Isoform-Specific Membrane Targeting Mechanism of Rac during FcγR-Mediated Phagocytosis: Positive Charge-Dependent and Independent Targeting Mechanism of Rac to the Phagosome

Ueyama, Takeshi; Eto, Mika; Kami, Keiichiro; Tatsuno, Toshihiko; Kobayashi, Toshihiro; Shirai, Yasuhito; Lennartz, Michelle R.; Takeya, Ryu; Sumimoto, Hideki; Saito, Naoaki

doi:10.4049/jimmunol.175.4.2381

Cited by 49 publications

(57 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Cell Culture-HEK293 cells (ATCC) were maintained in Eagle's minimal essential medium (Wako) containing 10% heat-inactivated FBS (Invitrogen), 100 M nonessential amino acids (Invitrogen), and antibiotics at 37°C in 5% CO 2 . RAW264.7 cells were described previously (36). For establishing clonally derived HEK293 lines with stable expression of human Nox2 and human Fc␥RIIa (HEK293 Nox2/Fc␥RIIa ), Nox2 in pcDNA3.1(Neo) and Fc␥RIIa in pcDNA3.1(Neo) were transfected into HEK293 cells using FuGENE 6 (Roche Applied Science) and followed by selection in the presence of 1 mg/ml G418 (Calbiochem).…”

Section: Methodsmentioning

confidence: 99%

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

Ueyama

Nakakita

Nakamura

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: p40phox acquires PI(3)P-binding capabilities through arachidonic acid-induced and H 2 O 2 -induced conformational changes in phagocytes. Results: In addition to conformational changes induced by H 2 O 2 in the cytoplasm, p40phox can acquire PI(3)P binding following membrane targeting. Conclusion: p40phox has novel mechanisms inducing its conformation changes, apart from p47 phox . Significance: This study demonstrates both p40 phox and p47 phox synchronously function as "carriers" and "adaptors" of Nox2-based NADPH oxidase assembly through their conformation changes.

show abstract

Section: Methodsmentioning

confidence: 99%

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

Ueyama

Nakakita

Nakamura

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition to stimulating PAK activity, negatively charged PA may function as a lipid anchor by binding positively charged residues at the C terminus of Rac1. Indeed, the polybasic region is required in addition to the C-terminal isoprenyl group of Rac1 for membrane targeting (Joseph et al, 1994;Kreck et al, 1996;Ueyama et al, 2005). The interaction of the polybasic region with DGK-derived PA could facilitate shuttling of the C-terminal isoprenyl group from the hydrophobic pocket in RhoGDI to the inner leaflet of the plasma membrane.…”

Section: Phosphatidic Acid and Rac Activitymentioning

confidence: 99%

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

Abramovici

Mojtabaie

Parks

et al. 2009

MBoC

101

View full text Add to dashboard Cite

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGK initiates RhoGDI release and Rac1 activation. In DGK-deficient fibroblasts PAK1 phosphorylation and Rac1-RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGK, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGK stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGK, and PA to the activation of Rac1: the PA generated by DGK activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility. INTRODUCTIONRho GTPases regulate gene transcription, cell cycle progression, vesicular traffic, and cell polarity (Jaffe and Hall, 2005;Ridley, 2006) but are best known for their ability to coordinate alterations in cellular actin networks that regulate cell morphology. Such changes are necessary for directed cell migration during embryogenesis, inflammation, wound healing, and tumor metastasis (Burridge and Wennerberg, 2004). In mammalian cells, Rac1 promotes actin polymerization and focal complex assembly, leading to lamellipodia protrusion and membrane ruffle formation; Cdc42 regulates filopodial extension; and Rho promotes the assembly of actin stress fibers and focal adhesions (Ridley et al., 1992;Nobes and Hall, 1995).All Rho GTPases cycle between inactive GDP-bound and active GTP-bound conformations; the active versions interact with specific downstream effectors to elicit distinct biological responses. This cycle is tightly regulated by guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP), and by GTPaseactivating proteins (GAPs), which inactivate Rho proteins by enhancing their intrinsic GTPase activity. A third class of proteins, guanine nucleotide dissociation inhibitors (GDIs), regulates Rho GTPase function in as many as three distinct ways. RhoGDI, the best characterized member, 1) prevents GDP dissociation, 2) inhibits intrinsic or GAP-stimulated GTP hydrolysis (Chuang et al., 1993b), and 3) regulates GTPase partitioning between the cytosol and plasma membrane (Olofsson, 1999). The latter task is accomplished by sequestering the GTPases as soluble cytosolic complexes in which the C-terminal membrane-tar...

show abstract

“…RAW 264.7 macrophages (Ueyama et al, 2005) and COS-7 cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin) at 37°C in 5% CO 2 .…”

Section: Cell Culturementioning

confidence: 99%

“…In recent work, we described mechanisms for translocation of p47 phox (Ueyama et al, 2004) and isoform-specific translocation of Rac to the phagosome (Ueyama et al, 2005). To understand and clarify targeting of p67 phox , an essential cytoplasmic Nox2 activator that acts through Rac, we used AA as a stimulant and explored the adaptor functions of p40 phox and p47 phox in recruitment of p67 phox to membranes.…”

Section: Introductionmentioning

confidence: 99%

“…Although relatively high concentrations of AA (50 -200 M) are required for activation of Nox2 both in vitro and in vivo, functional roles for cytosolic phospholipase A 2 (cPLA 2 ), which produces AA, in Nox2 activation have been demonstrated at the cellular level (Dana et al, 1998;Zhao et al, 2002;Shmelzer et al, 2003). Furthermore, recent studies have shown that the orchestration of low concentrations of AA produced by PLA 2 together with protein kinase C (PKC)-dependent phosphorylation promotes translocation of p47 phox and enhances ROS production by Nox2 (Shiose and Sumimoto, 2000;Peng et al, 2003).In recent work, we described mechanisms for translocation of p47 phox (Ueyama et al, 2004) and isoform-specific translocation of Rac to the phagosome (Ueyama et al, 2005). To understand and clarify targeting of p67 phox , an essential cytoplasmic Nox2 activator that acts through Rac, we used AA as a stimulant and explored the adaptor functions of p40 phox and p47 phox in recruitment of p67 phox to membranes.…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

Self Cite

101

View full text Add to dashboard Cite

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47 phox , p67 phox , p40 phox , and Rac) translocate and associate with the membrane-spanning flavocytochrome b 558 , leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40 phox that regulate its translocation to membranes upon stimulation. GFP-p40 phox translocates to early endosomes, whereas GFP-p47 phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67 phox does not translocate to membranes when expressed alone, but it is dependent on p40 phox and p47 phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40 phox or GFP-p47 phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67 phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40 phox or p47 phox . Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40 phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40 phox and p47 phox function as diverse p67 phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms. INTRODUCTIONIn phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al., 1989;Rotrosen and Leto, 1990;Kuribayashi et al., 2002;Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994;Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.Chronic granulomatous disease...

show abstract

Isoform-Specific Membrane Targeting Mechanism of Rac during FcγR-Mediated Phagocytosis: Positive Charge-Dependent and Independent Targeting Mechanism of Rac to the Phagosome

Cited by 49 publications

References 49 publications

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Contact Info

Product

Resources

About

Isoform-Specific Membrane Targeting Mechanism of Rac during FcγR-Mediated Phagocytosis: Positive Charge-Dependent and Independent Targeting Mechanism of Rac to the Phagosome

Cited by 49 publications

References 49 publications

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

Contact Info

Product

Resources

About

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox