Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin.

Abstract

The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation-dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3zeta and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation-dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3zeta, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585-712). Expression of this fragment suppresses receptor-induced cofilin-PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions.

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Direct stimulation of receptor-controlled
phospholipase D1 by phospho-cofilin
Li Han1,5,6, Matthias B Stope1,5,
Maider Lo ´pez de Jesu ´s1,5, Paschal A Oude
Weernink1, Martina Urban1,
Thomas Wieland2, Dieter Rosskopf1,
Kensaku Mizuno3, Karl H Jakobs1and
Martina Schmidt1,4,*
1Institut fu ¨r Pharmakologie, Universita ¨tsklinikum Essen, Essen,
Germany,2Institut fu ¨r Experimentelle und Klinische Pharmakologie und
Toxikologie, Fakulta ¨t fu ¨r Klinische Medizin Mannheim der Universita ¨t
Heidelberg, Mannheim, Germany,3Department of Biomolecular
Sciences, Graduate School of Life Sciences, Sendai, Miyagi, Japan and
4Department of Molecular Pharmacology, University of Groningen,
Groningen, The Netherlands
The activity state of cofilin, which controls actin dy-
namics, is driven by a phosphorylation–dephosphoryla-
tion cycle. Phosphorylation of cofilin by LIM-kinases
results in its inactivation, a process supported by 14-3-3f
and reversed by dephosphorylation by slingshot phospha-
tases. Here we report on a novel cellular function for
the phosphorylation–dephosphorylation cycle of cofilin.
Wedemonstratethatmuscarinic
stimulation of phospholipase D1 (PLD1) is controlled by
LIM-kinase, slingshot phosphatase as well as 14-3-3f, and
requires phosphorylatable cofilin. Cofilin directly and
specifically interacts with PLD1 and upon phosphorylation
by LIM-kinase1, stimulates PLD1 activity, an effect mi-
micked by phosphorylation-mimic cofilin mutants. The
interaction of cofilin with PLD1 is under receptor control
and encompasses a PLD1-specific fragment (aa 585–712).
Expression of this fragment suppresses receptor-induced
cofilin–PLD1 interaction as well as PLD stimulation and
actin stress fiber formation. These data indicate that till
now designated inactive phospho-cofilin exhibits an active
cellular function, and suggest that phospho-cofilin by its
stimulatory effect on PLD1 may control a large variety of
cellular functions.
The EMBO Journal (2007) 26, 4189–4202. doi:10.1038/
sj.emboj.7601852; Published online 13 September 2007
Subject Categories: signal transduction
Keywords: cofilin; LIM-kinase1; phospholipase D; slingshot;
14-3-3
receptor-mediated
Introduction
The dynamic nature of actin filament assembly/disassembly
and its cellular organization is regulated by several actin-
binding and -regulatory proteins, including the actin-depoly-
merizingfactor(ADF)/cofilin
Bamburg and Wiggan, 2002; Pollard and Borisy, 2003;
DesMarais et al, 2005). Members of the ADF/cofilin family
(hereafter referred to as cofilin) are now considered to be
pivotal regulators of actin filament dynamic-dependent pro-
cesses, including cell motility, neuronal pathfinding, mem-
brane dynamics, establishment of cell polarity, cell division
and apoptosis, and their dysfunction seems to contribute to
the progression of diseases as diverse as cancer, Alzheimer’s
dementia and ischemic kidney disease (Bamburg and
Wiggan, 2002; Chua et al, 2003; Wang et al, 2006). Cofilin
binds to both G- and F-actin, but due to a higher affinity for
ADP-bound subunits, the off-rate of actin monomers from the
pointed end of actin filaments is increased; in addition, cofilin
severs actin filaments and thus directly generates free actin
barbed ends (Condeelis, 2001; DesMarais et al, 2005).
The cofilin–actin interaction is tightly controlled by phos-
phocycling. Phosphorylation of cofilin at serine 3, located in
the actin-binding domain, by the LIM (Lin-11/Isl-1/Mec-3)
and the TES (testicular protein) kinases results in its inactiva-
tion, and the Unphosphorylatable S3A cofilin mutant as well
as the phosphorylation-mimic cofilin mutants (S3D cofilin
and S3E cofilin) have been widely used to define the role of
cofilin in cellular responses (Bamburg, 1999; Huang et al,
2006). The two LIM-kinases, LIM-kinase1 and LIM-kinase2,
are expressed in most tissues (Foletta et al, 2004; Acevedo
et al, 2006) and are activated by Rho GTPases, through their
effectors, Rho-kinase and p21-activated protein kinases
(Edwards and Gill, 1999; Kaibuchi et al, 1999). Recently,
interaction of LIM-kinase1 and cofilin with the scaffold
protein 14-3-3z has been reported (Birkenfeld et al, 2003),
leading to accumulation of inactive, phosphorylated cofilin
(Gohla and Bokoch, 2002). Dephosphorylation of cofilin
results in its reactivation, and is catalyzed by the cofilin-
specific phosphatases of the slingshot family (Niwa et al,
2002; Kaji et al, 2003; Ohta et al, 2003) and chronophin
(Gohla et al, 2005; Huang et al, 2006). The activity of the
slingshot phosphatases is apparently also controlled by multi-
ple signaling pathways, including Ca2þ, cyclic AMP and
phosphatidylinositol 3-kinase (for recent review see Huang
et al, 2006). It has been also shown that the slingshot
phosphatase 1L not only dephosphorylates cofilin but also
LIM-kinase1, resulting in its inactivation, and that the phos-
phatase activity is regulated by F-actin and 14-3-3z (Nagata-
Ohashi et al, 2004; Soosairajah et al, 2005).
Phospholipase D (PLD) enzymes, PLD1 splice variants
and PLD2, hydrolyze phosphatidylcholine of cell membranes
to phosphatidic acid (PA), in response to stimuli, and are
considered to be involved in a large variety of early and late
cellular responses,including
family(Bamburg,1999;
calciummobilization,
Received: 6 September 2006; accepted: 2 August 2007; published
online: 13 September 2007
*Corresponding author. Department of Molecular Pharmacology,
University of Groningen, A. Deusinglaan 1, Groningen 9713 AV,
The Netherlands. Tel.: þ31 50 363 3322; Fax: þ31 50 363 6908;
E-mail: m.schmidt@rug.nl
5These authors contributed equally to this work
6Present address: Department of Infection Control, Chinese Military
Institute of Disease Control & Prevention, Beijing 100071, China
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secretion, superoxide production, endocytosis, exocytosis,
vesicle trafficking, glucose transport, mitogenesis and apop-
tosis (Exton, 2002). The rise in cellular PA, in particular by
PLD1, has also been reported to induce stress fiber formation
in suitable cell types (Ha and Exton, 1993; Cross et al, 1996;
Kam and Exton, 2001; Porcelli et al, 2002; Komati et al, 2005).
As PA-activatable phosphatidylinositol-4-phosphate 5-kinase
(PIP 5-kinase) generates phosphatidylinositol 4,5-bispho-
sphate (PIP2) (reviewed in Oude Weernink et al, 2004a),
known to act as a PLD cofactor and to associate with a
plethora of actin-binding proteins that regulate actin dy-
namics (Exton, 2002; Yin and Janmey, 2003; Hilpela et al,
2004), such mechanisms may act in concert to promote actin
cytoskeleton reorganization. PLD and PIP 5-kinase are now
also recognized as effectors of Rho-dependent Rho-kinase
(Schmidt et al, 1999; Oude Weernink et al, 2000, 2004b; Kam
and Exton, 2001; Cummings et al, 2002; Yamazaki et al,
2002). Vice versa, components of the actin regulatory ma-
chinery have been found to affect PLD. PLD activity, primar-
ily PLD2, is negatively regulated by b-actin and the actin-
binding protein a-actinin (Park et al, 2000; Lee et al 2001).
Meanwhile, it has been reported that G-actin inhibits PLD1 as
well, but that F-actin has the opposite effect, suggesting that
specifically PLD1 may act as an actin dynamic responsive
cellular element (Kusner et al, 2002). Here we report on a
novel molecular link between the actin cytoskeleton and
PLD1. We demonstrate that receptor-induced and Rho/Rho-
kinase-dependent activation of PLD1 is under control of LIM-
kinase1 and its substrate, cofilin. Cofilin directly interacts
with and stimulates in its phosphorylated state PLD1. Our
data, thus, indicate for the first time that phospho-cofilin,
until now considered to be biologically inactive, is an active
cellular component, which by its signaling to PLD1 can
regulate essential cellular functions known to be under con-
trol of PLD.
Results
Involvement of cofilin in muscarinic receptor signaling
to PLD
The Rho effector, Rho-kinase, mediates PLD stimulation by G-
protein-coupled muscarinic
(mAChRs) in HEK-293 and N1E-115 neuroblastoma cells
(Schmidt et al, 1999; Kam and Exton, 2001; Cummings
et al, 2002). As PLD enzymes neither directly interact with,
nor are phosphorylated by Rho-kinase (Schmidt et al, 1999),
we considered the involvement of the Rho-kinase effector,
LIM-kinase, in mAChR signaling to PLD. As shown in
Figure 1A, overexpression of LIM-kinase1 in HEK-293 cells
strongly increased PLD stimulation by the mAChR agonist,
carbachol, whereas expression of kinase-deficient D460A
LIM-kinase1 had the opposite effect. The carbachol-induced
and LIM-kinase1-reinforced PLD stimulation was almost fully
blunted by adenoviral expression of lipase-inactive K898R
PLD1, whereas lipase-inactive K758R PLD2 had no effect.
Similar data were obtained for mAChR regulation of PLD
activity in N1E-115 neuroblastoma cells (data not shown).
These data suggested that the receptor-induced PLD stimula-
tion not only involves Rho-kinase, but probably also its
substrate, LIM-kinase, and that the PLD isozyme responsible
for the increased PLD activity is PLD1. Consequently, we
studied whether LIM-kinase1 may phosphorylate PLD1.
acetylcholine receptors
However, as shown in Figure 1B, purified recombinant LIM-
kinase1 did neither phosphorylate purified GST-tagged PLD1
nor PLD2. LIM-kinase1 did also not bind to the PLD enzymes
(data not shown). The purified LIM-kinase1 was active, as it
phosphorylated, as expected, wild-type cofilin, but not its
mutant, S3A cofilin (Figure 1B).
These data prompted us to study whether the LIM-kinase
substrate, cofilin, may be involved in PLD stimulation. As
illustrated in Figure 1C, overexpression of wild-type cofilin in
HEK-293 cells greatly enhanced, by about 2.5-fold, stimula-
tion of PLD by the mAChR agonist, carbachol, whereas
expression of Unphosphorylatable S3A cofilin suppressed
PLD stimulation by about 50%. In contrast, expression of
wild-type and S3A cofilin did not alter PLD stimulation by the
phorbol ester, phorbol 12-myristate 13-acetate (PMA), which
is Rho- and Rho-kinase independent in HEK-293 cells (Vo?
et al, 1999), and which was also not affected by expression of
LIM-kinase1 variants (data not shown). To confirm that
cofilin mediates mAChR-induced PLD stimulation, human
HEK-293 and mouse N1E-115 cells were transfected with
siRNA pSUPER expression plasmids, which direct the synth-
esis of siRNAs targeting either human or mouse cofilin,
respectively (Nishita et al, 2005; Kiuchi et al, 2007). As
shown in Figure 2, these maneuvers greatly reduced the
cellular content of cofilin in both cell types. Silencing of
cellular cofilin in HEK-293 and N1E-115 neuroblastoma cells
reduced the carbachol-induced PLD stimulation by about 60
and 80%, respectively (Figure 2). In contrast, knockdown of
cofilin expression did not alter PLD stimulation by PMA in
either cell type.
These findings together suggested that cofilin, possibly in
its phosphorylated state, may control the receptor stimulation
of PLD. To substantiate this assumption, we first studied
whether receptor activation leads to cofilin phosphorylation,
and whether this phosphorylation is under control of a
cofilin-specific phosphatase and the scaffold protein, 14-3-
3z. As illustrated in Figure 3A, agonist activation of the
mAChR induced a strong, but rather transient phosphoryla-
tion of cofilin in HEK-293 cells. The stimulatory effect of
carbachol reached its maximum at 15s and rapidly declined
thereafter. Expression of slingshot phosphatase 1L completely
abolished phosphorylation of cofilin, both in the basal state
and after stimulation by carbachol (Figure 3B). In contrast,
expression of 14-3-3z and inactive slingshot phosphatase 1L
(not shown) strongly increased phosphorylation of cofilin in
unstimulated cells, and there was no additional increase by
the receptor activation. We then examined the effects of the
slingshot and 14-3-3z on mAChR stimulation of PLD activity.
Expression of slingshot phosphatase 1L significantly de-
creased PLD stimulation by carbachol, whereas inactive
slingshot phosphatase 1L and 14-3-3z enhanced PLD stimula-
tion by carbachol (Figure 3C, left panel). In contrast, PLD
stimulation by epidermal growth factor (EGF), which similar
to PLD stimulation by PMA, is independent of Rho and Rho-
kinase in these cells (Vo? et al, 1999), was not altered by
expression of these proteins controlling the phosphorylation
state of cofilin (Figure 3C, right panel). In line with a role of
cofilin phosphorylation in PLD stimulation, expression of the
phosphorylation-mimic S3D cofilin mutant strongly increased
the carbachol-induced PLD stimulation, both in HEK-293 and
N1E-115 neuroblastoma cells (Figure 4A). We next studied
whether the expression of phosphorylation-mimic S3D cofilin
Phospho-cofilin stimulation of PLD1
L Han et al
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could counteract the inhibitory effect of cofilin silencing on
the PLD response. As shown in Figure 4B, expression of
phosphorylation-mimic S3D mouse cofilin in cofilin-silenced
HEK-293 cells considerably rescued PLD stimulation by car-
bachol.
Cofilin binds to and alters subcellular localization of
PLD1
For analysis of cofilin–PLD interaction, studies were per-
formed both in vitro with purified components and in intact
cells. As illustrated in Figure 5A, purified wild-type His-
tagged cofilin strongly bound to GST-tagged PLD1. In con-
trast, binding of Unphosphorylatable S3A cofilin to GST-
tagged PLD1 was hardly detectable, similar as binding of
wild-type or S3A cofilin to PLD2. Thus, cofilin can directly
and specifically interact with PLD1 and this interaction
apparently requires the phosphorylatable serine 3 of cofilin.
To examine whether cofilin also interacts with PLD1 in intact
cells, we transfected HEK-293 cells with PLD1 or PLD2 and
cofilin mutants for immunofluorescence laser confocal mi-
croscopy analysis. As reported before in other cell types
(Bamburg, 1999; Bamburg and Wiggan, 2002; Exton, 2002),
we found that PLD1 localized to intracellular compartments
and the plasma membrane, whereas PLD2 exclusively loca-
lized to the plasma membrane (Figure 5B, panels a and d);
the cofilin mutants were found to be localized to the plasma
membrane and intracellular compartments (Figure 5B, panels
g and h). Coexpression of wild-type cofilin altered subcellular
localization of PLD1 (Figure 5B, panel b). In cells coexpres-
sing PLD1 and wild-type cofilin, PLD1 was found primarily at
the plasma membrane. Coexpression of S3A cofilin and PLD1
caused only a minor subcellular redistribution of PLD1
(Figure 5B, panel c). In contrast to PLD1, the plasma mem-
brane localization of PLD2 was not altered by coexpression of
wild-type or S3A cofilin (Figure 5B, panels e and f). Thus,
cofilin can specifically alter subcellular localization of PLD1.
Control
GST-PLD2
Control
Cofilin
S3A Cofilin
GST-PLD1
GST
S3A Cofilin
Cofilin
Control
S3A Cofilin
Cofilin
Control
Ctr WTS3A
0.2
0.4
0.6
0.8
0
ControlLIM-kinase1
*
*
*
*
*
Stimulated PtdEtOH
formation (% of phospholipids)
0.1
0.2
0.3
0.4
0
*
*
Stimulated PtdEtOH
formation (% of phospholipids)
0.3
0.6
0.9
1.2
1.5
0
PMACarb
32P autoradiography
K758R
PLD2
K898R
PLD1
D460A
LIM-K1
LacZ
LIM-kinase1
— LIM-kinase1
— PLD1
— PLD2
+PLD1 +PLD2
ControlVirus
— PLD1
— PLD2
— Cofilin
— Cofilin
Figure 1 Cofilin, the sole substrate of LIM-kinase, regulates stimulation of PLD1 by the M3mAChR. HEK-293 cells were transfected with
kinase-deficient LIM-kinase1 (D460A LIM-K1) or wild-type LIM-kinase1, either alone or with adenoviruses encoding LacZ, lipase-inactive
K898R PLD1 or lipase-inactive K758R PLD2 (A), or transfected with empty vector (Control), wild-type cofilin and Unphosphorylatable S3A
cofilin (C). After 48h, stimulated [3H]PtdEtOH accumulation was determined in the presence of 1mM carbachol (Carb) (A, C) or 100nM PMA
(C). Data shown are means7s.e. (n¼3–4). The immunoblots demonstrate expression of LIM-kinase1, PLD enzymes and cofilin in cell lysates.
(B) [g-32P]ATP phosphorylation (32P autoradiography) of GST, GST-tagged PLD1, GST-tagged PLD2 (full-length each) and wild-type cofilin and
S3A cofilin by LIM-kinase1. Data are representative of three to four similar experiments. *Po0.05.
Phospho-cofilin stimulation of PLD1
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To substantiate that cofilin interacts with PLD1 also in
intact cells, co-immunoprecipitation studies were performed.
As illustrated in Figure 5C, left panel, cofilin and phospho-
cofilin were co-immunoprecipitated with PLD1 from lysates
of HEK-293 cells coexpressing PLD1 and cofilin, demonstrat-
ing their in vivo interaction. Most important, stimulation of
the mAChR with carbachol for 15s strongly enhanced the
amount of cofilin and phospho-cofilin co-immunoprecipi-
tated with PLD1. In contrast, cofilin and phospho-cofilin
poorly co-immunoprecipitated with PLD2, and there was no
effect of carbachol (Figure 5C, right panel).
Phosphorylation of cofilin is essential for stimulation of
PLD1
We then determined whether cofilin not only interacts with
PLD1, but also controls its activity, and whether such regula-
tion is dependent on the phosphorylation state of cofilin in
vitro. For this, wild-type and Unphosphorylatable S3A cofilin
were first treated with LIM-kinase1, in the absence and
presence of MgATP, to allow for cofilin phosphorylation,
and then the cofilin proteins were added to purified recombi-
nant PLD enzymes for measurement of enzyme activity.
Addition of purified recombinant wild-type or S3A cofilin
pretreated with LIM-kinase1, in the absence or presence of
MgATP had no effect on the activity of the PLD2 enzyme
(Figure 6A, right bar graph). Furthermore, similar to buffer
control, S3A cofilin was without effect on PLD1 activity.
Wild-type cofilin pretreated with LIM-kinase1, but without
MgATP, increased the activity of PLD1 only slightly, by about
50% (Figure 6A, left bar graph), whereas wild-type cofilin
pretreated without LIM-kinase1 was without effect on PLD1
activity (data not shown). In contrast, wild-type cofilin
phosphorylated by LIM-kinase1 (in the presence of MgATP;
Figure 6A,
activity, by about three-fold (Figure 6A, left bar graph). To
corroborate the hypothesis that it is the phosphorylated
cofilin, which stimulates PLD1, purified recombinant phos-
phorylation-mimic cofilin mutants were added to purified
32P autoradiography) strongly enhanced PLD1
HEK-293 cells
0.1
0.2
0.3
0.4
0
*
Stimulated PtdEtOH
formation (% of phospholipids)
Carb
0.3
0.6
0.9
1.2
0
PMA
— Cofilin
— Cofilin
siRNA hCofilin
Control
siRNA hCofilin
Control
N1E-115 cells
siRNA mCofilin
Control
siRNA mCofilin
Control
1
2
3
4
PMA
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0
*
Stimulated PtdEtOH
formation (% of phospholipids)
Carb
siRNA hCofilin
Control
siRNA mCofilin
Control
Figure 2 Depletion of cellular cofilin reduces PLD stimulation by carbachol. HEK-293 cells were transfected with human cofilin siRNA pSUPER
plasmid (siRNA hCofilin), or with the empty pSUPER vector (Control) (A). N1E-115 neuroblastoma cells were transfected with mouse cofilin
siRNA pSUPER plasmid (siRNA mCofilin), or with the empty pSUPER vector (Control) (B). After 48h, stimulated [3H]PtdEtOH accumulation
was determined in the presence of 1mM carbachol (Carb) or 100nM PMA. Data shown are means7s.e. (n¼3–4). The immunoblots
demonstrate endogenous expression of cofilin in lysates of cells transfected with empty pSUPER vector (Control) or the indicated siRNA
pSUPER plasmids. *Po0.05.
Phospho-cofilin stimulation of PLD1
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recombinant PLD1. Similar to wild-type cofilin phosphory-
lated by LIM-kinase1, the phosphorylation-mimic S3D and
S3E cofilin mutants strongly enhanced PLD1 activity, by
about three-fold (Figure 6B). Thus, cofilin not only directly
and specifically interacts with PLD1, but also strongly in-
creases its activity, and this activation is dependent on the
phosphorylation state of cofilin.
Identification of the PLD1 region responsible for
interaction with cofilin
To identify the PLD1 region involved in cofilin binding, we
constructed GST-tagged PLD1 fragments (Figure 7A and B)
and analyzed their ability to bind purified cofilin. The F-3
fragment of PLD1, encompassing the amino acids 585–712,
was found to exclusively bind wild-type cofilin (Figure 7C).
None ofthe PLD1 fragments
Unphosphorylatable S3A cofilin. Furthermore, there was no
binding of cofilin to the corresponding PLD2 fragments (Lee
analyzed, bound
et al, 2001; Chae et al, 2005) (Figure 7C). These data identify
the PLD1-specific region encoded by the amino acids 585–712
to be important for the direct interaction with cofilin. To
study whether cofilin phosphorylation alters binding to
PLD1, binding of the phosphorylation-mimic S3D cofilin
to the F-3 fragment of PLD1 was studied. As illustrated in
Figure 7D, S3D cofilin bound to the F-3 fragment, but some-
what less than wild-type cofilin. Cellular cofilin phosphory-
lated by treatment of HEK-293 cells for 15s with carbachol,
also bound to the PLD1 F-3 fragment and to full-length PLD1,
but again somewhat less than cofilin (data not shown). Thus,
phosphorylation of cofilin strongly increases PLD1 activity,
but not binding of cofilin to PLD1.
Inhibition of PLD stimulation and actin stress fiber
formation by the cofilin-binding F-3 fragment
As cofilin directly interacts with the F-3 fragment of PLD1, we
used this fragment to interfere with the mAChR-induced
Slingshot 1L
C393S slingshot 1L
14-3-3ζ
Control
Slingshot 1L
C393S slingshot 1L
14-3-3ζ
Control
Control
Slingshot 1L
Control
C393S
slingshot 1L
Control
14-3-3ζ
0.1
0.2
0.3
0.4
0.5
0
0.1
0.2
0.3
0.4
0.5
0
*
*
*
Stimulated PtdEtOH
formation (% of phospholipids)
Carb EGF
Incubation time (s)
–+–+–+–+
P-Cofilin
Cofilin
15 3060 120
Carbachol
–+–+–+
P-Cofilin
Cofilin
Slingshot 1L
14-3-3ζ
Control
Carbachol
Figure 3 Regulation of the cellular phosphorylation state of cofilin and its impact on the PLD response. HEK-293 cells were stimulated without
(?) or with (þ) 1mM carbachol for the indicated periods of time (A), or transfected with empty vector (Control), c-myc-tagged wild-type
slingshot 1L, inactive slingshot 1L (C393S slingshot 1L) or VSV-G-tagged 14-3-3z, and stimulated with carbachol for 15s (B). Phosphorylation
of cofilin (P-Cofilin) and the total cellular content of cofilin were detected in cell lysates with anti-phospho-cofilin and anti-cofilin antibodies,
respectively. Data are representative of three experiments. (C) Stimulated [3H]PtdEtOH accumulation was determined in the presence of 1mM
carbachol (Carb) or 100ng/ml EGF. Data shown are means7s.e. (n¼3–4). The immunoblots show expression of c-myc-tagged slingshot 1L
and myc-tagged inactive slingshot 1L (C393S slingshot 1L), or VSV-G-tagged 14-3-3z. *Po0.05.
Phospho-cofilin stimulation of PLD1
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cofilin-PLD1 interaction, as well as PLD stimulation and actin
stress fiber formation. Expression of the F-3 fragment in HEK-
293 cells strongly and specifically reduced PLD stimulation
by carbachol, without affecting PLD stimulation by PMA
(Figure 8A). In contrast, expression of the F-1 fragment of
PLD1, that does not interact with cofilin, did not alter PLD
stimulation by carbachol. The inhibitory effect of the F-3
fragment could be completely rescued by coexpression of
wild-type cofilin (Figure 8A). These data indicate that the
PLD1-specific F-3 fragment specifically interferes with the
activation of PLD1, most probably by attenuating the mAChR-
induced association of cofilin with PLD1. Indeed, the en-
hanced co-immunoprecipitation of cofilin with PLD1 induced
by activation of the mAChR by carbachol was completely
abolished by expression of the F-3 fragment (Figure 8B).
Cofilin is known to profoundly affect actin dynamics
(Bamburg, 1999; DesMarais et al, 2005), and also PLD1 has
been reported to alter the organization of the actin cytoske-
leton (Ha and Exton, 1993; Cross et al, 1996; Kam and Exton,
2001; Porcelli et al, 2002; Komati et al, 2005). To determine
whether interaction of cofilin with PLD1 may affect Rho-
dependent actin dynamics, we studied stress fiber formation
in human bladder carcinoma (J82) cells coexpressing the M3
mAChR and the PLD1-specific F-3 fragment. As expected,
activation of the M3 mAChR by carbachol induced the
formation of stress fibers (Figure 9, panel b). Coexpression
of the F-3 fragment completely suppressed stress fiber for-
mation (Figure 9B, panels b and d), suggesting that inter-
action of cofilin with PLD1 is involved in the regulation of the
actin cytoskeleton architecture.
Discussion
Cofilin plays a prominent role in the regulation of actin
dynamics and is involved in the initiation of actin-driven
processes, including cell motility, neuronal pathfinding, as-
sembly of cell polarity, cell division and apoptosis (Bamburg,
1999; Chua et al, 2003; Pollard and Borisy, 2003; DesMarais
et al, 2005). Cofilin can actually exert two opposing effects on
the actin filament assembly/disassembly machinery: (1)
barbed-end formation and subsequent actin polymerization,
as well as (2) actin depolymerization. At present, the precise
contribution of these cofilin-mediated mechanisms to actin
reorganization is rather undefined and may be even different
in various cell types (for recent review see DesMarais et al,
2005). Cellular functions of cofilin are tightly controlled by a
unique phosphorylation–dephosphorylation cycle, involving
specific kinases (LIM- and TES-kinases) and cofilin-specific
phosphatases (slingshot phosphatases and chronophin), as
well as the scaffold protein 14-3-3z. Overall, phosphorylation
of cofilin at serine 3 results in its inactivation, and reactiva-
tion is catalyzed by its dephosphorylation (reviewed in
Huang et al, 2006).
PLD enzymes generating PA from phosphatidylcholine, in
particular PLD1, have also been found to regulate actin
organization and to induce stress fiber formation in a cell-
dependent manner (Ha and Exton, 1993; Cross et al, 1996;
Kam and Exton, 2001; Porcelli et al, 2002; Komati et al, 2005).
PLD-driven regulation of the actin cytoskeleton architecture
likely occurs in concert with PA-responsive PIP 5-kinase (for
recent review see Oude Weernink et al, 2004a). As it has
become evident that various components of the actin regula-
tory network conversely affect PLD activity (Park et al, 2000;
Lee et al 2001; Kusner et al, 2002), this reciprocal modulation
of PLD activity and actin cytoskeleton dynamics, together
with the observation that both PLD and actin dynamics are
regulated by Rho GTPases and their effectors (Edwards and
Gill, 1999; Kaibuchi et al, 1999; Exton, 2002), points to
concerted mechanisms in cellular actions, involving acute,
localized alterations in actin architecture.
Here we report that the pivotal actin regulatory protein
cofilin, specifically interacts with and controls in its phos-
phorylated state the activity of PLD1. By this, we define a
novel cellular function for the phosphorylation–dephosphor-
ylation cycle of cofilin. While non-phosphorylated cofilin
controls actin dynamics, the phosphorylated cofilin is inac-
tive in this regard (Bamburg, 1999; DesMarais et al, 2005;
Huang et al, 2006). In contrast, it is apparently the phos-
phorylated cofilin, which preferentially stimulates PLD1 ac-
tivity. Furthermore, stimulation of PLD1 by cofilin is under
control of G-protein-coupled muscarinic receptors. Several
lines of evidence support this notion. First, by expression in
HEK-293 cells N1E-115 cells
0.05
0.10
0.15
0.20
0
*
Carb
0.1
0.2
0.3
0.4
0.5
0
*
Stimulated PtdEtOH
formation (% of phospholipids)
Carb
S3D Cofilin
Control
S3D Cofilin
Control
0.1
0.2
0.3
0.4
0.5
0
*
*
Stimulated PtdEtOH
formation (% of phospholipids)
Carb
S3D Cofilin
Control
siRNA hCofilin
Both
HEK-293 cells
Figure 4 Phosphorylation-mimic S3D cofilin potentiates PLD1 sti-
mulation by carbachol and rescues PLD1 stimulation in cofilin-
depleted cells. HEK-293 cells or N1E-115 cells were transfected with
phosphorylation-mimic S3D cofilin, or with empty vector (Control)
(A), or transfected with either phosphorylation-mimic S3D mouse
cofilin, human cofilin siRNA pSUPER plasmid (siRNA hCofilin), or
both constructs together (B). After 48h, stimulated [3H]PtdEtOH
accumulation was determined in the presence of 1mM carbachol
(Carb). Data shown are means7s.e. (n¼3) (A), or are representa-
tive of three experiments (B). *Po0.05.
Phospho-cofilin stimulation of PLD1
L Han et al
The EMBO Journal VOL 26|NO 19|2007
&2007 European Molecular Biology Organization
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Page 7

intact cells, we found that factors known to increase cofilin
phosphorylation, such as LIM-kinase1, inactive slingshot
phosphatase 1L and 14-3-3z, enhanced receptor-mediated
stimulation of PLD1, whereas expression of wild-type sling-
shot phosphatase 1L, which abolished cofilin phosphoryla-
tion, and the Unphosphorylatable S3A cofilin mutant had the
opposite effect on PLD stimulation. This regulation of PLD
activity was specific for the G-protein-coupled receptors,
known to be controlled by Rho and the LIM-kinase activator,
Rho-kinase (Schmidt et al, 1999), whereas PLD stimulation
by PMA and EGF, which is independent of Rho and Rho-
kinasein HEK-293and N1E-115
(Vo? et al, 1999), was not altered. Most important, silencing
of cellular cofilin largely prevented PLD stimulation by
neuroblastoma cells
WB: α-P-Cofilin
WB: α-Cofilin
IP: α α-PLD1
–
IP: α α-PLD2
–++
Lysate
Carbachol
— 20 kDa
— 20 kDa
— Cofilin
10µm
10µm
10µm 10µm10µm
10µm
10µm10µm
g
PLD1
h
d
c
b
PLD2
a
e
f
S3A Cofilin
Cofilin
GST
GST-PLD1
GST-PLD1
+
–
+
–
–
+
α-PLD1
α-His
α-PLD2
α-His
S3A Cofilin
Cofilin
GST
GST-PLD2
GST-PLD2
+
–
+
–
–
+
Cofilin
0.25
0.50
0.75
1.00
0
GST-PLD1 GST-PLD2
Bound cofilin mutants
(fold control)
S3A cofilin
Cofilin
S3A cofilin
Figure 5 Direct cofilin–PLD1 interaction is reflected by cofilin-induced subcellular redistribution of PLD1, but not PLD2. (A) Immobilized GST,
GST-tagged PLD1 and GST-tagged PLD2 were incubated with recombinant His6-tagged wild-type or unphosphorylatable S3A cofilin overnight at
41C. Specifically bound proteins were separated by SDS–PAGE, transferred onto nitrocellulose membrane and detected by immunoblotting with
anti-PLD and anti-His antibodies. Bar graph illustrates mean7s.e.m. (n¼3), with the amount of wild-type cofilin bound to GST-PLD1 set to 1
(Control). (B) HEK-293 cells were transfected with PLD1 (a–c), PLD2 (d–f), HA-tagged wild-type cofilin (b, e, g) or with HA-tagged
unphosphorylatable S3A cofilin (c, f, h), either alone (a, d, g, h) or with the indicated combinations. After 48h, immunofluorescence laser
confocal microscopy was performed as described in the Materials and methods section. Yellow color: merge of red (PLD) and green (cofilin)
colors. Data are characteristic of three similar experiments. Scale bar, 10mm. (C) HEK-293 cells were transfected with wild-type PLD1 or PLD2.
After 48h, the cells were treated for 15s without (?) or with (þ) 1mM carbachol, followed by cell lysis and immunoprecipitation with
anti-PLD antibodies. The PLD immunoprecipitates (IP) and total lysates were resolved by SDS–PAGE and probed with anti-phospho-cofilin
(a-P-Cofilin) or anti-cofilin antibodies (a-cofilin) as indicated. The results shown are representative of 3–4 experiments. WB, Western blot.
Phospho-cofilin stimulation of PLD1
L Han et al
&2007 European Molecular Biology OrganizationThe EMBO JournalVOL 26|NO 19|2007 4195

Page 8

carbachol in both cell types, whereas expression of phos-
phorylation-mimic S3D cofilin had the opposite effect.
Second, mAChR activation induced a very rapid association
of cofilin specifically with PLD1 and a phosphorylation of
cofilin. Third, in vitro studies with purified components
showed that cofilin specifically bound to PLD1. Evidence
for such a specific cofilin–PLD1 interaction was also observed
by expression of these proteins in intact cells, where cofilin
induced a specific subcellular redistribution of PLD1. Finally,
cofilin phosphorylated by LIM-kinase1 as well as two phos-
phorylation-mimic cofilin mutants (S3D and S3E) strongly
and specifically increased enzyme activity of PLD1.
Phosphorylation of cofilin as studied in detail for the M3
mAChR in HEK-293 cells, was a very rapid and transient
response, indicating that cellular phosphocycling of cofilin is
indeed tightly controlled by kinases and phosphatases
(Huang et al, 2006). Several previous studies designed to
analyze the regulation of cofilin dephosphorylation may not
have been aware of such rapid induction of cofilin phosphor-
ylation (Zhan et al, 2003; Nagata-Ohashi et al, 2004; Nebl
et al, 2004; Nishita et al, 2004; Wang et al, 2005). The activity
of the slingshot phosphatases is controlled by cellular Ca2þ,
in part mediated by the Ca2þ/calmodulin-dependent protein
phosphatase calcineurin, cyclic AMP and the Ras effector,
phosphatidylinositol 3-kinase (Zhan et al, 2003; Nagata-
Ohashi et al, 2004; Nebl et al, 2004; Nishita et al, 2004;
Wang et al, 2005). As the M3mAChR not only induces PLD
stimulation in HEK-293 cells, but also increases intracellular
Ca2þconcentration and cyclic AMP, and induces activation of
Ras GTPases (Evellin et al, 2002; Lo ´pez de Jesu ´s et al, 2006),
such mechanisms may contribute to the rapid dephosphoryla-
tion of cofilin. The rapid and transient phosphorylation of
cofilin perfectly reflects its well-established pivotal role in
the regulation of actin filament dynamics (Bamburg, 1999;
Bamburg and Wiggan, 2002; Pollard and Borisy, 2003;
DesMarais et al, 2005). Similarly interesting, this receptor-
induced cofilin phosphorylation profile is in line with the rapid
and very transient PLD stimulation by G-protein-coupled
receptors, including muscarinic receptors (Schmidt et al,
1995; reviewed in Exton, 2002). Recently, it has been reported
that tubulin specifically interacts with the F-3 fragment of
PLD2, and that inhibition of receptor-induced PLD2 stimula-
tion, probably by direct interaction with tubulin, may contri-
bute to the rapid decline of the PLD2 response (Chae et al,
2005). Thus, different PLD isozymes may use and associate
with different cytoskeletal proteins for rapid deactivation.
Finally, we analyzed the region in PLD1 responsible for
cofilin binding. For this, PLD1 fragments were generated
essentially as the corresponding PLD2 fragments described
before (Lee et al, 2001; Chae et al, 2005). Cofilin and
phosphorylated cofilin cofilin were found to specifically
bind to the F-3 fragment of PLD1, encompassing the amino
acids 585–712. Although phosphorylation-mimic cofilin
strongly increased PLD1 activity, binding to PLD1 was not
enhanced. The somewhat reduced affinity of phospho-cofilin
may contribute to the rapid termination of PLD1 stimulation
by muscarinic receptors. In line with the binding on the
full-lengthPLDenzymes,
Unphosphorylatable S3A cofilin to any of the PLD1 frag-
ments, and also no binding of cofilin to PLD2 fragments.
These data indicate that this region in PLD1 is necessary and
sufficient for cofilin binding. This cofilin-binding F-3 frag-
ment was then used to further probe the role of cofilin–PLD1
interaction for receptor signaling to PLD and actin stress fiber
formation. Expression of the F-3 fragment profoundly and
specifically suppressed PLD stimulation by the M3 AChR,
an effect fully reversed by expression of cofilin. This was
there wasno binding of
LIM-kinase1
–+ –+
<
<
<<
205 _
66_
(kDa)
116 _
20_
97¯
CS α-GST CS
α-HisCS
CofilinS3A Cofilin
α-GST
Cofilin
S3ACofilin
S3A Cofilin
Cofilin
PLD1
PLD1
PLD2
PLD2
LIM-kinase1
6.0
0
1.0
2.0
3.0
4.0
5.0
–ATP
+ATP
–ATP
+ATP
PtdEtOH (nmol× ×30 min–1× ×mg–1)
0.4
0.8
1.2
1.6
2.0
0
—Cofilin
PLD1 PLD2
0
1.0
2.0
3.0
4.0
5.0
6.0
PtdEtOH (nmol× ×30 min–1× ×mg–1)
S3A Cofilin
S3D Cofilin
S3A Cofilin
S3A Cofilin
Cofilin
Cofilin
Cofilin
Without
S3A Cofilin
S3E Cofilin
S3E Cofilin
S3D Cofilin
Without
Without
Cofilin
Coomassie blue staining
PLD1
32P autoradiography
Figure 6 Phospho-cofilin stimulates PLD1. (A) With LIM-kinase1,
phosphorylated (þATP) and unphosphorylated (?ATP) wild-type
or unphosphorylatable S3A cofilin were incubated with GST-tagged
PLD1 or GST-tagged PLD2 to measure PLD activity. The upper blots
show purified GST-tagged LIM-kinase1, GST-tagged PLD1 and PLD2
and His6-tagged wild-type and S3A cofilin by Coomassie blue
staining (CS), and immunoblotting with anti-GST and anti-His
antibodies, respectively, as well as the specific phosphorylation of
wild-type cofilin with [g-32P]ATP (32P autoradiography) in the
absence (?) and presence (þ) of LIM-kinase1. (B) GST-tagged
PLD1 was incubated without and with recombinant unphosphor-
ylatable S3A cofilin, wild-type cofilin, phospho-mimetic S3D cofilin
or phospho-mimetic S3E cofilin to measure PLD activity. Purified
His6-tagged cofilin mutants are presented by Coomassie blue stain-
ing. The results shown are representative of 3–4 experiments.
Phospho-cofilin stimulation of PLD1
L Han et al
The EMBO Journal VOL 26|NO 19|2007
&2007 European Molecular Biology Organization
4196

Page 9

accompanied by a similar inhibition of receptor-induced
association of cofilin with PLD1, indicating that indeed this
PLD1 fragment binds cofilin also in intact cells. Expression of
the cofilin-binding F-3 fragment also abrogated M3mAChR-
induced stress fiber formation. These findings together in-
dicate that cofilin-induced actin rearrangements (Chan et al,
2000; Endo et al, 2003; Ghosh et al, 2004; Marcoux and Vuori,
2005) may be partly mediated by PLD1 and its product PA,
which have been shown to participate in receptor-induced
stress fiber formation in various cell types (Ha and Exton,
1993; Cross et al, 1996; Kam and Exton, 2001; Porcelli et al,
2002; Komati et al, 2005).
In conclusion, we report here on a novel molecular link
between the actin cytoskeleton and PLD1, and provide evi-
dence that inactive phospho-cofilin exerts an unexpected
biological function. By its stimulation of PLD1, known to
regulate many early and late cellular functions, from calcium
mobilization, glucose transport, mitogenesis to apoptosis
(Exton, 2002), phospho-cofilin is most likely an active signal-
ing component, and may control essential cellular functions.
Materials and methods
Cell culture, transfection, plasmid construction and
adenoviruses
HEK-293 and N1E-115 cells grown to near confluence on 145-mm
culture dishes were transfected as reported (Schmidt et al, 2001;
Lo ´pez de Jesu ´s et al, 2006). PLD1 and PLD2 (each subcloned into
pCGN), wild-type LIM-kinase1 and kinase-deficient D460A LIM-
kinase1 (each subcloned into pUCD2), wild-type cofilin, Unpho-
sphorylatable S3A cofilin and phosphorylation-mimic S3D cofilin
(each subcloned into pcDL-SRa), slingshot phosphatase 1L, inactive
C393S slingshot phosphatase 1L, VSV-G (vesicular stomatitis virus
glycoprotein)-tagged 14-3-3z, as well as the M3 mAChR (each
subcloned into pCDNA3.1/myc-His) were kindly provided by Drs
MA Frohman, A Morris, K Mizuno, T Uemura, H Betz and CJ van
Koppen, respectively. Typically 50–100mg of DNA for cell transfec-
tion were used, except for Unphosphorylatable S3A cofilin
amounting to 150mg of DNA. For transfection of the cells
with siRNA pSUPER plasmids (Brummelkamp et al, 2002), cells
on 35-mm dishes were transfected with 3ml Lipofectamine 2000 in
1ml Opti-MEM containing 6–9mg of human cofilin siRNA or mouse
cofilin siRNA. If phosphorylation-mimic S3D mouse cofilin was
cotransfected with human cofilin siRNA, 6mg of the vector DNAwas
used in a total amount of 12mg. Human cofilin siRNA pSUPER
PX
PHCRI CRIILOOP CRIII CRIV
NC
aa1331
F-1
F-2
F-loop
F-3
F-4
F-5
F-6
497
584
712
825
9271036
PLD1
PX
PH CRI CRIICRIII CRIV
CN
aa1314
F-1
F-2
F-3
F-4
F-5
F-6
475
612
723
826
934
PLD2
F-1
F-2
F-3
F-4
F-5
F-6
F-loop
GST
F-1
F-2
F-3
F-4
F-5
F-6
PLD21DLP
F-1
F-2
F-3
F-4
F-5
F-6
GST
F-1
F-2
F-3
F-4
F-5
F-6
F-loop
GST
2DLP
1DLP
S3D Cofilin
F-3 binding
Loading
Cofilin
Cofilin
Cofilin
S3A Cofilin
— Cofilin
WB: α-Cofilin
Cofilin
Figure 7 Interaction of cofilin with the F-3 fragment of PLD1. (A) A schematic representation of the highly conserved regions in PLD1 and
PLD2. PX, phox; PH, pleckstrin homology; CRI–CRIV, conserved regions I to IV. (B) Purified GSTand GST-tagged fragments of PLD1 and PLD2,
visualized by Coomassie blue staining. (C) Binding of purified recombinant wild-type or unphosphorylatable S3A cofilin to GST-tagged
fragments of PLD1 or PLD2 was determined as described under Materials and methods. (D) Binding of purified recombinant wild-type or
phospho-mimetic S3D cofilin to GST-tagged F-3 fragment of PLD1. The lower blot shows the loading control by Western blot (WB). The results
shown are representative of 2–4 experiments.
Phospho-cofilin stimulation of PLD1
L Han et al
&2007 European Molecular Biology OrganizationThe EMBO JournalVOL 26|NO 19|2007 4197

Page 10

plasmid (target sequence GGAGGATCTGGTGTTTATC) and mouse
cofilin siRNA pSUPERplasmid
GACCTGGTGTTCATC) were constructed as described previously
(Nishita et al, 2005; Kiuchi et al, 2007). Expression of the encoded
proteins was verified by immunoblotting of cell lysates with specific
antibodies. Assays were performed 48h after transfection. To
generate the c-myc-tagged F-1 and F-3 fragments of hPLD1b,
HindIII to XbaI 842 and 442-bp fragments from pGEX4T1 were
subcloned into pCDNA3.1, respectively (Invitrogen). Adenoviruses
encoding lipase-inactive PLD mutants were generated essentially as
described before (He et al, 1998; Ru ¨menapp et al, 2001). Briefly,
K898R human PLD1b or K758R mouse PLD2 XbaI–SmaI fragments
from pCGN were subcloned into pAdTrack-CMV XbaI–EcoRV,
followed by homologous recombination with the pAdEasy vector
(both vectors kindly provided by Dr B Vogelstein), the constructs
were linearized and transfected into HEK-293 cells using LipofectA-
MINE (Invitrogen). The vector constructs were verified by sequen-
cing. After several cycles of amplification, the adenoviruses were
purified using a CsCl2gradient and titration with the Adeno-X Rapid
titer kit, according to the manufacturer’s instructions (BD Bio-
sciences). HEK-293 cells were infected with adenoviral bacterial
b-galacosidase LacZ serving as a control (a kind gift from Dr T
Eschenhagen), or adenoviral-encoded PLD mutants, 48h before
PLD assays, at a multiplicity of infection of 50. Before the assay,
infected cells were visualized by fluorescence microscopy (Zeiss
Axiovert S100).
(targetsequenceGGAG
Purification of proteins and protein binding assays
To obtain GST-tagged PLD1, PLD2 (cDNAs kindly provided by Drs A
Morris and MA Frohman) and His6-tagged phosphorylation-mimic
S3E cofilin, Spodoptera frugiperda cells were infected with appro-
priate recombinant baculoviruses. GST/flag-tagged LIM-kinase1
(kindly provided by Dr K Mizuno) was expressed in HEK-TsA201
cells. The recombinant proteins were purified with glutathione
Sepharose (Lo ´pez de Jesu ´s et al, 2006). GST-tagged PLD2 fragments
F-1–F-6, His6-tagged wild-type cofilin, unphosphorylatable S3
cofilin and phosphorylation-mimic S3D cofilin (cDNAs kindly
provided by Drs SH Ryu and K Mizuno) were expressed in
Escherichia coli, and purified with glutathione Sepharose or Ni-
NTA Superflow Agarose beads (Lo ´pez de Jesu ´s et al, 2006). To
obtain the soluble and untagged proteins, the immobilized cofilin
proteins were eluted with 400mM imidazole, and for protease
digestion of GST-tagged proteins, the washed beads were resus-
pended in thrombin buffer (50mM Tris/HCl, pH 8.6, 150mM NaCl,
2.5mM CaCl2, 0.1% 2-mercaptoethanol), and incubated for 2h at
room temperature with thrombin (followed by overnight incubation
at 41C). GST-tagged PLD1 fragments were generated essentially as
the corresponding PLD2 fragments described before (Lee et al, 2001;
Chae et al, 2005). Briefly, fragment 1 (F-1, aa 1–331, 64kDa)
encompassed the PX- and the PH-domains, fragment 2 (F-2, aa 332–
497, 45kDa) the conserved regions CRI and CRII, which are
followed by the PLD1-specific loop (F-loop, aa 498–585, 37kDa).
Fragment 3 (F-3, aa 585–712, 41kDa) comprised the region between
the PLD1-specific loop and the conserved region CRIII, fragment 4
(F-4, aa 713–825, 40kDa) the CRIII region, fragment 5 (F-5, aa 826–
927, 38kDa) the CRIV region, and fragment 6 (F-6, aa 928–1036,
39kDa) encompassed the entire COOH-terminus of human PLD1b
(Ensembl Protein Report; www.ensembl.org). The fragments were
amplified with Pfu polymerase (Fermentas) from a human PLD1b
vector, using modified PCR primers harboring an artificial SalI-
(sense) or NotI-site (antisense). The PCR fragments were ligated in
the SalI/NotI-restricted multiple cloning site of the bacterial
+Cofilin
+Cofilin
— α-myc
Control
F-3
+Cofilin
Control
F-1
WB: α
IP: α α-PLD1
Carbachol
— Cofilin
— Cofilin
––
++
Lysate
F-3
–
+ –
+
0.1
0.2
0.3
0
Control F-3
*
Stimulated PtdEtOH
formation (% of phospholipids)
F-1
Control
0.1
0.2
0.3
0
0.3
0.6
0.9
1.2
0
Control F-3
PMA Carb Carb
Figure 8 The F-3 fragment of PLD1 specifically reduces PLD stimulation by carbachol and interaction of PLD1 with cofilin. (A) HEK-293 cells
were transfected with empty vector (Control), F-1 or F-3, either alone or together with wild-type cofilin. After 48h, stimulated [3H]PtdEtOH
accumulation was determined in the presence of 1mM carbachol (Carb) or 100nM PMA. Data shown are means7s.e. (n¼3–4). The
immunoblots show expression of c-myc-tagged F-3 and F-1 fragments in cell lysates. (B) HEK-293 cells were transfected with wild-type PLD1
alone or together with the F-3 fragment. After 48h, the cells were treated for 15s without (?) or with (þ) 1mM carbachol, followed by cell
lysis and immunoprecipitation with the anti-PLD1 antibody. The PLD1 immunoprecipitates (IP) and total lysates were resolved by SDS–PAGE
and probed with anti-cofilin antibodies (a-cofilin) as indicated. The results shown are representative of 3–4 experiments. WB, Western blot.
Phospho-cofilin stimulation of PLD1
L Han et al
The EMBO JournalVOL 26|NO 19|2007
&2007 European Molecular Biology Organization
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Page 11

expression vector pGEX4T1 (Amersham). The vector clones were
verified by sequencing. The resulting GST-tagged hPLD1b fragments
were expressed in E. coli, and purified with glutathione Sepharose
(Lo ´pez de Jesu ´s et al, 2006). Purification of the proteins was
analyzed by SDS–PAGE. GST, GST-tagged PLD1, PLD2 (full length
each) and their corresponding fragments were incubated with
purified wild-type, unphosphorylatable S3 cofilin or phosphoryla-
tion-mimic S3D cofilin overnight at 41C, and the ratio of beads to
cofilin (approximately 5–30mg each) was assessed by calibration on
the basis of Coomassie-stained gels. After three washes, the beads
were resolved in Laemmli buffer, subjected to SDS–PAGE and
transferred to nitrocellulose for Western blotting to visualize bound
recombinant cofilin proteins using anti-His (Santa Cruz) or anti-
cofilin antibodies (Cell Signaling Technology).
Confocal laser scanning microscopy and fluorescence
microscopy
HEK-293 cells transfected with PLD1 or PLD2 either alone or
together with HA-tagged wild-type or Unphosphorylatable S3A
cofilin were grown on coverslips (Falcon) pretreated with 0.1mg/ml
poly-D-lysine (Stope et al, 2004). After 48h, the cells were rinsed
with Moscona, containing 13.6mM NaCl, 4mM KCl, 12mM
NaHCO3, 10mM
D-glucose, 0.36mM NaH2PO4 and 0.18mM
KH2PO4, pH 7.4, fixed and permeabilized with ethanol/acetone
(1/1, v/v) for 10min at RT, followed by two washes with Moscona.
Unspecific binding was blocked for 15min by incubation with
Moscona/BSA (Moscona supplemented with 0.5% BSA). The cells
were rinsed and incubated for 1h at RT with anti-PLD antibodies
(dilution 1:50; kindly provided by Dr S Bourgoin) or with an anti-
HA antibody (12CA5, dilution 1:20; Roche Molecular Biochem-
icals). Afterwards, the cells were rinsed three times with Moscona/
BSA, incubated for 45min in darkness with the corresponding
fluorescence-conjugated secondary antibodies (Alexa-633 red for
PLD, Alexa-488 green for cofilin; dilution 1:200; MoBiTec) at RT.
The secondary antibodies were washed out in darkness with
Moscona/BSA, and the cell specimens were mounted in Moscona
supplemented with 90% (v/v) glycerol and 1.0% (v/v) p-
phenylenediamine. Confocal immunofluorescence imaging was
performed using Zeiss LSM 510 Axiovert 100M confocal laser
scanning microscopy system (Plan-Neofluor ?40/1.3 oil objective,
488/633nm excitation). Confocal laser microscopy images were
processed with the ImageProPlus software (Version 4.5; Media
cybernatics) equipped with a Gaussian filter module (Stope et al,
2004). J82 bladder carcinoma cells transfected with M3mAChR
alone or together with F-3 fragment of PLD1 (50mg DNA each)
were stained for actin filaments with tetramethyl-rhodamine
Basal
Control
Carbachol
F-3
20 µm
a
c
d
b
20 µm
20 µm
20 µm
B
25
50
75
100
0
*
Stress fiber formation
(% positive cells)
F-3
Control
Figure 9 The F-3 fragment of PLD1 abolishes stress fiber formation in J82 cells. (A) J82 cells were transfected with the M3mAChR alone or
together with the PLD1-specific F-3 fragment. Forty-eight hours after transfection, cells stimulated without (Basal) or with 1mM carbachol for
5min were examined for the actin cytoskeleton by TRITC–phalloidin staining. Arrows indicate carbachol-induced stress fiber formation. Scale
bar, 20mm. (B) Carbachol-induced stress fiber formation in M3mAChR-positive cells in the absence (Control) or presence of the F-3 fragment
(F3). Data shown are means7s.e. (n¼3). *Po0.05.
Phospho-cofilin stimulation of PLD1
L Han et al
&2007 European Molecular Biology OrganizationThe EMBO Journal VOL 26|NO 19|2007 4199

Page 12

isothiocyanate
(Ru ¨menapp et al, 1999). Fluorescent images were obtained using
Zeiss Axiovert S100 microscope.
(TRITC)-conjugatedphalloidin (Sigma-Aldrich)
Phosphorylation of recombinant proteins and
phosphorylation of cellular cofilin
To study phosphorylation of recombinant proteins by LIM-kinase1,
immobilized LIM-kinase1 (1.5mM) was equilibrated with phosphor-
ylation buffer (25mM Tris/HCl, pH 7.4, 5mM MgCl2, 1mM EDTA,
0.1mM EGTA, 1mM dithiothreitol) and mixed with 10–50mg
recombinant GST, GST-tagged PLD1, GST-tagged PLD2, wild-type
or Unphosphorylatable S3A cofilin. After preincubation for 5min at
371C (or at 251C to measure cofilin phosphorylation), the reaction
was started by adding 100mM Tris/HCl, pH 7.5, 25mM MgCl2,
1mM CaCl2, 250mM [g-32P]ATP (0.75MBq per reaction tube) at a
dilution of 1:5. After 15min (or 45min for the cofilin phosphoryla-
tion), the reaction was stopped by addition of Laemmli sample
buffer and heating for 5min at 951C. The proteins were separated by
SDS–PAGE, and phosphorylated proteins detected by autoradiogra-
phy using Kodak X-Omat AR films. To measure phosphorylation of
cellular cofilin, transfected cells were incubated for the indicated
periods of time at 371C with 1mM carbachol, followed by cell lysis
in a buffer containing 1% SDS and 10mM Tris/HCl, pH 7.4, and five
passages through a 25-gauge needle (Keiper et al, 2004). The lysates
were clarified by centrifugation, followed by determination of
protein concentration and incubation in Laemmli buffer for 10min
at 951C. After SDS–PAGE and transfer to nitrocellulose membranes,
phosphorylated cofilin and the total cellular content of cofilin were
detected with anti-phospho-cofilin (anti-P-Cofilin) and anti-cofilin
antibodies, respectively (Cell Signaling Technology).
Immunoprecipitation and immunoblotting
Cells were transfected with the expression plasmids indicated in the
figure legends and grown on 60-mm culture dishes in serum-free
medium. Cells were rinsed in Hank’s balanced salt solution and
incubated without and with carbachol for 15s at 371C. Then, the
cells were washed twice with ice-cold phosphate-buffered saline,
containing 137mM NaCl, 2.7mM KCl, 6.5mM NaH2PO4, 1.5mM
KH2PO4, 0.9mM CaCl2, 0.5mM MgCl2and 100mM orthovanadate,
pH 7.2. Meanwhile, the anti-PLD antibodies (7mg each) were gently
mixed with protein A–Sepharose (20ml per reaction tube) in
immunoprecipitation buffer containing 50mM Tris/HCl, pH 7.5,
150mM NaCl, 10mM sodium pyrophosphate, 50mM NaF, 1mM
EGTA, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1%
sodium dodecylsulfate, for 1h at 41C. Cells were scraped in 500ml
ice-cold immunoprecipitation buffer, supplemented with 1mM
orthovanadate, 1mM phenylmethylsulfonylfluoride, 10mg/ml leu-
peptin and 25mg/ml aprotinin, transferred to precooled reaction
tubes, vortexed for 10s and incubated on ice for at least 10min.
Lysates were clarified by centrifugation, the supernatants (4mg of
protein/reaction tube) were gently mixed overnight at 41C and
precipitates were washed four times with immunoprecipitation
buffer. Precipitated proteins were incubated in Laemmli buffer for
10min at 951C and separated by SDS–PAGE. Bound phosphorylated
cofilin and cofilin were detected by immunoblotting with specific
cofilin antibodies. Analysis of the total cellular cofilin content in the
lysates served as control. For detection of c-myc, LIM-kinase1, VSV-
G, PLD1 and PLD2, equal amounts of protein from cell lysates were
separated by SDS–PAGE on 5 or 15% acrylamide gels. After a
transfer to nitrocellulose membranes and a 1-h incubation with the
appropriate antibodies, the proteins were visualized by enhanced
chemiluminescence.
Measurement of PLD activity
PLD activity was measured in transfected cells labeled with
[3H]oleic acid (5Ci/mmol, PerkinElmer Life Sciences) as formation
of the specific PLD product, [3H]phosphatidylethanol ([3H]PtdE-
tOH), for 30min at 371C in the presence of ethanol. Basal PLD
activity amounted to 0.0870.03 [3H]PtdEtOH formation expressed
as the precentage of total labeled phospholipids in HEK-293,
cells and to 0.0170.002 [3H]PtdEtOH formation expressed as
the precentage of total labeled phospholipids in N1E-115 cells,
respectively. As basal PLD activity was not altered by any
maneuver, agonist-induced PLD stimulation is expressed as
stimulated [3H]PtdEtOH formation (Ru ¨menapp et al, 2001). To
study the role of phosphorylated cofilin on recombinant PLD
activity, GST-tagged LIM-kinase1 bound to glutathione Sepharose
beads (30mg) and His6-tagged wild-type or S3A cofilin (1mg each)
were incubated together in the presence or absence of 1mM MgATP
for 45min at 251C. LIM-kinase1 was removed by centrifugation for
3min at 2400r.p.m. at 41C. The supernatant containing phosphory-
lated or unphosphorylated cofilin was incubated with GST-tagged
PLD1 or PLD2 immobilized on glutathione Sepharose beads (20–
30mg per reaction tube), [3H]PtdCho/PIP2vesicles (1-palmitoyl-2-
[9,10-3H]palmitoyl-glycerophosphocholine (89Ci/mmol, PerkinEl-
mer Life Sciences) mixed with PIP2(Roche Applied Science) in a
molar ratio of 8:1), 2% (v/v) ethanol and stimulatory agents for
30min at 301C (Schmidt et al, 1999; Wilde et al, 2002). Under
identical experimental conditions, the activity of GST-tagged PLD1
immobilized on glutathione Sepharose beads (20–30mg per reaction
tube) was measured in the presence or absence of recombinant
wild-type cofilin, Unphosphorylatable S3A cofilin, phospho-mi-
metic S3D cofilin or phospho-mimetic S3E cofilin (2.5mg per
reaction tube). Data shown in the figures are either representative
experiments or means7s.e.m. of n independent experiments, each
performed in triplicate. Comparisons between means were either
with the Student’s paired t-test or one-way analysis of variance test,
and a difference was regarded significant at Po0.05.
Acknowledgements
We thank Y Mahlke and S Schimanski for exceptional technical
assistance, supported by assistance from K Baden, M Hagedorn, H
Geldermann, D Petermeyer and A Ko ¨tting-Dorsch. C Heneweer and
M Thie supported the microscopic studies. The gifts of various
plasmids and antisera from R Agami, H Betz, S Bourgoin, T
Eschenhagen, MA Frohman, A Morris, CJ van Koppen, SH Ryu,
B Vogelstein and T Uemura are greatly appreciated. This work
was supported by grants from the Deutsche Forschungsgemein-
schaft,theFonds derChemischen
Forschungsfo ¨rderung Essen and the Fakulta ¨t fu ¨r Klinische Medizin
Mannheim. M Lo ´pez de Jesu ´s is recipient of a Basque Government
Fellowship. M Schmidt is recipient of a Rosalind Franklin
Fellowship from the University of Groningen.
Industrie,theInterne
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