Tài liệu Báo cáo khoa học: Peroxiredoxin II functions as a signal terminator for H2O2-activated phospholipase D1 doc

A novel interaction with peroxiredoxin II PrxII, an enzyme that eliminates cellular H2O2, which is a known stimulator of PLD1, was identified by PLD1-affinity pull-down and MS.. Functional

for H2O2-activated phospholipase D1

Nianzhou Xiao, Guangwei Du and Michael A Frohman

Department of Pharmacology and the Center for Developmental Genetics, University Medical Center at Stony Brook, NY, USA

Mammalian phospholipase D (PLD) is a

signal-trans-ducing enzyme that hydrolyzes PtdCho to generate the

membrane-bound lipid signal phosphatidic acid (PA)

(reviewed in [1,2]) PA is a second messenger and can

be further converted into diacylglycerol PLD is

indi-rectly activated in response to cellular stimulation by

various extracellular agonists including hormones,

growth factors, neurotransmitters, adhesion molecules,

cytokines and physical stimuli The direct mechanism

by which PLD is activated involves physical

interac-tion with protein kinase C (PKC) and the

ADP-ribosy-lation factor (ARF) and RhoA small GTPase families

A well-studied role for PLD in stimulation of

NADPH during respiratory oxidative burst has been

described by may groups [3–6] (reviewed in [7]) PLD

functions both directly, by generating PA, which binds

to and stimulates the p47(phox) component of the

NADPH oxidase complex [5,8], and by conversion

of some of the PA into diacylglycerol Diacylglycerol

recruits PKC to the plasma membrane, which is also required for NADPH activation [9,10] Once NADPH oxidase is activated, it generates H2O2, which can func-tion to kill intracellular bacteria and play pro-apopto-tic or anti-apoptopro-apopto-tic roles depending on the cellular context In addition, H2O2 stimulates PLD activity by

a poorly understood, probably indirect, mechanism involving tyrosine kinases and PKC [11–13] This cre-ates a runaway positive feedback cycle: PLD activation promotes H2O2 production and PKC recruitment, which leads to even more PLD activity This paper reports the identification of a cellular mechanism by which this positive feedback cycle may be regulated and terminated

In addition to the extensively documented interac-tions between PLD1 and the proteins (PKC, ARF and RhoA) that stimulate it directly [14–18], interactions involving other proteins, such as actin, protein kinase N, casein-kinase-2-like serine kinase and amphiphysin,

Keywords

hydrogen peroxide; peroxiredoxin II;

phosphatidic acid; phospholipase D1; PMA

Correspondence

M Frohman, Center for Developmental

Genetics, 438 CMM, Stony Brook, NY

11794-5140, USA

Fax: +1 631 632 1692

Tel: +1 631 632 1476

E-mail: michael@pharm.sunysb.edu

(Received 4 May 2005, revised 3 June

2005, accepted 8 June 2005)

doi:10.1111/j.1742-4658.2005.04809.x

Phospholipase D1 (PLD1) is a signal-transduction regulated enzyme which regulates several cell intrinsic processes including activation of NAPDH oxidase, which elevates intracellular H2O2 Several proteins have been reported to interact with PLD1 in resting cells We sought to identify pro-teins that interact with PLD1 after phorbol 12-myristate 13-acetate (PMA) stimulation A novel interaction with peroxiredoxin II (PrxII), an enzyme that eliminates cellular H2O2, which is a known stimulator of PLD1, was identified by PLD1-affinity pull-down and MS PMA stimulation was con-firmed to promote physical interaction between PLD1 and PrxII and to cause PLD1 and PrxII to colocalize subcellularly Functional significance

of the interaction was suggested by the observation that over-expression of PrxII specifically reduces the response of PLD1 to stimulation by H2O2 These results indicate that PrxII may have a signal-terminating role for PLD1 by being recruited to sites containing activated PLD1 after cellular stimulation involving production of H2O2

Abbreviations

HA, hemagglutinin; MobA, molybdopterin guanine dinucleotide biosynthesis protein AI; PA, phosphatidic acid; PLD, phospholipase D; PKC, protein kinase C; PMA, phorbol-12-myristate 13-acetate; PrxII, peroxiredoxin II.

have found that PLD1 undergoes regulated

transloca-tion from perinuclear membrane vesicles to the plasma

membrane and back as part of the cellular response to

agonist signaling [23], raising the possibility that PLD1

acquires different protein partners once it becomes

acti-vated and⁄ or as it transits through the cell In this

report, we describe one such interaction with

peroxi-redoxin II (PrxII) which may serve as a mechanism for

signal termination when the function of PLD1 involves

stimulating increased production of H2O2

Results

PLD activation pattern after stimulation with

phorbol-12-myristate 13-acetate (PMA)

PMA directly activates PKC and stimulates PLD1 by

direct and indirect mechanisms (reviewed in [2,24]) We

reported previously that stimulation of COS-7 cells

with PMA for 2 h causes PLD1 to translocate from

perinuclear membrane vesicles to the plasma membrane

[23] However, the timing of PLD1 activation in this

system was not examined, and in other cell types,

PLD1 activation can be quite rapid To maximize the

likelihood of identifying proteins newly interacting with

PLD1 in its activated state, we first examined how long

it takes PLD to reach peak activity after stimulation

PMA stimulation of PLD1 activation was examined

using CHO cell lines stably transfected with

Tet-indu-cible PLD1 expression constructs with which

hemagglu-tinin (HA)-tagged PLD1 can be expressed efficiently

Butan-1-ol was added to the cultures for a 2-min

win-dow to obtain brief, successive snapshots of PLD

acti-vity through the accumulation of phosphatidylbutanol

at different times after the initiation of the PMA

stimu-lation (Fig 1) PLD activity increased rapidly (within

5 min) after the addition of 100 nm PMA and reached

peak levels at 10 min, which was subsequently taken as

the standard time for which to activate PLD1 using

PMA for the purpose of identifying and examining

sti-mulus-dependent protein interactions

Identification of PrxII as a PMA-promoted

PLD1-interacting protein

HA-tagged PLD1 was induced in the CHO cells, and

affinity pull-down performed before and after 10 min

of 100 nm PMA stimulation In brief, the resting stage

cells and stimulated cells were harvested and exposed

to anti-HA beads to pull-down HA-PLD1 and proteins potentially in complex with it The protein samples were separated by SDS⁄ PAGE (12% gel) followed by silver staining Several bands exhibited different levels

of intensity between the two samples (Fig 2) Interest-ing bands were processed for MS analysis at the SUNY-Stony Brook CASM facility The most promin-ent band ( 22 kDa; indicated by the arrow in Fig 2) was identified as PrxII (Fig 3) PrxII is a 22 kDa pro-tein that belongs to the peroxiredoxin family [25] Peroxiredoxins use redox-active cysteines to reduce peroxides and thus protect many types of enzyme from oxidation [26] Peroxiredoxin antioxidant activity is linked to many signaling pathways, including enhance-ment of natural killer cell activity, cell proliferation and differentiation, heme metabolism, immune response and apoptosis [26] Peroxiredoxin activity and function have been associated with human disease in many contexts, in particular carcinogenesis and aging [27–29]

To rule out the possibility of inadvertent contamin-ation during the sample processing for MS analysis, the PLD1 affinity precipitation experiment was repea-ted and analyzed using western immunoblotting (Fig 4) HA-PLD1 and PrxII were visualized using antibody to HA and a rabbit anti-PrxII IgG, respec-tively PLD1 induction did not affect PrxII concentra-tion (Fig 4A), and PLD1 pulled-down approximately fivefold more PrxII after PMA stimulation (Fig 4B) The amount of PrxII pulled-down by PLD1 from unstimulated cells varied from none (as shown in Fig 2) to small amounts (as shown in Fig 4) In all

Fig 1 PLD activity time course upon PMA stimulation In vivo transphosphatidylation PLD assays were performed as described in Experimental procedures Cells were stimulated with PMA for 0, 3,

8, 28 and 58 min before the addition of butan-1-ol, followed by an additional 2 min of culture and assay termination using ice-cold methanol The experiment was performed three times with similar results.

cases, however, a substantial increase in PrxII

pull-down was observed after cellular stimulation

PrxII complexes with and precipitates PLD1

in an in vitro binding assay

To confirm the PLD1–PrxII protein interaction

revealed by HA-PLD1 affinity precipitation, we

per-formed an in vitro assay using recombinant PrxII and

PLD1 His6-tagged PrxII protein was expressed and

purified from Escherichia coli, mixed with sf9 cell

lysates containing baculoviral-generated

GluGlu-tag-ged PLD1, pulled-down using Ni⁄ nitrilotriacetate ⁄

agarose, and analyzed using SDS⁄ PAGE and western

blotting (Fig 5) To address nonspecific binding,

pull-down of PLD1 by Ni⁄ nitrilotriacetate ⁄ agarose alone

was determined (Fig 5, lane 3), and also the extent to

which it was pulled-down by His6-tagged

molybdopterin guanine dinucleotide biosynthesis

pro-tein A (MobA; lane 2) MobA is a 21 kDa nucleic

acid-binding protein [30,31] which would not be a

likely candidate to interact with PLD family members

MobA-His6 protein was expressed and purified from

E coli (kindly provided by J Daniels, SUNY-Stony Brook) Only a very small amount of GluGlu-PLD1 protein was pulled-down by the Ni⁄ nitrilotriacetate ⁄ ag-arose alone More nonspecific pull-down was observed

in the presence of MobA, which is expected as PLD1

Fig 3 MS-MS identification of PrxII as a PMA-stimulated PLD1-interacting protein Protein bands were excised from the polyacryl-amide gel as described in Experimental procedures and processed for MS analysis MS-MS identified a 22-kDa protein, PrxII, at score

103 (protein scores greater than 71 are significant P < 0.05) The arrow denotes the PrxII score Peptides that matched with PrxII sequences are shown in italic underline A single hit of significant (85) but lesser significance was observed for another protein As only one hit was observed, this candidate was not pursued further.

Fig 4 PMA promotes interaction of PLD1 with PrxII CHO cells inducibly expressing HA-PLD1 and variably stimulated with PMA were immunoprecipitated using anti-HA matrix and analyzed by western blotting as described in Experimental Procedures The HA-PLD1 and endogenous PrxII proteins were imaged using a rat monoclonal antibody to HA and rabbit anti-PrxII serum, respectively (A) HA-PLD1 and PrxII in the whole cell lysate (B) PLD1 (120 kDa) and PrxII (22 kDa) probed by their specific antibodies Representa-tive of three experiments IP, immunoprecipitation; Tet, doxy-cycline.

Fig 2 Identification of a protein that exhibits enhanced interaction

with PLD1 after PMA stimulation HA-tagged PLD1 was induced in

CHO cells, and affinity pull-down performed before and after a

10-min PMA stimulation The protein samples were analyzed by

SDS ⁄ PAGE (12% gel) followed by silver staining The arrow

indi-cates the band that was determined to be PrxII in MS as described

subsequently The results are representative of three independent

experiments.

is a hydrophobic, sticky protein [1] After correction

for the Ni⁄ nitrilotriacetate ⁄ agarose nonspecific

pull-down and normalization to the relative amounts of

PrxII and MobA used in the assay (Fig 5A, bottom

panel), quantitation of the amount of PLD1

pulled-down revealed that His6-PrxII pulled-down PLD1

40-fold more efficiently than His6-MobA (Fig 5B)

PLD1 and PrxII colocalize after cellular

stimulation

Lysis of cells before affinity pull-down creates the

opportunity for proteins normally localized in different

subcellular compartments to associate and hence

gen-erate false positive results [32] We thus used

immuno-fluorescent detection and confocal microscopy to

examine the PLD1 and PrxII subcellular patterns of

localization PMA was used to stimulate CHO cells in

whereas PrxII localized to both the cytoplasm and possibly some membrane vesicles of unknown identity that did not contain PLD1 (Fig 6, top row) In con-trast, in cells stimulated with PMA for 10 min, whereas PLD1 still exhibited perinuclear localization, PrxII now localized to both the cytoplasm and the perinuclear vesicles containing PLD1 (Fig 6, lower panel) Thus, PLD1 and PrxII partially colocalize before lysis in PMA-stimulated cells, suggesting that the interaction between them is physiological rather than an artifact of lysis These results also support, using a visual rather than molecular biochemical approach, the proposal that PMA triggers increased interaction between PLD1 and PrxII

PrxII over-expression inhibits H2O2-stimulated PLD1 activity

A priori, the interaction between PrxII and PLD1 may affect PLD1 activation directly in a general context, or PrxII effects on PLD1 activity may be restricted just

to the context in which H2O2 is the agonist responsible for stimulating PLD1 activation To explore this, we examined the consequences of PrxII over-expression

on PLD1 activation in the context of stimulation by PMA in comparison with stimulation by H2O2 PMA stimulates PLD1 by activating PKC, which is a direct activator of PLD1 and does not require H2O2 to medi-ate its stimulation [15]

PrxII was over-expressed in CHO cells inducibly expressing PLD1 An in vivo PLD assay was per-formed in which the cells were stimulated with either PMA or H2O2 for 10 min (Fig 7) PrxII over-expres-sion did not affect basal PLD1 activity or PMA-stimu-lated PLD1 activation, but completely ablated stimulation of PLD1 by H2O2 This shows that PrxII can function as a negative regulator of PLD1 activa-tion by H2O2

Discussion

We describe here the identification of a novel PLD1-interacting protein, PrxII Although PLD1-PLD1-interacting proteins have been identified previously, this is the first report of an interaction that is promoted in the context

of PLD1 activation Our original goal was to identify novel proteins that interact with PLD1 as it transits from perinuclear vesicles to the plasma membrane and back through endosomes In our previous report on

B

Fig 5 In vitro pull-down of PLD1 by PrxII GluGlu-PLD1 and

puri-fied His6-PrxII and His6-MobA proteins were prepared as described

in Experimental procedures, mixed as indicated, and pulled-down

using Ni ⁄ nitrilotriacetate ⁄ agarose (A) Analysis of pull-down by

SDS ⁄ PAGE (8% gel for GluGlu-PLD1; 12% gel for His 6 -PrxII and

His 6 -MobA) followed by western blotting using antibodies to His 6

and GluGlu (B) Quantification of band intensity using Odyssey

soft-ware Assay backgrounds (lane 3 in A) were subtracted from lanes

1 and 2, and then the PLD1 IP values (top row, lanes 1 and 2)

nor-malized to the amounts of PrxII and MobA used for the

immuno-precipitation (bottom row, lanes 1 and 2) The scale of the y-axis

was set such that the amount of PLD1 immunoprecipitated by

MobA is equal to 1 Representative of three independent

experi-ments IB, immunoblot.

PLD1 translocation, we showed that PLD1 can be

found at the plasma membrane of COS-7 cells 2 h

after the initiation of PMA stimulation, and that the

translocation is facilitated by PLD1 activation [23] In

this study, we searched for proteins that interact with

PLD1 in a stimulation-dependent manner at the peak

of PLD1 activity (10 min, Fig 1), which precedes

PLD1 translocation to the plasma membrane (Fig 6)

Other stimulation-dependent interactions that occur

after PLD1 translocates to the plasma membrane may

occur and await discovery

What might be the functional significance of a

stimu-lation-dependent PLD1–PrxII interaction? As described

in the introduction and illustrated in Fig 8, functional

interaction of the NADPH oxidase complex, H2O2and PLD1 forms a positive feedback loop When PLD1 is activated, it hydrolyzes PtdCho to produce PA PA sti-mulates the NADPH oxidase complex to generate H2O2 [5,33,34] H2O2is a small, diffusible molecule and func-tions in part as a second messenger Both its production and its elimination (signal termination) are important

Resting

PMA-Stimulated

Fig 6 PLD1 and PrxII exhibit partial

colocal-ization after PMA stimulation

Immunofluo-rescent detection of HA-PLD1 and

endogenous PrxII in resting and

PMA-stimu-lated CHO cells (63 ·) Rabbit antibody to

PrxII and an Alexa-647-labeled goat

anti-rabbit IgG secondary were used to visualize

PrxII Mouse monoclonal antibody to HA

and an Alexa 488-labeled goat anti-mouse

secondary were used to visualize HA-PLD1.

Fig 7 PrxII over-expression inhibits PLD1 activation by H 2 O 2 but

not PLD1 activation by PMA PrxII-pCR3 was over-expressed in

CHO cells inducibly expressing PLD1 A PLD in vivo assay was

per-formed to record PLD activity during the first 10 min of stimulation

by H 2 O 2 (0.4 m M ) or PMA (0.1 m M ) The experiment was performed

in triplicate and is representative of three experiments with similar

outcomes Sudent’s t-test was used to establish significance. Fig 8 Working model for PLD1–PrxII function interaction

Extracel-lular stimulation establishes a positive feedback loop wherein acti-vation of PLD1 generates PA, which leads to stimulation of NADPH oxidase complex generation of H2O2, which further stimulates PLD1 generation of PA H 2 O 2 can participate in many signaling pathways, including both pro-apoptotic and anti-apoptotic ones PrxII is proposed here to function as a signal terminator, eliminating

H 2 O 2 through oxidation and thereby decreasing PLD1 activity in addition to inhibiting other H2O2effector pathways.

activation remains incompletely defined [11–13] Thus,

PA production leads to H2O2 production, which

sti-mulates more PA production As described here, the

identification of PrxII as a stimulation-dependent

PLD1-interacting protein suggests that it would

func-tion as a signal terminator, as the oxidafunc-tion of H2O2in

the local region surrounding PLD1 would reduce the

activity of the indirect pathways that stimulate PLD1

activity, which would then lower the concentrations of

PA (which is rapidly metabolized, once made), and in

turn dampen NADPH oxidase activity Support for this

hypothesis comes from the previously described

obser-vation that the p29 peroxiredoxin can be found in

association with NADPH in neutrophils [36]

Does PrxII interact only with PLD1? A second

iso-form, PLD2 [37], can be found at the plasma

mem-brane in many cell types [37,38] and has been reported

to stimulate NADPH in vascular smooth muscle cells

[39] Although we have not examined whether PLD2

and PrxII physically interact, this observation suggests

that PrxII may play a role in the regulation of both

isoforms On the other hand, PLD1 appears to be the

relevant isoform in other cell types, such as neutrophils

stimulated through the Fcc receptor [40]

Having established that PrxII can function as a

sig-nal terminator in an artificial situation, i.e CHO cells

inducibly over-expressing PLD1 in the presence of

PrxII, the next step will be to demonstrate that

endo-genous concentrations of PrxII function as PLD signal

terminators in relevant cell types, such as neutrophils

Experimental procedures

General reagents

Cell culture media (Opti-MEM-I, Dulbecco’s modified

Eagle’s medium and F-12) were obtained from Gibco-BRL

(Gaithersburg, MD, USA), fetal bovine serum was from

Clontech (Mountain View, CA, USA), and complete

Grace’s Medium, LipofectAmine Plus and Cellfectin

rea-gent for cell transfection from Invitrogen (Carlsbad, CA,

USA) Antibiotics were obtained as follows: doxycycline

(Sigma, St Louis, MO, USA), gentamicin (Fisher,

Pitts-burgh, PA, USA), penicillin⁄ streptomycin (Cellgro,

Hern-don, VA, USA), and zeocin and blasticidine (Invitrogen)

l-Dipalmitoyl PtdCho [choline-methyl-3H] ([3H]PtdCho)

was purchased from American Radiolabeled Chemicals,

Inc (St Louis, MO, USA), and protease inhibitor cocktail

from Roche (Basel, Switzerland) The following antibodies

polyclonal antibody to PrxII was a gift from S.G Rhee (NIH, Bethesda, MD, USA) MobA protein (generated using bacterial expression and purified using nickel resin affinity chromatography and HPLC) was a gift from

J Daniels (Stony Brook University) All other reagents were of analytical grade unless otherwise specified

Cell culture and transfection

Cell cultures (except Sf9 cells) were maintained in a humi-dified atmosphere containing 5% (v⁄ v) CO2at 37
C as des-cribed previously [23] CHO T-REx PLD1 stable cell lines were maintained in conditioned complete medium [F-12 with 10% (v⁄ v) tetracycline-free fetal bovine serum, 300 lgÆmL)1 zeocin, 10 lgÆmL)1 blasticidine, 100 UÆmL)1 penicillin and

100 lgÆmL)1streptomycin] For transient transfection, cells

at 80% confluence were switched into Opti-MEM I, and transfected with 1 lg DNA per (3–4)· 105cells using Lipo-fectAmine Plus At 3 h after transfection, the medium was replaced with conditioned complete medium

Sf9 cells were maintained in a humidified atmosphere at

27
C in complete Grace’s medium as described previously [1] To transfect the cells, 9· 105Sf9 cells were seeded in 35-mm tissue culture plate in fresh medium A mixture of PLD1-GluGlu-pFASTBAC DNA and Cellfectin reagent was added to the cells, which were then incubated at 27
C for 5 h At 72 h after transfection, virus-containing medium was collected, and the titer determined

In vivo PLD assay

Nearly confluent PLD1-induced CHO stable cells cultured

in 35 mm plates were labeled with 4 lCiÆmL)1[3H]myristic acid and serum-starved overnight (F-12 medium), using previously described methods [41] At 24 h after labeling, PMA (100 nm) was added to the cells for various lengths of time The medium was then spiked with butan-1-ol to a final concentration of 0.3%, incubated for another 2 min, and collected into 600 lL ice-cold methanol Total cellular lipids were then extracted and analyzed on Whatman LK5DF silica gel 150A TLC plates using previously pub-lished protocols [23] PLD activity was expressed as the ratio of [3H]phosphatidylbutanol to total3H-labeled lipids

Affinity pull-down assay

Nearly confluent PLD1-induced CHO stable cells were grown on 150-mm plates and serum-starved overnight The cells were stimulated with PMA (100 nm) for 10 min, harvested, and lysed in a mixture containing 60 mm

n-octyl-b-glucopyranoside, 0.5 mm EDTA, 50 mm

Tris⁄ HCl, pH 7.5, 150 mm NaCl, and 1 · protease

inhib-itor cocktail at 37
C for 20 min The lysate was spun down

at 50 000 g for 15 min The supernatant was mixed with

pre-equilibrated anti-HA affinity matrix and rocked at 4
C

for 2 h The lysate⁄ matrix was pelleted and washed three

times with the lysis buffer To elute the matrix-bound

pro-teins, 1 mgÆmL)1 HA peptide (Roche) was incubated with

the matrix at 37
C for 20 min, and the supernatant

collec-ted after brief centrifugation Then 2· SDS ⁄ PAGE

sam-ple-loading buffer containing urea (8 m) was added for

subsequent analysis by SDS⁄ PAGE

Immunofluorescent analysis

Cells were cultured on 35 mm tissue culture plates

contain-ing coverslips After transfection and⁄ or treatment with

reagent, the cells were washed five times with ice-cold

NaCl⁄ Pi, fixed in 2% (v⁄ v) formaldehyde for 10 min, and

permeabilized with 0.1% (v⁄ v) Triton X-100 in NaCl ⁄ Pifor

10 min at room temperature The fixed cells were washed

three times in NaCl⁄ Pi, blocked with 5% (w⁄ v) BSA and

5% (v⁄ v) goat serum for 1 h, and then incubated with

pri-mary antibody in NaCl⁄ Picontaining 5% (v⁄ v) goat serum

for 1 h After being washed three times, the cells were

stained with secondary antibodies in NaCl⁄ Pi with 5%

(v⁄ v) goat serum for 1 h in the dark After another wash,

the cells were mounted on slides using mounting medium

(Vector, Burlingame, CA, USA) Images were captured

using confocal microscopy (Leica Microsystems Inc.)

Recombinant protein expression in E coli

A recombinant PrxII-pQE construct containing a His6 tag

at the C-terminus was generously given by Professor

Rong-Nan Huang, National Central University, Taiwan [42] The

PrxII-His6protein was transformed into E coli XL10-Gold

and induced with 1 mm isopropyl thio-b-d-galactoside at

room temperature for 4 h, D 0.6 The cells were

harves-ted by centrifuging and lysed in 5 mL lysis buffer (10 mm

Tris⁄ HCl, pH 7.5, 300 mm NaCl, 10 mm imidazole,

5 mgÆmL)1lysozyme, 10 lgÆmL)1RNase, 5 lgÆmL)1DNase)

per 50 mL culture cells After a brief sonication, the lysate

was centrifuged at 10 000 g for 20 min, and the supernatant

recovered All steps were performed at 4
C

Purification of PrxII-His6protein

The supernatant recovered as described above was mixed with

pre-equilibrated Ni⁄ nitrilotriacetate ⁄ agarose (Qiagen,

Valen-cia, CA, USA), and rocked at 4
C for 1 h The lysate ⁄ Ni ⁄

nitrilotriacetate bead mixture was transferred to a poly prep

chromatography column and the agarose packed The

column was washed twice with wash buffer (10 mm Tris⁄ HCl,

pH 7.5, 300 mm NaCl, 20 mm imidazole), and PrxII-His6 protein was eluted using 10 mm Tris⁄ HCl, pH 7.5, containing

300 mm NaCl and 250 mm imidazole Protein concentration and integrity were confirmed by SDS⁄ PAGE (12% gel)

Baculoviral production of PLD1

The PLD1-GluGlu-pFASTBAC construct was used to pre-pare PLD1 protein as described previously [1] In brief, recombinant baculoviruses were generated in the Bac-to-Bac baculovirus expression system (Life Science) To express PLD1, exponentially growing Sf9 cells (200–300 mL cells at

a density of 1· 106

ÆmL)1) were seeded in a 500-mL spinner flask and infected with recombinant baculoviruses at a mul-tiplicity of 10 The infected cells were grown for 48 h and pelleted at 2000 g for 5 min After being washed, the pellet was lysed with 5 mL lysis buffer (1% Nonidet P40, 20 mm Tris⁄ HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, 20 lm leupeptin, 0.1 mm phenylmethanesulfonyl fluoride, 0.1 mm benzamidine) by incubation on ice for 30 min The lysate was centrifuged at 50 000 g for 30 min, and the supernatant recovered for use in the in vitro PLD1 and PrxII interaction assays All steps were performed at 4
C

In vitro PLD1 and PrxII binding assay

Purified His6-PrxII, His6-MobA, and PLD1 were prepared

as described above PrxII or MobA were added to aliquots

of PLD1-containing lysate and mixed well Ni⁄ nitrilotriace-tate⁄ agarose (Qiagen) was added to the protein mixture and rocked for 1 h Elution steps were performed as des-cribed above All steps were performed at 4
C

Western blotting

Protein samples were separated by SDS⁄ PAGE (8% or 12% gel) and transferred to nitrocellulose membrane in semidry transfer buffer (25 mm Tris, 250 mm glycine, 15% methanol) using the Panther Semidry Electroblotter apparatus The blots were incubated with Odyssey blocking buffer [1% (w⁄ v) casein in Tris-buffered saline] and primary antiserum [in 1% (w⁄ v) casein in Tris-buffered saline ⁄ Tween 20], fol-lowed by washing with Tris-buffered saline⁄ Tween 20 four times, 5 min each time The blot was then incubated in fluor-escent secondary antibody in the dark After a wash with Tris-buffered saline⁄ Tween 20 and NaCl ⁄ Pi, blots were scanned using an Odyssey machine (LI-COR Inc., Lincoln,

NE, USA) to image the resulting signals

Silver staining

After SDS⁄ PAGE, gels were washed briefly with double-distilled water and fixed in 50% (v⁄ v) ethanol, 10% (v ⁄ v)

each for 10 min, respectively, and washed twice with

dou-ble-distilled water, for 10 min each time The gels were

placed in silver solution [1% (w⁄ v) silver solution in

dou-ble-distilled water] and incubated for another 10 min,

fol-lowed by brief rinsing in double-distilled water and

incubation in developer [5% (v⁄ v) ProteoSilver Developer

I, 0.1% ProteoSilver Developer 2, in double-distilled water]

until the desired staining intensity was achieved

Proteo-Silver Stop reagent was used to stop the reaction

Acknowledgements

We are grateful to laboratory members for helpful

dis-cussions and critical reading of the manuscript, to

J Daniels (Stony Brook University) for the gift of

MobA protein, and to S.-G Rhee (NIH) for the gift

of several critical reagents including antiserum to

PrxII This work was supported by NIH GM60452

and DK64166 to M.A.F

References

1 Du G, Morris AJ, Sciorra VA & Frohman MA (2002)

G-protein-coupled receptor regulation of phospholipase

D Methods Enzymol 345, 265–274

2 McDermott M, Wakelam MJ & Morris AJ (2004)

Phos-pholipase D Biochem Cell Biol 82, 225–253

3 Regier DS, Greene DG, Sergeant S, Jesaitis AJ &

McPhail LC (2000) Phosphorylation of p22phox is

mediated by phospholipase D-dependent and

-indepen-dent mechanisms Correlation of NADPH oxidase

activ-ity and p22phox phosphorylation J Biol Chem 275,

28406–28412

4 Agwu DE, McPhail LC, Sozzani S, Bass DA & McCall

CE (1991) Phosphatidic acid as a second messenger in

human polymorphonuclear leukocytes Effects on

acti-vation of NADPH oxidase J Clin Invest 88, 531–539

5 Palicz A, Foubert TR, Jesaitis AJ, Marodi L & McPhail

LC (2001) Phosphatidic acid and diacylglycerol directly

activate NADPH oxidase by interacting with enzyme

components J Biol Chem 276, 3090–3097

6 Dewald B, Payne TG & Baggiolini M (1984) Activation

of NADPH oxidase of human neutrophils Potentiation

of chemotactic peptide by a diacylglycerol Biochem

Biophys Res Commun 125, 367–373

7 Gomez-Cambronero J & Keire P (1998) Phospholipase

D: a novel major player in signal transduction Cell

Signal 10, 387–397

8 Park JW (1996) Phosphatidic acid-induced translocation

of cytosolic components in a cell-free system of

ment of protein kinase C in the phosphorylation of 46 kDa proteins which are phosphorylated in parallel with activation of NADPH oxidase in intact guinea-pig poly-morphonuclear leukocytes Biochim Biophys Acta 888, 332–337

10 Bey EAet al (2004) Protein kinase C delta is required for p47phox phosphorylation and translocation in acti-vated human monocytes J Immunol 173, 5730–5738

11 Servitja JM, Masgrau R, Pardo R, Sarri E & Picatoste

F (2000) Effects of oxidative stress on phospholipid sig-naling in rat cultured astrocytes and brain slices

J Neurochem 75, 788–794

12 Natarajan V, Taher MM, Roehm B, Parinandi NL, Schmid HH, Kiss Z & Garcia JG (1993) Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide J Biol Chem 268, 930–937

13 Min DS, Kim EC & Exton JH (1998) Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts J Biol Chem 273, 29986–29994

14 Hammond SM, Altshuller YM, Sung TC, Rudge SA, Rose K, Engebrecht J, Morris AJ & Frohman MA (1995) Human ADP-ribosylation factor-activated phos-phatidylcholine-specific phospholipase D defines a new and highly conserved gene family J Biol Chem 270, 29640–29643

15 Hammond SM, Jenco JM, Nakashima S, Cadwallader

K, Gu Q, Cook S, Nozawa Y, Prestwich GD, Frohman

MA & Morris AJ (1997) Characterization of two alter-nately spliced forms of phospholipase D1 Activation of the purified enzymes by phosphatidylinositol 4,5-bisphos-phate, ADP-ribosylation factor, and Rho family mono-meric GTP-binding proteins and protein kinase C-alpha

J Biol Chem 272, 3860–3868

16 Du G, Altshuller YM, Kim Y, Han JM, Ryu SH, Morris AJ & Frohman MA (2000) Dual requirement for rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling Mol Biol Cell 11, 4359–4368

17 Zhang Y, Altshuller YM, Hammond SM, Hayes F, Morris AJ & Frohman MA (1999) Loss of receptor reg-ulation by a phospholipase D1 mutant unresponsive to protein kinase C EMBO J 18, 6339–6348

18 Yamazaki Met al (1999) Interaction of the small G protein RhoA with the C terminus of human phospho-lipase D1 J Biol Chem 274, 6035–6038

19 Kusner DJ, Barton JA, Wen KK, Wang X, Rubenstein

PA & Iyer SS (2002) Regulation of phospholipase D activity by actin Actin exerts bidirectional modulation

of mammalian phospholipase D activity in a

polymeri-zation-dependent, isoform-specific manner J Biol Chem

277, 50683–50692

20 Oishi K, Takahashi M, Mukai H, Banno Y, Nakashima

S, Kanaho Y, Nozawa Y & Ono Y (2001) PKN

regu-lates phospholipase D1 through direct interaction

J Biol Chem 276, 18096–18101

21 Ganley IG, Walker SJ, Manifava M, Li D, Brown HA

& Ktistakis NT (2001) Interaction of phospholipase D1

with a casein-kinase-2-like serine kinase Biochem J 354,

369–378

22 Lee C, Kim SR, Chung JK, Frohman MA, Kilimann

MW & Rhee SG (2000) Inhibition of phospholipase D

by amphiphysins J Biol Chem 275, 18751–18758

23 Du G, Altshuller YM, Vitale N, Huang P,

Chasserot-Golaz S, Morris AJ, Bader MF & Frohman MA (2003)

Regulation of phospholipase D1 subcellular cycling

through coordination of multiple membrane association

motifs J Cell Biol 162, 305–315

24 Jenkins GM & Frohman MA (2005) Phospholipase D:

a lipid centric review Cell Mol Life Sci, in press

25 Lim MJ, Chae HZ & Rhee SG, Yu DY, Lee KK &

Yeom YI (1998) The type II peroxiredoxin gene family

of the mouse: molecular structure, expression and

evolu-tion Gene 216, 197–205

26 Wood ZA, Poole LB & Karplus PA (2003)

Peroxire-doxin evolution and the regulation of hydrogen

per-oxide signaling Science 300, 650–653

27 Neumann CA, Krause DS, Carman CV, Das S, Dubey

DP, Abraham JL, Bronson RT, Fujiwara Y, Orkin SH

& Van Etten RA (2003) Essential role for the

peroxi-redoxin Prdx1 in erythrocyte antioxidant defence and

tumour suppression Nature 424, 561–565

28 Kim SH, Fountoulakis M, Cairns N & Lubec G (2001)

Protein levels of human peroxiredoxin subtypes in

brains of patients with Alzheimer’s disease and Down

syndrome J Neural Transm Suppl 223–235

29 Noh DY, Ahn SJ, Lee RA, Kim SW, Park IA & Chae

HZ (2001) Overexpression of peroxiredoxin in human

breast cancer Anticancer Res 21, 2085–2090

30 McLuskey K, Harrison JA, Schuttelkopf AW, Boxer

DH & Hunter WN (2003) Insight into the role of

Escherichia coliMobB in molybdenum cofactor

bio-synthesis based on the high resolution crystal structure

J Biol Chem 278, 23706–23713

31 Guse A, Stevenson CE, Kuper J, Buchanan G, Schwarz

G, Giordano G, Magalon A, Mendell RR, Lawson DM

& Palmer T (2003) Biochemical and structural analysis

of the molybdenum cofactor biosynthesis protein

MobA J Biol Chem 278, 25302–25307

32 Sambrook J & Russell DW (2001) Molecular Cloning: A Laboratory Manual.3rd Edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

33 Rhee SG, Chang TS, Bae YS, Lee SR & Kang SW (2003) Cellular regulation by hydrogen peroxide J Am Soc Nephrol 14, S211–S215

34 Bokoch GM & Diebold BA (2002) Current molecular models for NADPH oxidase regulation by Rac GTPase Blood 100, 2692–2696

35 Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, Chock

PB & Rhee SG (1997) Epidermal growth factor (EGF)-induced generation of hydrogen peroxide Role in EGF receptor-mediated tyrosine phosphorylation J Biol Chem 272, 217–221

36 Leavey PJ, Gonzalez-Aller C, Thurman G, Kleinberg

M, Rinckel L, Ambruso DW, Freeman S, Kuypers SA

& Ambruso DR (2002) A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phos-pholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity J Biol Chem 277, 45181–45187

37 Colley WC, Sung TC, Roll R, Jenco J, Hammond SM, Altshuller Y, Bar-Sagi D, Morris AJ & Frohman MA (1997) Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization Curr Biol 7, 191–201

38 Du G, Huang P, Liang BT & Frohman MA (2004) Phospholipase D2 localizes to the plasma membrane and regulates angiotensin II receptor endocytosis Mol Biol Cell 15, 1024–1030

39 Shaw SS, Schmidt AM, Banes AK, Wang X, Stern DM

& Marrero MB (2003) S100B-RAGE-mediated augmen-tation of angiotensin II-induced activation of JAK2 in vascular smooth muscle cells is dependent on PLD2 Diabetes 52, 2381–2388

40 Melendez AJ, Bruetschy L, Floto RA, Harnett MM & Allen JM (2001) Functional coupling of FcgammaRI to nicotinamide adenine dinucleotide phosphate (reduced form) oxidative burst and immune complex trafficking requires the activation of phospholipase D1 Blood 98, 3421–3428

41 Morris AJ, Frohman MA & Engebrecht J (1997) Mea-surement of phospholipase D activity Anal Biochem

252, 1–9

42 Chang KN, Lee TC, Tam MF, Chen YC, Lee LW, Lee

SY, Lin PJ & Huang RN (2003) Identification of galec-tin I and thioredoxin peroxidase II as two arsenic-bind-ing proteins in Chinese hamster ovary cells Biochem J

371, 495–503