Peroxiredoxin II functions as a signal terminator
for H
2
O
2
-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 H
2

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:
(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 H
2
O
2
. 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 H
2
O
2
, 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 H

O
2
.
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-

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.
Regulation of PLD1 by interaction with PrxII N. Xiao et al.
3930 FEBS Journal 272 (2005) 3929–3937 ª 2005 FEBS
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. His
6
-tagged PrxII protein was expressed and
purified from Escherichia coli, mixed with sf9 cell
lysates containing baculoviral-generated GluGlu-tag-

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.
N. Xiao et al. Regulation of PLD1 by interaction with PrxII
FEBS Journal 272 (2005) 3929–3937 ª 2005 FEBS 3931
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 His
6
-PrxII pulled-down PLD1 40-
fold more efficiently than His
6

O
2
-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 H
2
O
2
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 H
2
O
2
. PMA
stimulates PLD1 by activating PKC, which is a direct
activator of PLD1 and does not require H
2
O
2
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

fied His
6
-PrxII and His
6
-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.
Regulation of PLD1 by interaction with PrxII N. Xiao et al.
3932 FEBS Journal 272 (2005) 3929–3937 ª 2005 FEBS
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

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 mM) or PMA (0.1 mM). 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 H
2
O

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 oxidation of H
2
O
2
in
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

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) CO
2
at 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
)1
streptomycin]. For transient transfection, cells
at 80% confluence were switched into Opti-MEM I, and
transfected with 1 lg DNA per (3–4) · 10
5
cells 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 · 10
5

cells were stimulated with PMA (100 nm) for 10 min,
harvested, and lysed in a mixture containing 60 mm
Regulation of PLD1 by interaction with PrxII N. Xiao et al.
3934 FEBS Journal 272 (2005) 3929–3937 ª 2005 FEBS
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 ⁄ P
i
, fixed in 2% (v ⁄ v) formaldehyde for 10 min, and
permeabilized with 0.1% (v ⁄ v) Triton X-100 in NaCl ⁄ P
i
for
10 min at room temperature. The fixed cells were washed
three times in NaCl ⁄ P

)1
RNase, 5 lgÆmL
)1
DNase)
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-His
6
protein
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-His
6
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 · 10
6
ÆmL

[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 ⁄ P
i
, 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)
N. Xiao et al. Regulation of PLD1 by interaction with PrxII
FEBS Journal 272 (2005) 3929–3937 ª 2005 FEBS 3935
acetic acid and 40% (v ⁄ v) double-distilled water for 2 h at
room temperature. The gels were then incubated in 30%
ethanol, double-distilled water and sensitization solution
[1% (w ⁄ v) ProteoSilver Sensitizer in double-distilled water]
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

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
NADPH oxidase: mechanism of activation and effect of
diacylglycerol. Biochem Biophys Res Commun 229,
758–763.
9 Ohtsuka T, Okamura N & Ishibashi S (1986) Involve-
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

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-

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.