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Diacylglycerol kinase αmediates HGF-induced Rac
activation and membrane ruffling by regulating
atypical PKC and RhoGDI
Federica Chianale
a,1
, Elena Rainero
a,1
, Cristina Cianflone
a
, Valentina Bettio
a
, Andrea Pighini
a
, Paolo E. Porporato
a
,
Nicoletta Filigheddu
a
, Guido Serini
b,c
, Fabiola Sinigaglia
a
, Gianluca Baldanzi
a
, and Andrea Graziani
a,2
a
Department of Clinical and Experimental Medicine and Biotecnologie per la Ricerca Medica Applicata, University Amedeo Avogadro of Piemonte Orientale,
28100 Novara, Italy;
b
Department of Oncological Sciences and Division of Molecular Angiogenesis, Istituto per la Ricerca e la Cura del Cancro, Institute for
Cancer Research and Treatment, University of Torino School of Medicine, 10060 Candiolo, Italy; and
c
Center for Complex Systems in Molecular Biology and
Medicine, University of Torino, 10100 Torino, Italy
Edited* by Lewis Clayton Cantley, Harvard Medical School, Boston, MA, and approved December 14, 2009 (received for review July 29, 2009)
Diacylglycerol kinases (DGKs) convert diacylglycerol (DAG) into
phosphatidic acid (PA), acting as molecular switches between
DAG- and PA-mediated signaling. We previously showed that Src-
dependent activation and plasma membrane recruitment of DGKα
are required for growth-factor-induced cell migration and ruffling,
through the control of Rac small-GTPase activation and plasma
membrane localization. Herein we unveil a signaling pathway
through which DGKαcoordinates the localization of Rac. We show
that upon hepatocyte growth-factor stimulation, DGKα, by produc-
ing PA, provides a key signal to recruit atypical PKCζ/ι(aPKCζ/ι)in
complex with RhoGDI and Rac at ruffling sites of colony-growing
epithelial cells. Then, DGKα-dependent activation of aPKCζ/ιmedi-
ates the release of Rac from the inhibitory complex with RhoGDI,
allowing its activation and leading to formation of membrane ruf-
fles, which constitute essential requirements for cell migration.
These findings highlight DGKαas the central element of a lipid sig-
naling pathway linking tyrosine kinase growth-factor receptors to
regulation of aPKCs and RhoGDI, and providing a positional signal
regulating Rac association to the plasma membrane.
cell migration
|
growth factors
|
phosphatidic acid
Cell migration, central to many biological and pathological
processes such as cancer metastatic progression, is a multistep
cycle involving extension of protrusions and formation of stable
attachments near the leading edge, followed by translocation of the
cell body forward (1). The protrusive activity occurring at the
leading edge depends on the spatial and temporal coordination
between cell substrate adhesion and actin reorganization. Rho-
family small GTPases coordinate the recruitment at the leading
edge of downstream effectors, thereby mediating the formation of
ruffles and lamellipodia. Their GTP-bound state is tightly regulated
by both guanine nucleotide exchange factors (GEFs), which stim-
ulate GTP loading, and GTPase activating proteins (GAPs), which
catalyze GTP hydrolysis. Moreover, Rho-family GTPases are re-
gulated by guanine nucleotide dissociation inhibitors (GDIs), which
antagonize both GEFs and GAPs and mediate the cycling of Rho
proteins between the cytosol and the membrane (2, 3).
Atypical protein kinase C ζand ι(aPKCζ/ι), unlike classical
and novel PKCs, feature a C1-like domain which does not bind
to either diacylglycerol or phorbol esters, and have recently been
proposed as key transducers for establishment of cell polarity
and migration (4).
Diacylglycerol kinases (DGKs), which convert diacylglycerol
(DAG) to phosphatidic acid (PA), comprise a family of 10 distinct
enzymes grouped into fiveclasses, each featuring distinct regulatory
domains and a highly conserved catalytic domain preceded by two
cysteine-rich C1-like domains. An increasing body of evidence
indicates that DGKs, by acting as terminators of diacylglycerol-
triggered signaling, contribute to regulating C1 domain-containing
proteins, such as classical and novel PKCs and the Rac-GAP
β-chimaerin (5). Conversely, by generating PA, DGKs regulate
several signaling proteins, including serine kinases, small-GTPase-
regulating proteins, and lipid-metabolizing enzymes (reviewed in
refs. 6 and 7). Thus, by regulating in a reciprocal manner the level of
both DAG and PA lipid second messengers, DGK enzymes may act
as terminators of DAG-mediated signals as well as activators of
PA-mediated ones. We previously showed that DGKαis activated
by growth factors on recruitment to the plasma membrane through
its Src-mediated phosphorylation on Tyr
335
. Activation of DGKα
mediates growth-factor-induced cell migration and proliferation in
epithelial, endothelial, and lymphoma cells (8–12). Moreover, we
demonstrated that in epithelial cells, DGKαis required for hep-
atocyte growth factor (HGF) -induced membrane ruffling by regu-
lating Rac membrane targeting and activation (13). However, the
molecular mechanisms by which DGKαregulates Rac function still
remain to be elucidated.
Here we unveil a previously undescribed signaling pathway
linking HGF receptor to Rac activation and targeting at the
plasma membrane of epithelial cells, where it drives the formation
of membrane protrusions. Our data highlight that upon HGF-
induced plasma membrane recruitment and activation of DGKα,
PA recruits at the plasma membrane and activates aPKCζ/ι,in
complex with RhoGDI and Rac. Then, Rac is released from the
inhibitory complex with RhoGDI, allowing its activation and for-
mation of membrane ruffles.
Results
DGKαRegulates Rac by Directing Its Recruitment to the Plasma
Membrane. MDCK epithelial cells grow in discrete colonies and
on HGF stimulation undergo scatter, involving cell spreading,
dissolution of intercellular adhesions, and migration of cells away
from one another. At early time points from HGF stimulation,
cells at the periphery of a colony reorganize the actin cytoske-
leton and form dynamic protrusions of the outer plasma mem-
brane known as ruffles, mediated by membrane targeting and
activation of Rac (14, 15). We previously showed that down-
regulation of DGKαaffected both HGF-induced membrane
ruffling and recruitment of Rac to the plasma membrane (13).
Here, by live-cell imaging, we report that HGF-induced rapid
and transient recruitment of EGFP-Rac at sites of intense ruf-
Author contributions: F.C., E.R., F.S., G.B., and A.G. designed research; F.C., E.R., C.C., V.B.,
A.P., G.S., and G.B. performed research; F.C., E.R., C.C., V.B., A.P., P.E.P., N.F., G.S., G.B., and
A.G. analyzed data; G.S. contributed new reagents/analytic tools; and F.C., E.R., and A.G.
wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1
F.C. and E.R. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: graziani@med.unipmn.it.
This article conta ins supporting in formation online at www.pnas.org/cgi/content/full/
0908326107/DCSupplemental.
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fling was impaired upon DGK inhibition (Fig. S1 and Movies S1
and S2), achieved by cell treatment with 1 μM R59949. R59949 is
a DGK-specific inhibitor interacting with the catalytic domain,
endowed with a strong preference for α,β, and δisoforms (16,
17). We previously showed that overexpression of DGKαre-
verted R59949-mediated inhibition of HGF signaling (8).
Thus, we investigated the mechanisms by which DGKαmight
regulate Rac-specific localization. In the absence of growth-fac-
tor stimulation, constitutively active RacV12 mutant localized
mainly in the cytoplasm and at cell-cell adhesions, whereas its
expression was not sufficient to induce ruffling at the leading
edge of cells at the periphery of a colony, according to previous
observations (15). HGF cell treatment increased the percentage
of cells featuring concomitant membrane ruffles and RacV12 at
the outer plasma membrane in a DGK-dependent manner (Fig.
S2). In addition, we compared endogenous Rac localization in
MDCK cells transiently transfected with either DGKαwild type
(DGKαWT) or a constitutively active membrane-bound DGKα
mutant (myr-DGKα). Indeed, the expression of myr-DGKα, but
not of DGKαWT, promoted a 2.5-fold increase of the percent-
age of cells displaying membrane ruffles, along with Rac
recruitment to the plasma membrane at ruffling sites (Fig. 1).
Overall, these data indicate that DGKαactivation provides a
crucial signal which is both necessary and sufficient to direct Rac
membrane localization at the leading edge, regardless of its GTP
loading, thereby promoting ruffle formation.
DGKαRegulates RhoGDI Membrane Recruitment. An increasing
body of evidence indicates that RhoGDI, which in unstimulated
cells associates with GDP-bound Rac in a cytosolic complex,
regulates Rac targeting to specific adhesion sites at the plasma
membrane through largely unidentified mechanisms (2). Once at
the plasma membrane, Rac is released from the inhibitory com-
plex with RhoGDI and is eventually activated by a Rac-GEF (2,
18). To verify whether growth factors trigger Rac localization at
the plasma membrane through interaction with RhoGDI, we
transiently transfected MDCK cells with a Rac mutant, RacR66E,
unable to bind to RhoGDI (19). Indeed, RacR66E failed to be
recruited at ruffling sites upon HGF cell treatment whereas HGF-
induced membrane ruffle formation was not impaired, indicating
that endogenous Rac is properly activated (Fig. 2). These data
demonstrate that RhoGDI physical interaction with Rac mediates
growth-factor-induced Rac targeting at ruffling sites.
Thus, we investigated whether HGF, although inducing mem-
brane ruffles, was able to recruit RhoGDI to the leading edge and
whether this process required DGKαactivity. We show that HGF
increased the percentage of cells displaying RhoGDI at ruffling
sites, which was impaired by both pharmacological inhibition (Fig.
3A) and siRNA-mediated silencing of DGKα(Fig. S3A). In
addition, in time-lapse experiments, HGF-induced rapid and
transient recruitment of RhoGDI to ruffling sites required DGK
activity (Fig. S4 and Movies S3 and S4).
Moreover, we investigated whether DGKαconstitutive activa-
tion at the plasma membrane was sufficient to recruit RhoGDI.
Thus, the expression of myr-DGKα, but not of DGKαWT, was
sufficient to recruit RhoGDI at ruffling sites in almost 80% of
transfected cells (Fig. 3Band Fig. S3B). Altogether, these findings
demonstrate that stimulation of DGKαenzymatic activity pro-
vides a crucial signal which is necessary and sufficient to direct the
recruitment of RhoGDI, which in turn is required for Rac tar-
geting at protrusion sites and ruffle formation.
DGKαRegulates Membrane Ruffling by Modulating aPKCζ/ιFunction.
The molecular mechanisms regulating RhoGDI localization are
still largely unknown, as it lacks membrane-interacting domains.
PKCζ, reported to be regulated by direct binding to PA but not to
DAG (20), associates with and phosphorylates RhoGDI, thereby
allowing its dissociation from Rac (21). Indeed, in MDCK cells, a
small amount of the whole cellular aPKCζ/ιwas constitutively
associated with RhoGDI (see below, Fig. 6). Thus, we raised the
hypothesis that DGKαmight control RhoGDI by providing the
lipid-regulating aPKCζ/ι.
To obtain an appreciable down-regulation of cellular aPKCs,
we silenced both PKCζand PKCιby different combinations of
their respective specific siRNAs (Fig. 4A). We observed that
aPKCζ/ιsilencing impaired HGF-induced membrane ruffle for-
mation, as well as both RhoGDI and Rac recruitment to the
outer plasma membrane (Fig. 4B). The aPKCζ/ιcatalytic activity
was actually required, as aPKCζ/ιinhibition by cell treatment
with PKCζ/ιpseudosubstrate peptide impaired HGF-induced
membrane ruffle formation and Rac plasma membrane trans-
location (Fig. S5A). Similarly, inhibition of PKCζby expression
of PKCζdominant-negative mutant (PKCζKW) impaired HGF-
induced RhoGDI translocation to the outer plasma membrane
Rac
DGK WT
Rac+DGK
+actin
myr-DGK
ZOOM
DGK
0
20
40
60
80
% of cells with Rac
at ruffles
DGKWT
myrDGK
***
Fig. 1. DGKαprovides the signal directing Rac to the nascent ruffle. MDCK
cells, transfected with either DGKαWT or myr-DGKα, were cultured over-
night in the absence of serum, fixed, and stained for Rac (green), myc tag
(red), and actin (blue). Arrows indicate DGKαWT- or myr-DGKα-transfected
cells. (Scale bar, 10 μm.) C, 30 transfected cells, scored for the presence of Rac
at ruffling sites. n= 6, with SEM; ***P= 0.0002.
ZOOM
mCherry-
RacR66E
control HGF
mCherry-
RacR66E
+actin
RacR66E
at pl. membr.
% of cells
*
control
HGF
0
20
40
60
80
ruffles
Fig. 2. Rac plasma membrane targeting requires Rac/RhoGDI interaction.
MDCK cells were transiently transfected with mCherry-RacR66E, treated with
10 ng/mL HGF for 15 min, fixed, and stained for actin (green). (Scale bar,
24 μm.) C, 30 transfected cells, scored for the presence of ruffles or RacR66E
plasma membrane localization. n= 3, with SEM; *P= 0.0057.
Chianale et al. PNAS
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(Fig. S5B). Moreover, aPKCζ/ιwas also required for myr-DGKα-
induced membrane ruffle formation and Rac and RhoGDI tar-
geting to the outer plasma membrane (Fig. 4Cand Fig. S5C).
Together these data indicate that, upon HGF stimulation or
myr-DGKαexpression, aPKCζ/ιmediates membrane ruffle for-
mation and recruitment of the molecular machinery necessary
for polarized extension of plasma membrane protrusions.
We then investigated whether DGKαwas involved in the regu-
lation of aPKCζ/ιdownstream of HGF signaling. Indeed, we ob-
served that HGF induced the recruitment of aPKCζ/ιto membrane
ruffles in a DGKα-dependent manner. In fact, both specificdown-
regulation of DGKαby two different siRNAs (Fig. 5Aand Fig.
S6A) and its pharmacological inhibition by R59949 cell treatment
(Fig. S6B) completely abolished HGF-induced recruitment of
aPKCζ/ιto the outer plasma membrane, indicating that enzymatic
activity of DGKa was required.
Moreover, expression of myr-DGKαwas sufficient to recruit
aPKCζ/ιto ruffling sites, as the percentage of cells displaying
this localization of aPKCζ/ιincreased 3-fold in myr-DGKα-
expressing cells compared to DGKαWT-expressing ones (Fig. 5B
and Fig. S6C). Accordingly, cell treatment with PA but not with
lysophosphatidic acid (LPA) or DAG was sufficient to promote
concurrent cortical actin rearrangements and plasma membrane
translocation of aPKCζ/ι, Rac, and RhoGDI (Fig. 5Cand Fig.
S7). Moreover, cell treatment with exogenous PA, but not with
DAG, induced aPKCζ/ιactivation, as revealed by the increase in
phosphorylation of the catalytic domain Thr410 (Fig. 5D).
Together, these data strongly support the idea that the gen-
eration of PA by DGKαis a crucial signal driving the recruitment
and activation of the molecular machinery necessary for exten-
sion of membrane protrusions.
As accumulating evidence suggests that the small GTPase Cdc42
acts upstream of PKCζand regulates it through the Par3/Par6
complex, thus driving cell polarity and directional migration (22),
we further verified whether DGKαmight regulate aPKCζ/ιvia
Cdc42. Indeed, the expression of Cdc42N17, although impairing
membrane ruffles induced by myr-DGKα, did not affect aPKCζ/ι
plasma membrane recruitment (Fig. S8). Thus, although Cdc42 is
required for ruffle formation induced by constitutive generation of
PA at the plasma membrane, it is not required for the recruitment
of aPKCζ/ι. Overall, these data indicate that DGKαacts upstream
of aPKCζ/ι, in a Cdc42-independent manner, by regulating its
recruitment to rufflingsitesthroughproductionofPA.
A
control R59949 HGF+R59949
RhoGDI
RhoGDI
+actin
HGF
*
ctr
% of cells with
RhoGDI at the
pl. membr.
*
RHH+R
0
10
20
30
40
50
B
ZOOM
DGK WT
RhoGDI
RhoGDI+DGK
+actin
myr-DGK
DGK
Fig. 3. DGKαregulatesRhoGDI targeting to the plasma membrane. (A) MDCK
cells were stimulated with 10 ng/mL HGF for 5 min in the presence or absence
of 1 μM R59949, fixed, and stained for RhoGDI (green) and actin (blue). Arrows
indicate RhoGDI at membrane ruffles. (Scale bar, 24 μm.) C, 70 cells, scored for
RhoGDI localization at ruffling sites. n= 3, with SEM; *P<0.05. (B) MDCK cells,
transfected with either DGKαWT or myr-DGKα,were cultured overnight in the
absence of serum, fixed, and stained for RhoGDI (green), myc tag (red), and
actin (blue). Arrowheads indicate transfected cells. (Scale bar, 10 μm.)
A
B
C
Fig. 4. aPKCζ/ιmediates both HGF- and myr-DGKα-induced extension of
membrane protrusions. (A) MDCK cells were transfected with three combi-
nations of PKCζ(79, 80) and PKCι(1–3) specific siRNAs. Whole-cell lysates
were analyzed for levels of aPKCζ/ιexpression by western blot. (B) MDCK
cells were transfected as in A, treated with 10 ng/mL HGF for 15 min, fixed,
and stained for Rac or RhoGDI and actin. C, 110 cells, analyzed for the
presence of ruffles and Rac or RhoGDI at the plasma membrane. n= 4 (Rac
and RhoGDI), n= 8 (ruffles), with SEM; **P<0.005, ***P<0.0005. (C) MDCK
cells were transiently transfected either with myr-DGKαalone or cotrans-
fected with myr-DGKαand PKCζKW, cultured overnight in the absence of
serum, fixed, and stained for myc and flag tags, actin, and Rac or RhoGDI. C,
20 transfected cells, scored for the presence of ruffles and Rac or RhoGDI at
protrusion sites. n= 4, with SEM; **P<0.001, ***P<0.0001.
4184
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We then investigated whether, by regulating the function of
aPKCζ/ι,DGKαmight control the dissociation of Rac/RhoGDI
complex. We immunoprecipitated RhoGDI from MDCK cells sta-
bly expressing either the control vector (MDCK/empty vector) or a
kinase-defective dominant-negative mutant of DGKα(DGKαDN).
Rac coimmunoprecipitated with RhoGDI both in MDCK/empty
vector and in MDCK/DGKαDN unstimulated cells. Upon HGF
stimulation, Rac was released from the complex with RhoGDI in
MDCK/empty vector cells, whereas expression of DGKαDN pre-
vented both dissociation of the complex (Fig. 6 and Fig. S9)andRac
activation (13). Collectively, the data presented above identify
DGKαas an upstream regulator of aPKCζ/ι.DGKα,byproducing
PA, provides the crucial signal for the recruitment at the plasma
membrane and activation of aPKCζ/ι, in complex with RhoGDI and
Rac, which finally dissociates in a DGKα-dependent manner,
thereby allowing Rac activation.
Finally, we tested the hypothesis that DGKαmediates the for-
mation of membrane ruffles by regulating aPKCζ/ι.Thus,we
verified whether transient expression of membrane-bound con-
stitutively active myr-PKCζcould overcome DGKαinhibition, as
expected for a downstream effector. Indeed, myr-PKCζ-expressing
cells featured an altered morphology with membrane protrusions
and ruffles, even in the absence of stimulation, and in 60% of them
RhoGDI was localized at the outer plasma membrane. Prolonged
(1-h) pharmacological inhibition of DGK activity by R59949 did
not affect either formation of membrane protrusions or RhoGDI
localization to the outer plasma membrane (Fig. 7), confirming that
aPKCζ/ιis responsible, downstream of DGKα, for the recruitment
of RhoGDI at the leading edge and consequent activation of the
molecular machinery necessary for extension of membrane ruffles.
Overall, our findings highlight that DGKα, by producing PA,
provides a crucial signal to recruit aPKCζ/ιand RhoGDI/Rac
complex and to activate aPKCζ/ι, thereby allowing Rac mem-
brane targeting and activation and consequent actin cytoskeleton
remodeling preliminary to cell migration.
Discussion
Epithelial cells at the periphery of a colony initiate migration by
extending membrane protrusions whose formation relies on re-
cruitment and activation of Rac to their tips, to harness and
localize actin polymerization (23). Spatially restricted activation of
Rac at nascent ruffles is triggered by coordinated signals provided
by both growth factors and adhesion receptors. Moreover, the
observation that in epithelial cells the expression of a constitutively
active form of Rac is not per se sufficient to promote ruffle for-
mation (15, 24) strongly indicates that a further localization signal,
A
B
C
D
Fig. 5. DGKαregulates aPKCζ/ιfunction. (A) MDCK cells were transfected
either with control siRNA or DGKαsiRNA c1 and c2, treated with 10 ng/mL
HGF for 15 min, fixed, and stained for aPKCζ/ι(green) and actin (red). (Scale
bar, 10 μm.) (B) MDCK cells, transfected either with DGKαWT or myr-DGKα,
were cultured overnight in the absence of serum, fixed, and stained for
aPKCζ/ι(green), myc tag (red), and actin (blue). Arrowheads indicate trans-
fected cells. (Scale bar, 10 μm.) (C) MDCK cells were stimulated with either
250 μM C8-PA, C8-DAG, or C6-LPA or left untreated, and fixed and stained
for actin (red) and aPKCζ/ι(green). Arrowheads indicate cortical actin rear-
rangements, while the arrow indicates aPKCζ/ιmembrane localization.
(Scale bar, 19 μm.) (D) MDCK cells were stimulated with either 250 μM C8-PA,
10% FCS medium (as positive control), 250 μM C8-DAG, or left untreated.
Whole-cell lysates were analyzed by western blot and the intensity of
phospho-aPKCζ/ιbands was quantified by densitometry. For each condition,
nine replicate points were performed in four independent experiments. The
densitometry of each band was normalized as the percentage of the den-
sitometry mean of control points in the same experiment, and is shown in
the histogram, with SEM; **P<0.005. A representative picture is shown.
-H
emptyvector
DGKDN
-H
RhoGDI
IB: RhoGDI
IB: Rac
IP:
IB: aPKC /
IB: RhoGDI
IB: aPKC /
IB: Rac
IB: myc
input
-H
emptyvector
DGKDN
-H
Fig. 6. DGKαregulates Rac/RhoGDI complex dissociation. MDCK/empty
vector or MDCK/DGKα-DN cells were stimulated with 50 ng/mL HGF for
15 min. Cell lysates were immunoprecipitated for RhoGDI and analyzed by
western blot.
Chianale et al. PNAS
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CELL BIOLOGY
provided by growth factors, is required to generate RacGTP
accumulation at protrusion sites. Starting from our initial obser-
vation that DGKαis required for HGF-induced RacV12 mem-
brane targeting, we unveil a signaling pathway by which HGF
regulates Rac localization to nascent ruffles through membrane
recruitment of DGKα, aPKCζ/ι, and RhoGDI.
We previously showed that, upon HGF stimulation, activation
and membrane recruitment of Rac, as well as ruffle formation,
are fully dependent on DGKα(13). Our finding that expression
of a myristoylated mutant of DGKαpromoted ruffle formation
and Rac recruitment at protrusion sites, in the absence of growth
factor, strongly suggests that activation of DGKαat the plasma
membrane provides a crucial signal to regulate Rac function.
Previous evidence underscored the role of RhoGDI in regu-
lating the dynamics of Rac targeting to the plasma membrane,
where Rac dissociates from the inhibitory complex with RhoGDI
to interact with its downstream effectors (2, 18). Our observation
that in HGF-treated cells RhoGDI is recruited to nascent ruffles,
and that a Rac mutant unable to bind to RhoGDI is not, provides
further support to the claim that the interaction between Rac and
RhoGDI is necessary for Rac membrane targeting. Moreover, the
present data demonstrate that growth factors promote Rac
membrane localization by regulating RhoGDI targeting. We show
that DGKαat the plasma membrane provides a key lipid signal
necessary and sufficient to recruit RhoGDI. Furthermore, the
failure to detect DGKαin a complex with RhoGDI and Rac
suggests that DGKα, rather than acting as a scaffolding protein,
may recruit RhoGDI through its enzymatic activity. However,
RhoGDI does not feature any clear domain responsible for
membrane binding, suggesting that the lipid signal generated
by DGKαmay recruit RhoGDI through an interacting lipid-
binding protein.
Recent evidence indicates that PKCζassociates with RhoGDI
and regulates the dissociation of Rac/RhoGDI complex, thereby
allowing Rac activation (21). Moreover, PKCιregulates invasive
behavior by activating Rac in lung cancer cells (25). Interestingly,
PA, the lipid product of DGKαactivity, has been reported to bind
directly to PKCζand to stimulate its enzymatic activity (20),
whereas it was recently shown that DGKαenhances PKCζ-
mediated phosphorylation of p65/Rel (26). Here we show that
upon HGF stimulation, DGKα-generated PA is a necessary and
sufficient signal to recruit aPKCζ/ιat protrusion sites and activate
it, thereby promoting Rac and RhoGDI membrane targeting.
Kuribayashi et al. showed that PKCζ, associated in a complex
with RhoGDI, mediates its phosphorylation on threonine, thereby
allowing Rac release (21). Based on this observation and on our
data, we expected that Rac release from the complex with RhoGDI
depended on DGKαactivity. Consistently, in thepresent report, we
show that DGKαenzymatic activity mediated HGF-induced Rac
release from RhoGDI, although we could not detect any threonine
phosphorylation of endogenous RhoGDI. Finally, these results
suggest the following working model: Upon HGF stimulation, Src-
mediated activation of DGKα, once targeted to the plasma mem-
brane, results in accumulation of PA, whichdirects the recruitment
of aPKC/RhoGDI/Rac complex at protrusion sites, likely through
the interaction of DGKα-produced PA with the C1-like domain of
aPKCζ/ι, which is finally activated. Thus, aPKCζ/ιis identified as
the direct downstream effector of DGKα. Once at the plasma
membrane, aPKCζ/ιmay allow Rac release from RhoGDI, likely
through RhoGDI phosphorylation. This model is confirmed by the
finding that expression of myr-PKCζ, in the absence of growth
factors, recapitulates both RhoGDI recruitment and protrusion
formation in a DGKα-independent manner, providing further
support to the tenet that DGKαregulates RhoGDI and Rac by
acting upstream of aPKCζ/ι(Fig. 8).
aPKC is recruited to the plasma membrane through binding to
active Cdc42 (27) or by directed interaction with ceramide in a
Cdc42-independent manner (28). Our finding that Cdc42 is dis-
pensable for aPKCζ/ιrecruitment induced by myr-DGKαsuggests
that Cdc42 function is not required for lipid-mediated aPKCζ/ι
membrane targeting, albeit necessary for ruffle formation down-
stream of DGKα.
As RhoGDI also controls Cdc42, we may speculate that acti-
vation of DGKαprovides a localization signal driving plasma
membrane recruitment of aPKCs in complex with RhoGDI and
Rac or Cdc42, eventually leading to the activation of Cdc42 and
Rac, which may further recruit aPKCs in a Cdc42-dependent
manner. This might establish a feed-forward mechanism that
allows the full activation of the molecular machinery driving
actin polymerization and consequent formation of membrane
protrusions.
PAK1-mediated serine phosphorylation of RhoGDI promotes
the selective release of Rac downstream of Cdc42 activation
(29). Recently, DGKζwas shown to regulate PAK1-mediated
phosphorylation of RhoGDI, leading to selective activation of
Rac but not of Cdc42 (30). Conversely, PKCζ-dependent
phosphorylation of RhoGDI promotes the release of both Rac
and Cdc42 (21). Thus, it seems that DGKαand DGKζ, by reg-
ulating, respectively, aPKCζ/ιand PAK1, are involved in two
myr-aPKC /RhoGDI
RhoGDI
ZOOM
Myr-aPKC /+
RhoGDI+actin
control
R59949
0
20
40
60
80
100
protrusions
Pl.membr.
RhoGDI
% of cells
vehicle
R59949
Fig. 7. DGKαdoes not affect myr-PKCζ-induced events. MDCK cells were
transiently transfected with myr-PKCζ, grown overnight in the absence of
serum, and treated with 1 μM R59949 for 1 h. Cells were fixed and stained for
flag tag (red), RhoGDI (green), and actin (blue). Arrows indicate RhoGDI
staining at protrusion sites. (Scale bar, 24 μm.) C, 30 transfected cells, scored for
the presence of protrusions and RhoGDI at protrusion sites. n= 3, with SEM.
HGF
•actin
polymerization
•ruffling
•migration
Src
Src
P
RhoGDI
Rac
GDP
aPKC/
PA
PA
DGK
DGK
DAG
RhoGDI
aPKC/
Rac
GTP
P
PA
Fig. 8. Model proposed for Rac-localized activation at the leading edge
upon growth-factor stimulation. Upon growth-factor stimulation, DGKαis
activated in a Src-dependent manner and recruited to the plasma mem-
brane. The production of PA is the crucial signal to direct the recruitment of
aPKCζ/ι, in complex with RhoGDI and Rac. PKCζ/ι, in turn, mediates the dis-
sociation of Rac from the inhibitory complex with RhoGDI, which may
become prone to activation by a RacGEF.
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distinct molecular mechanisms leading to the control of small-
GTPase function.
In conclusion, our findings constitute a coherent demon-
stration that DGKα, upon Src-mediated activation, acts as a
positive transducer of growth-factor signaling by producing PA
rather than removing DAG. Here we unveil a PA-mediated
signaling pathway linking tyrosine kinase receptors to Rac acti-
vation, membrane ruffling, and cell migration. In particular, we
highlight a pivotal role for a DGKα-aPKCζ/ι-RhoGDI axis in the
regulation of the initial events leading to activation of Rac at the
leading edge of migrating cells.
Materials and Methods
Cell Stimulation. Before any treatment, cells were cultured overnight in the
absence of serum. In the case of R59949 cell treatment, a 15-min pretreat-
ment with R59949, or vehicle alone, was performed.
Immunofluorescence. Culturing, fixing, and staining detailed procedures are
described in SI Materials and Methods and ref. 13. Representative pictures
are shown in the figures.
Statistical Analysis. In experiments involving counting of cells displaying
ruffles, membrane protrusions, or localization of proteins at the outer plasma
membrane (not at cell-cell contacts), only colony-edge cells were considered.
The average number of cells scored for each condition in each experiment is
indicated in figure legends by “C.”Statistical analysis was performed con-
sidering nindependent experiments (indicated in the figure legends). The P
values were calculated by one-tailed Standard ttest.
Further details are provided in SI Materials and Methods.
ACKNOWLEDGMENTS. This work was supported by Associazione Italiana per
la Ricerca sul Cancro, Ministero dell’Istruzione, dell’Università e della Ricerca,
PRIN Program 2007, and Regione Piemonte (Ricerca Sanitaria Finalizzata).
F.C. is funded by Regione Piemonte (L.R. 4/2006 for brain drain containment,
D.R. 392-2007). We thank Paola Chiarugi, Celine DerMardirossian, Louise
Hodgson, Giorgio Scita, and Alex Toker for providing reagents.
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