutant potently inhibited chemoattractant-induced superoxide production. The expression of Cdc42N17 interfered with the GTP-loading of Rac and Ras and with the activation of the MAP-kinase pathway. A drastic reduction of chemoattractant-induced inositol-1,4,5-trisphosphate formation and calcium mobilization was observed, corroborating previous in vitro study results identifying PLC␤2 as a Rac/Cdc42 effector. Cdc42N17 was also found to inhibit the translocation of Ras-GRF2, a guanine nucleotide exchange factor for Ras and Rac but not for Cdc42. Thus, the dominant-inhibitory mutant Cdc42N17 was found to interfere at multiple levels in the signaling pathways. The pleiotropic inhibitory effects of Cdc42N17 illustrate the potential pitfalls of using dominant-inhibitory proteins to study the function of Ras-family GTPases. In this regard, a number of conclusions drawn from the use of dominant-inhibitory mutants in myeloid cells might have to be reconsidered. IntroductionTransendothelial migration of leukocytes and their accumulation at sites of inflammation are of particular importance for host defense against invading pathogens. Chemotactic factors, including the N-formyl peptides, C5a, and interleukin-8 (IL-8), which bind to specific heterotrimeric G-protein-coupled transmembrane receptors, regulate cell migration and activate microbicidal and cytotoxic functions through the release of proteolytic enzymes from specific granules and the generation of superoxide anions by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Activation of this enzyme proceeds through a multistep assembly of several components including the 2 transmembrane subunits of cytochrome b 558 (p22 phox and gp91 phox ) and 4 cytosolic proteins (p47 phox , p67 phox , p40 phox , and the small GTPase Rac). 1,2 The triggering and control of bactericidal functions require a coordinated activation of several signaling pathways, namely the activation of tyrosine kinases, phospholipases (PLC␤2, PLD, and PLA 2 ), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) cascades. 3 Low-molecular-weight GTPases are key regulators of a wide spectrum of cellular functions. [4][5][6][7] The binding of guanine nucleotide regulates all members of the Ras-superfamily. Activation of GTPases through guanosine 5Ј-diphosphate (GDP)-GTP exchange is prom...

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Section: Discussionmentioning

confidence: 99%

Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells

Rabiet

Tardif

Braun

et al. 2002

Blood

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“…The binding of glutathione S-transferase (GST)-C2A or GST-Rac (negative control) to several phospholipids and phosphoinositides was evaluated by a dot-blot assay, as described (18) with minor modifications. The GST fusion proteins were affinity-purified by the use of glutathione-agarose (Amersham Pharmacia), as described (11,19). Phospholipids were resuspended at 2 mg͞ml according to the manufacturer's specifications and were spotted (4 g) onto nitrocellulose sheets.…”

Section: Methodsmentioning

confidence: 99%

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Catz

Johnson

Babior

2002

Proc. Natl. Acad. Sci. U.S.A.

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Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3 -phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3 -phosphoinositides in vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3 -phosphoinositides in vivo. We show that the C2A domain of JFC1 is the only domain present in this protein that localizes to the plasma membrane in living cells. Moreover, the C2A domain of JFC1 binds 3 -phosphoinositides in vitro with similar specificity as that described for full-length JFC1, suggesting that the domain mediates the specific membrane localization of the full-length protein. Furthermore, the C2A domain of JFC1 colocalized with the pleckstrin homology domain of Akt in vivo, and both the JFC1 C2A domain and the full-length JFC1 dissociated from the membrane in the presence of PI 3-kinase specific inhibitors. We also show that the association of the C2A domain to the membrane is modulated by calcium. From these results we analyze possible mechanisms for the role of JFC1 in cellular trafficking. P hosphoinositides are specialized phospholipids known to localize to the inner leaflet of the plasma membrane, vesicles, and the nuclear envelope and participate in several signaling pathways (1). One of these phosphoinositides, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ], plays a central role in the regulation of several intracellular pathways, from actin cytoskeleton modulation and vesicle trafficking (2) to modulation of the transcription factor Tubby protein (3). Furthermore, PtdIns(4,5)P 2 is a precursor for the second messenger phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P 3 ] via the phosphoinositide 3-kinases ref. 4). PI 3-kinases and their products 3Ј-phosphorylated phosphoinositides (3Ј-phosphoinositides) are also involved in many crucial cell functions including vesicle trafficking (1, 2), cytoskeleton modulation, and cell survival (5). It is now clear that most of the functions regulated by phosphoinositides are mediated by adaptor proteins that share the ability to bind to PtdIns(4,5)P 2 and PI 3-kinase products with high affinity and specificity. These effectors are characterized by the presence of specialized phosphoinositide-binding domains. These domains include the pleckstrin homology (PH) domain (6), the FYVE finger (7), and the recently discovered PHOX homology (PX) domain (8). Furthermore, C2 domains have been shown to bind to phospholipids or phosphoinositides in the presence or absence of calcium (9). Proteins that contain C2 domains could generally be categorized as signal transduction mediators or membrane traffic effectors (10). The most extensively studied proteins included in the last group are the synaptotagmins (Syts). Syts are a group of transmembrane proteins that contain tan...

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“…The phospholipase-generated lipid messengers could activate protein kinase C or other protein kinases (58 -62), contributing to the phosphorylation of NADPH oxidase components. Phosphorylation of at least one oxidase component, p47 phox , is required for oxidase activation by the nonphysiological agonist phorbol myristate acetate, which activates protein kinase C isoforms (2,8).…”

Section: R59949 Inhibits the Lipid-induced Sedimentation Of Recombinamentioning

confidence: 99%

“…The activation of NADPH oxidase is initiated by receptor-ligand interaction and involves complex intracellular signaling events. These include the activation of protein kinases to phosphorylate cellular proteins and NADPH oxidase components (2,8) and the generation of various lipid second messengers (AA by phospholipase A 2 (9); DG by phospholipase C or PA phosphohydrolase; PA by phospholipase D or DG kinase (10,11)). In cell-free systems, these lipids can induce activation of the enzyme (12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

Palicz

Foubert

Jesaitis

et al. 2001

Journal of Biological Chemistry

112

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The enzyme NADPH oxidase is regulated by phospholipase D in intact neutrophils and is activated by phosphatidic acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed previously that cell-free NADPH oxidase activation by these lipids involves both protein kinase-dependent and -independent pathways. Here we demonstrate that only the protein kinase-independent pathway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA ؉ DG was ATP-independent and unaffected by the protein kinase inhibitor staurosporine, indicating the lack of protein kinase involvement. Both PA and DG were required for optimal activation to occur. The drug R59949 reduced activation of NADPH oxidase by either arachidonic acid or PA ؉ DG, with IC 50 values of 46 and 25 M, respectively. The optimal concentration of arachidonic acid or PA ؉ DG for oxidase activation was shifted to the right with R59949, indicating interference of the drug with the interaction of lipid activators and enzyme components. R59949 inhibited the lipid-induced aggregation/sedimentation of oxidase components p47 phox and p67 phox , suggesting a disruption of the lipidmediated assembly process. The direct effects of R59949 on NADPH oxidase activation complicate its use as a "specific" inhibitor of DG kinase. We conclude that the protein kinase-independent pathway of NADPH oxidase activation by PA and DG involves direct interaction with NADPH oxidase components. Thus, NADPH oxidase proteins are functional targets for these lipid messengers in the neutrophil.The NADPH oxidase (the respiratory burst enzyme) in phagocytic cells produces superoxide (O 2 . ) 1 by catalyzing electron transfer from NADPH to molecular oxygen upon cell stimulation (1-3). This enzyme plays important roles in host defense against infection and in tissue damage due to inflammation (1-5). In addition, NADPH oxidase-like enzymes are present in a variety of other cell types, where the oxygen radicals formed may have signaling roles (5, 6). The enzyme in phagocytes consists of the membrane-bound heterodimeric flavocytochrome b 558 (gp91 phox and p22 phox ) and four cytosolic proteins (p47 phox , p67 phox , p40 phox , Rac1/2) (2-4, 7). Components must assemble in the membrane for the enzyme to become active (2-4, 7). The activation of NADPH oxidase is initiated by receptor-ligand interaction and involves complex intracellular signaling events. These include the activation of protein kinases to phosphorylate cellular proteins and NADPH oxidase components (2, 8) and the generation of various lipid second messengers (AA by phospholipase A 2 (9); DG by phospholipase C or PA phosphohydrolase; PA by phospholipase D or DG kinase (10, 11)). In cell-free systems, these lipids can induce activation of the enzyme (12-17). AA exerts its effect by directly acting on enzyme components (18 -22). PA has been shown to partially activate purified flavocytochrome b 558 (23), suggesting it interacts with this protein. It is not known whether DG has any direct effe...

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Activation of the Leukocyte NADPH Oxidase by Protein Kinase C in a Partially Recombinant Cell-free System

Cited by 46 publications

References 36 publications

Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells

Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells

The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

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