The Actin-Bundling Protein Palladin
Is an Akt1-Specific Substrate
that Regulates Breast Cancer Cell Migration
Y. Rebecca Chin1and Alex Toker1,*
1Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
The phosphatidylinositol 3-kinase (PI3K) signaling
pathway is frequently deregulated in cancer. Down-
stream of PI3K, Akt1 and Akt2 have opposing roles
in breast cancer invasive migration, leading to meta-
static dissemination. Here, we identify palladin, an
actin-associated protein, as an Akt1-specific sub-
strate that modulates breast cancer cell invasive
migration. Akt1, but not Akt2, phosphorylates palla-
din at Ser507 in a domain that is critical for F-actin
bundling. Downregulation of palladin enhances
migration and invasion of breast cancer cells and
induces abnormal branching morphogenesis in
3D cultures. Palladin phosphorylation at Ser507 is
required for Akt1-mediated inhibition of breast can-
cer cell migration and also for F-actin bundling,
leading to the maintenance of an organized actin
cytoskeleton. These findings identify palladin as an
Akt1-specific substrate that regulates cell motility
and provide a molecular mechanism that accounts
for the functional distinction between Akt isoforms
in breast cancer cell signaling to cell migration.
Metastasis, one of the hallmarks of human solid tumors, is
orchestrated by multiple signaling pathways that regulate cell
proliferation, survival, metabolism, migration, and angiogenesis.
Recent studies have revealed that the phosphatidylinositol
3-kinase (PI3K)/Akt signaling cascade is one of the most fre-
quently deregulated pathways in cancer, particularly breast
carcinoma (Altomare and Testa, 2005; Engelman et al., 2006).
The Akt family members, Akt1 (also known as PKBa), Akt2
(PKBb), and Akt3 (PKBg), play pivotal roles in cellular functions
that are associated with all stages of cancer, including progres-
sion to metastasis (Chin and Toker, 2009; Woodgett, 2005).
Although both Akt1 and Akt2 promote cancer cell survival and
growth, they exert distinct effects on breast cancer cell invasive
migration and metastasis (Chin and Toker, 2009). In this
context, Akt1 has been shown to promote tumor induction
but, somewhat paradoxically, inhibit invasion and metastasis
(Hutchinson et al., 2004; Irie et al., 2005; Liu et al., 2006; Marou-
lakou et al., 2007; Yoeli-Lerner et al., 2005). Conversely, Akt2
enhances invasive migration and metastasis in vivo (Arboleda
et al., 2003; Irie et al., 2005). A number of distinct effector path-
ways have been shown to mediate the distinct effects of Akt1
and Akt2 on breast cancer cell invasion. Akt1 blocks breast
cancer cell migration by promoting degradation of the transcrip-
tion factor NFAT (nuclear factor of activated T cells) (Yoeli-
Lerner et al., 2005). Akt1 also attenuates cell migration by
regulating extracellular signal-regulated kinase/mitogen-acti-
vated protein kinase (ERK/MAPK) (Irie et al., 2005) and tuberous
sclerosis complex 2 (TSC2) pathways (Liu et al., 2006). In con-
trast, Akt2 but not Akt1 upregulates b1 integrins, thereby
promoting invasion of breast cancer cells in vitro as well as
metastasis in vivo (Arboleda et al., 2003). However, to date,
the immediate isoform-specific substrates that modulate cell
migration in an Akt isoform-specific manner have not been
Palladin is an actin-binding and crosslinking protein that
controls the organization of cellular actin networks (Dixon
et al., 2008). Palladin localizes in areas of actin stress fiber-
dense regions and focal adhesions (Parast and Otey, 2000).
Palladin also functions as a molecular scaffold by linking
several anchor proteins to actin fibers, including profilin (Bou-
khelifa et al., 2006), VASP (Boukhelifa et al., 2004), a-actinin
(Ro ¨nty et al., 2004), Eps8 (Goicoechea et al., 2006), and ezrin
(Mykka ¨nen et al., 2001). Studies have revealed palladin overex-
pression in human breast tumor tissues (Goicoechea et al.,
2009) and invasive rat mammary tumor cells (Wang et al.,
2004). However, the mechanisms that regulate the function of
palladin in cytoskeletal reorganization and cell motility remain
Here, we report the identification of palladin as a specific
substrate of Akt1. Akt1, but not Akt2, phosphorylates palladin
at Ser507 in vitro and in cells. Downregulation of palladin by
small hairpin RNA (shRNA) enhances invasive migration and
disrupts spheroid morphogenesis, indicating an antimigratory
and anti-invasive function for palladin in breast cancer cells.
Phosphorylation of palladin plays a critical role in inhibiting
breast cancer cell motility and promoting actin-bundling activity.
Taken together, these data identify palladin as an Akt isoform-
specific substrate that contributes to differential regulation of
breast cancer cell migration.
Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc. 333
Akt Phosphorylates Palladin at Ser507 In Vitro
and in Cells
Recent phosphoproteomic studies have revealed phosphoryla-
tion of palladin at Ser507 in a consensus sequence that con-
forms to the optimal Akt phosphorylation motif (RXRXXS/T)
(Obata et al., 2000; Olsen et al., 2006; Ville ´n et al., 2007)
(Figure 1A). To determine whether palladin is an Akt substrate,
we transfected hemagglutinin (HA)-tagged palladin into HeLa
cells and stimulated cells with insulin-like growth factor-1
(IGF-1) to activate endogenous PI3K and Akt. Immunoprecipi-
tated palladin was immunoblotted with an antibody that recog-
nizes the Akt consensus phosphorylation motif (Figure 1B).
attenuates IGF-1-induced palladin phosphorylation (Figure 1B).
HeLa cells were also cotransfected with a constitutively active
Figure 1. Akt Phosphorylates Palladin at Ser507
(A) Schematic of palladin showing the position of the putative Akt consensus phosphorylation site at Ser507. Amino acid sequences of Akt motifs in other known
Akt substrates are shown for comparison. The Ser507 Akt motif in palladin is evolutionarily conserved. Numbers on the left of sequences indicate the position of
Ser in the Akt motif. PP, polyproline; Ig, immunoglobulin-like domain.
(B) HeLa cells were transfected with HA-palladin or control vector for 9 hr, then serum starved for 12–16 hr. Cells were then stimulated with IGF-1 (100 ng ml?1)
for 20 min in the presence of Wortmannin (100 nM), Akt inhibitor SN30978 (5 mM), or DMSO. Cell extracts were immunoprecipitated with anti-HA antibody and
immunoblotted with the indicated antibodies.
(C) HeLa cells were transfected with wild-type HA-palladin (HA-palladin WT) or HA-palladin Ser507Ala mutant or control vector for 9 hr, followed by
serum starving for 12–16 hr. Cells were then stimulated with IGF-1 (100 ng ml?1) for 20 min. Palladin was precipitated from whole-cell lysates followed by
(D) HeLa cells were transfected with HA-palladin WT, HA-palladin Ser507Ala, or control vector in serum-free media for 24 hr. Anti-HA immunoprecipitates were
used in in vitro assays with recombinant active Akt1 (Recom. Akt1). The kinase reaction was terminated and samples were immunoblotted. All results are repre-
sentative of three independent experiments. See also Figure S1.
Palladin Phosphorylation Regulates Cell Migration
334 Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc.
myristoylated Akt1 allele (Myr-Akt1) and green fluorescent
protein (GFP)-tagged palladin. Palladin coimmunoprecipitates
with Myr-Akt1, indicative of an association (Figure S1A). Fur-
thermore, GFP-palladin is phosphorylated by Myr-Akt1 in cells,
suggesting that active Akt1 alone is sufficient to stimulate palla-
din phosphorylation. Other AGC kinases downstream of PI3K,
et al., 2008) and S6 kinase-1 (S6K1), which share the Akt con-
sensus phosphorylation motif, do not signal to palladin, since
treatment with the mTOR (mammalian target of rapamycin)
inhibitor rapamycin has no effect on palladin phosphorylation
Akt phosphorylates palladin at Ser507, since a Ser507Ala
mutant is not phosphorylated in IGF-1-stimulated cells when
compared to wild-type palladin (Figure 1C). To investigate
whether palladin is a direct substrate of Akt1, purified recombi-
nant wild-type and Ser507Ala palladin was incubated with
purified recombinant active Akt1 protein in an in vitro pro-
tein kinase assay. Akt1 efficiently phosphorylates wild-type pal-
ladin, whereas the Ser507Ala mutant is not phosphorylated
To determine the relevance of palladin phosphorylation by
Akt in breast cancer, we first evaluated palladin expression in
a panel of breast cancer cell lines. Figure 2A shows that palladin
is expressed in all breast cancer cell lines examined. As with
HeLa cells, palladin is phosphorylated in an IGF-1- or EGF-
and Akt-dependent manner in MDA-MB-231 and SKBR3 breast
cancer cell lines (Figure 2B). In immortalized nontumorigenic
Figure 2. Phosphorylation of Palladin in Breast Cancer Cells
(A) Analysis of palladin expression in various cell lines by immunoblotting.
(B)MDA-MB-231and SKBR3cells weretransfectedwithHA-palladinfor 9hrthenserumstarvedfor 12–16hr. Cellswerethenstimulated withIGF-1(100ngml?1)
for 20 min or EGF (20 ng ml?1) for 10 min in the presence of SN30978 (5 mM) or DMSO. Cell extracts were immunoprecipitated with anti-HA antibody followed by
(C) Serum-starved MCF10A and BT-549 cells were stimulated with EGF (20 ng ml?1) for 10 min in the presence of Wortmannin (100 nM) or SN30978 (5 mM), or
DMSO. Endogenous palladin was immunoprecipitated with anti-palladin antibody and immunoblotted with a-pAkt-MOTIF antibody.
(D) MCF10A cells infected with empty vector or retroviral vectors expressing the indicated HA-p110a variants were serum starved overnight. Phosphorylation of
endogenous palladin was detected as described in (C). * denotes a nonspecific band. All results are representative of three independent experiments.
Palladin Phosphorylation Regulates Cell Migration
Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc. 335
MCF10A breast epithelial cells, inhibition of PI3K/Akt signaling
by Wortmannin or SN30978 impairs EGF-mediated phosphory-
lation of endogenous palladin (Figure 2C). Similar results were
obtained in a phosphatase and tensin homolog (PTEN) null
breast cancer cell line, BT-549. Similarly, we evaluated phos-
phorylation of palladin in cells expressing the two oncogenic
hotspot mutations in the p110a catalytic subunit (PIK3CA)
(Saal et al.,2005). Both H1047R and E545K mutant p110aalleles
induce hyperactivation of Akt as measured by pSer473 phos-
phorylation (Figure 2D). Importantly, this is accompanied by
hyperphosphorylation of palladin with both mutant alleles.
Therefore, palladin is phosphorylated in breast cancer cells in
response to both physiological stimuli and oncogenic activation
of the PI3K pathway.
Palladin Is an Akt1-Specific Substrate
We next determined if palladin is an Akt isoform-specific sub-
strate. First, cells were cotransfected with GFP-palladin and
HA-Myr-Akt1 or HA-Myr-Akt2. In spite of the high amino acid
similarity between Akt isoforms, Akt1 but not Akt2 coimmuno-
precipitates with palladin in all three cell lines tested (Figure 3A).
Consistent with this, palladin is specifically phosphorylated
by Akt1 but not Akt2 in an in vitro kinase assay (Figure 3B).
In contrast, the Akt substrate GSK3b is efficiently phosphory-
lated by both Akt isoforms (Figure S2A). To evaluate isoform
specificity in cells, small interfering RNA (siRNA) targeting Akt1
or Akt2 were introduced into HeLa or SKBR3 cells. Knockdown
of Akt1 significantly attenuates growth factor-induced palladin
phosphorylation (Figure 3C). Conversely, depletion of Akt2 has
no effect. Similar results are observed in the PTEN-deficient
breast cancer cell line BT-549 (Figure 3C). These findings dem-
onstrate that palladin is an Akt1-specific substrate. Since Akt3
expression is minimal or below the detection limits in HeLa cells
(Zinda et al., 2001) and SKBR3 cells (Figure S2B), this Akt iso-
form cannot account for palladin phosphorylation in these cell
lines. Conversely, although Akt3 is expressed in BT-549 cells
(Figure S2C), it is unlikely to contribute to palladin regulation
since Akt1 shRNA completely eliminates phosphorylation.
To determine the domain or region in Akt1 that determines
isoform specificity toward palladin phosphorylation, Akt1 and
Akt2 chimeras were used (Zhou et al., 2006). Palladin phos-
phorylation is observed in cells expressing the Akt chimera
containing the PH and linker domains of Akt1 (1122), whereas
phosphorylation is significantly lower in the presence of Akt
chimera 2211, which contains PH and linker domains of Akt2
of Akt1 are important determinants for palladin phosphoryla-
tion. The Akt chimera 2111 contains the same domains as
the chimera 2211, with the exception of the linker region that
originates from Akt1. Palladin is phosphorylated efficiently in
cells expressing the chimera 2111, but not 2211 (Figure 3D).
This indicates that the linker region of Akt1 plays an important
role in determining the Akt isoform-specific function of palladin
Palladin Inhibits Breast Cancer Cell Invasive Migration
localization. In serum-starved cells, palladin is distributed evenly
in the cytoplasm with a minor colocalization with cortical actin
(Figure 4A). Upon IGF-1 stimulation, we observe an increased
accumulation and colocalization of palladin and actin at sites
of membrane ruffling. In contrast, this colocalization is reduced
upon inhibition of PI3K signaling with the inhibitor LY294002.
Wenext determined the function of palladin in modulating breast
cancer cell migration. ShRNA sequences specific to palladin
were generated (Ro ¨nty et al., 2007), and efficient silencing was
evaluated with a specific palladin antibody (Figure S3A). Chemo-
tactic cell motility was assessed using Transwell migration
assays. In both highly invasive (MDA-MB-231 and SUM-159-
PT) and poorly invasive (MCF-7) breast cancer cell lines as well
as MCF10A cells, knockdown of palladin results in enhanced
migration (Figure 4B), suggestive of an antimigratory function
of palladin in breast cancer cells. Consistent with this, expres-
sion of palladin in MCF-7 cells results in decreased migration
(Figure S3B). Moreover, MCF10A cells expressing wild-type
or oncogenic PIK3CA exhibit increased cell migration (Fig-
ure S3C). This is consistent with previous studies that have
demonstrated enhancement of MCF10A and MDA-MB-231 cell
migration mediated by oncogenic PIK3CA (Pang et al., 2009;
Zhang et al., 2008). Silencing of palladin enhances migration of
cells with both wild-type and mutant p110a alleles (Figure S3C).
Furthermore, in Matrigel invasion assays, palladin silencing also
results in increased invasion of breast cancer cells (Figure S3D).
This was further corroborated using 3D cultures. Whereas
control cells are able to form normal 3D spheroids, palladin
knockdown cells display abnormal branching morphogenesis
with protrusions invading into Matrigel (Figure 4C).
Akt1 Phosphorylation of Palladin Blocks Migration
of Breast Cancer Cells
As enhanced migration upon palladin silencing phenocopies
downregulation of Akt1 (Figure S4A), we next examined if
inhibition of migration by the Akt1 pathway is mediated through
palladin phosphorylation. In agreement with recent studies
(Yoeli-Lerner et al., 2005), expression of activated Myr-Akt1
blocks migration of MDA-MB-231 cells (Figure 5A). This effect
is rescued by silencing palladin with shRNA. To determine if
this occurs under physiological signaling conditions, IGF-1
stimulation was used to activate endogenous Akt, resulting in
decreased cell migration, as previously demonstrated (Yoeli-
Lerner et al., 2005) (Figure 5B). Downregulation of palladin in
serum-free conditions has minimal effect on migration (Fig-
ure 5B). In contrast, IGF-1-induced inhibition of migration is
completely rescued by palladin silencing. Similarly, combined
downregulation of palladin and Akt1 has little or no addictive
effect on migration (Figure 5C), further demonstrating that the
inhibitory effects of Akt1 on migration are mediated, at least in
part, by palladin. To determine if palladin phosphorylation by
Akt1 is critical for the migration phenotype, an shRNA-resistant
WT palladin allele was generated (HA-palladin WT*) along with
an Ser507Ala mutant (HA-palladin Ser507Ala*) and reintroduced
into palladin-depleted MDA-MB-231 cells. Whereas HA-palladin
WT* effectively reverses the migratory effect induced by palladin
shRNA, HA-palladin Ser507Ala* does not rescue (Figure 5D).
Inaddition,palladin phosphorylation ishigher innontumorigenic,
noninvasive MCF10A breast epithelial cells when compared to
Palladin Phosphorylation Regulates Cell Migration
336 Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc.
invasive and metastatic breast cancer cell lines SUM-159-PT
and MDA-MB-231 (Figure S4B). This observation is consistent
with an inhibitory function of palladin phosphorylation in cell
migration. Taken together, these data demonstrate that palladin
phosphorylation at Ser507 is required for Akt1-mediated inhibi-
tion of migration.
Figure 3. Palladin Is an Akt1-Specific Substrate
(A) HeLa, SKBR3, and MDA-MB-231 cells were transfected with HA-Myr-Akt1, HA-Myr-Akt2, or empty vector, along with GFP-palladin. Twenty-four hours after
transfection, cells were lysed and immunoprecipitated with anti-GFP. Whole-cell lysates and immunoprecipitates were subjected to immunoblotting.
(B) SKBR3 cells were transfected with HA-palladin WT in serum-free medium for 24 hr. Anti-HA immunoprecipitates were used as substrates in in vitro kinase
assays with recombinant active Akt1 or Akt2. The kinase reaction was terminated and samples were immunoblotted.
(C) HeLa and SKBR3 cells were cotransfected with HA-palladin and Akt1, Akt2, or control luciferase siRNA. Thirty-six hours after transfection, HeLa and SKBR3
cells were serum starved for 12 hr, then treated with IGF-1 (100 ng ml?1) for 20 min and EGF (20 ng ml?1) for 10 min, respectively. BT-549 cells were infected with
Akt1 or Akt2 shRNA lentiviral vector or empty vector for 72 hr, followed by serum starvation and then stimulation with EGF (20 ng ml?1) for 10 min. Lysates were
subjected to immunoprecipitation and immunoblot analysis.
(D) HeLa cells were infected with retroviral vectors expressing Akt chimeras, followed by transfection with GFP-palladin for 24 hr. Cell extracts were immuno-
precipitated with anti-GFP antibody and immunoblotted with the indicated antibodies. Akt has four domains: PH, linker, catalytic, and C-terminal regulatory
domains.Aktchimera 1122containsPHand linkerregions of Akt1pluscatalytic and regulatory domains of Akt2.Aktchimera 2211containsPHandlinker regions
of Akt2 plus catalytic and regulatory domains of Akt1. Akt chimera 2111 contains PH domain of Akt2 plus linker, catalytic, and regulatory domains of Akt1.
All results are representative of three independent experiments. See also Figure S2.
Palladin Phosphorylation Regulates Cell Migration
Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc. 337
Since Transwell assays measure directional migration, we
next evaluated the role of palladin phosphorylation in nondirec-
tional migration using time-lapse video microscopy. MDA-MB-
231 cells were plated on NIH 3T3 cell-secreted extracellular
matrix (ECM), and migration was monitored 1 hr subsequent to
plating. Control cells spread uniformly on ECM and migrate
slowly (Figures 6A and 6B and Movie S1). In contrast, silencing
of palladin results in a distinct morphology with reduced areas
of spreading and diminished continuity of lamellipodia. In addi-
tion, these cells have multiple protrusions resulting in a stellate
appearance and also display significantly increased random
migration (Figures 6A and 6B and Movie S2). Reintroduction of
wild-type palladin resistant to silencing (WT*) reverses the
morphology and motility to that observed with control cells
Figure 4. Palladin Inhibits Breast Cancer Cell Invasive Migration
(A) MDA-MB-231 cells were stimulated with IGF-1 (100 ng ml?1) for 20 min
in the presence of DMSO or LY294002 (10 mM). Immunofluorescence was
performed using anti-palladin antibody and Alexa Fluor 488-conjugated
phalloidin. Cell nuclei were labeled with DAPI.
(B) MDA-MB-231, MCF7, SUM-159-PT, and MCF10A cells were infected
with palladin shRNA lentiviral vector or empty vector for 48 hr, followed by
Transwell migration assays. Relative migration (y axis) = ratio of the
number of migrated cells in test versus control. Error bars represent
mean ±SEM. Total cell lysates were subjected to immunoblot analysis.
(C) MCF10A cells infected with palladin shRNA lentiviral vector or empty
pLKO vector were grown in 3D cultures for 8 days. Lysates were immuno-
blotted withtheindicated antibodies.Allresultsarerepresentativeofthree
independent experiments. See also Figure S3.
ing HA-palladin Ser507Ala* display the stellate morphology
and enhanced motility that mimics that observed in palla-
din-depleted cells (Figures 6A and 6B and Movie S4). These
results demonstrate that phosphorylation of palladin regu-
lates both random and directional migration of breast cancer
Palladin Phosphorylation Modulates Cytoskeletal
Organization and F-Actin Bundling
Palladin has been shown to directly bind F-actin (Dixon et al.,
2008). The Akt phosphorylation site Ser507 resides in a
domain that is critical for crosslinking actin filaments into
bundles. We therefore tested if palladin phosphorylation
modulates actin organization. In control cells, the actin cyto-
skeleton is highly organized (Figure 7A). However, palladin
silencing results in disruption of actin stress fibers. Reintro-
reverses the highly organized actin cytoskeletal phenotype,
indicating an important role of palladin phosphorylation
in actin dynamics (Figure 7A). To further dissect the mecha-
nism by which palladin modulates in actin reorganization,
cosedimentation assays were performed. Lysates of palla-
din-depleted MDA-MB-231 cells expressing HA-palladin
variants were incubated with purified F-actin, followed by
differential centrifugation to cosediment palladin with actin
filaments and actin bundles. Wild-type and Ser507Ala
mutant palladin cosedimented equally efficiently with actin
filaments (Figure 7B), suggesting that phosphorylation of
palladin is not essential for actin binding. Conversely, in bundling
assays, wild-type palladin cosediments efficiently, but this is
reproducibly reduced with the Ser507Ala mutant. These results
indicate that palladin phosphorylation functions to control
F-actin crosslinking, consistent with the enhanced migration
phenotype observed upon palladin silencing.
Since invasive cancer cells form actin-rich invadopodia to
degrade the ECM during invasion (Artym et al., 2009), we exam-
inedif palladin phosphorylation regulates formation ofinvadopo-
dia. Palladin silencing significantly increases the percentage of
MDA-MB-231 cells with invadopodia (Figure S5), indicating
that palladin inhibits invadopodial formation. However, introduc-
tion of both wild-type (WT*) and Ser507Ala* mutant palladin
alleles reverses the percentage of invadopodia-containing cells
Palladin Phosphorylation Regulates Cell Migration
338 Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc.
to values observed with control cells (Figure S5). These results
indicate that although palladin functions to control invadopodia
formation, Ser507 phosphorylation does not play a role in this
phenotype. Instead, the role of palladin phosphorylation by
Akt1 may be restricted to the regulation of F-actin crosslinking,
as discussed above.
The essential role of Akt in cancer cell growth and survival has
made it an attractive target for the development of anticancer
therapeutics (Hennessy et al., 2005). However, recent studies
that have revealed opposing functions of Akt1 and Akt2 in the
regulation of carcinoma migration leading to metastatic dissem-
ination have necessitated a reevaluation of inhibition of Akt in
cancer therapy (Chin and Toker, 2009; Sawyers, 2006). Thus
far, Akt isoform-specific substrates and molecular mechanisms
that are responsible for this distinction have remained elusive.
Indeed, despite over 150 Akt substrates that have been charac-
terized to date, only a few have been evaluated for isoform spec-
ificity. These include the cell cycle regulators p21 CIP1 (He ´ron-
Milhavet et al., 2006) and SKP2 (Gao et al., 2009) that are
Akt1-specific targets, whereas MDM2 (Brognard et al., 2007)
and AS160 (Bouzakri et al., 2006; Gonzalez and McGraw,
2009) are specifically phosphorylated by Akt2. However, none
of these accounts for the differential effects of Akt isoforms on
In the present study, we have identified palladin, an actin-
associated protein, as an Akt1 substrate. Phosphorylation of
palladin by PI3K and Akt1 signaling by physiological stimuli
such as IGF-1, as well as by genetic mutations in the pathway,
such as PTEN loss and oncogenic PIK3CA, supports a role for
this Akt1 target in both physiological and pathophysiological
signaling. The results presented here also provide evidence for
a functional role for palladin in invasive migration of breast
cancer cells and highlight the critical role of palladin phosphory-
lation in regulating migration and actin bundling. Despite the
high sequence and structural homology among Akt isoforms,
Akt1, but not Akt2, interacts with and phosphorylates palladin.
Substrate selectivity of isoforms can be achieved by several
potential mechanisms, including subcellular compartmentaliza-
tion, binding to distinct scaffolding molecules, and differential
activation or regulation by extracellular stimuli. Indeed, it has
recently been shown that in insulin-stimulated adipocytes, Akt2
preferentially accumulates at the plasma membrane and specif-
ically phosphorylates the substrate AS160 to regulate GLUT4
trafficking (Gonzalez and McGraw, 2009). However, differential
compartmentalization of Akt isoforms is unlikely to account for
theexclusivity ofpalladin phosphorylation byAkt1, sincepurified
Akt1, but not Akt2, phosphorylates palladin in a cell-free system.
It is more likely that an intrinsic molecular determinant or
Figure 5. Phosphorylation of Palladin at Ser507 by Akt1 Inhibits Cell Migration
(A) MDA-MB-231 cells infected with palladin shRNA or empty lentiviral vectors were transfected with HA-Myr-Akt1 or control vector. Twenty-four hours after
transfection, cells were subjected to Transwell migration assays, and lysates were immunoblotted with the indicated antibodies.
(B) MDA-MB-231 cells were infected with palladin shRNA lentiviral vector or empty vector. Forty-eight hours after infection, cells were serum starved overnight,
then stimulated with IGF-1 (100 ng ml?1) for 18 hr, followed by Transwell migration assays. Total cell lysates were subjected to immunoblot analysis.
(C) MDA-MB-231 cells were infected with palladin shRNA and/or Akt1 shRNA lentiviral vectors or empty vector. Forty-eight hours after infection, cells were
subjected to Transwell migration assays, and lysates were immunoblotted.
(D) MDA-MB-231 cells were infected with palladin shRNA lentiviral vector or empty vector for 48 hr. Cells were then transfected with shRNA-resistant (*) HA-pal-
ladin WT* or HA-palladin Ser507Ala mutant* or control vector. Twenty-four hours after transfection, cells were subjected to Transwell migration assays. Cell
lysates were immunoblotted with anti-HA and anti-actin. All Transwell migration data are represented as mean ±SEM. All results are representative of three inde-
pendent experiments. See also Figure S4.
Palladin Phosphorylation Regulates Cell Migration
Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc. 339
‘‘docking motif’’ on Akt1 promotes its association with palladin.
The data from the Akt chimera studies indicate that the linker
region of Akt1 plays an important role in determining the isoform
specificity of palladin phosphorylation. Interestingly, Field and
colleagues have identified the same region as an important
determinant in the ability of Akt1 and Akt2 to form dorsal ruffles
during fibroblast migration (Zhou et al., 2006). The precise resi-
dues within the linker region that are responsible for this isoform
specificity are yet to be determined. Regardless, these data
point to an important function of the linker region in modulating
palladin phosphorylation by the Akt1 pathway.
Our finding that palladin has an antimigratory role in breast
cancer cells differs from a recent study in which palladin knock-
down by siRNA inhibited invasive migration in Transwell assays
(Goicoechea et al., 2009). The distinction between the two
studies could be explained by the different migration conditions
or chemoattractants used. In the present study, results obtained
with Transwell assays were corroborated using time-lapse video
microscopy as well as 3D cultures. In all cases, cells expressing
palladin shRNA exhibited highly migratory and invasive pheno-
types in four distinct breast cancer cell lines. Most importantly,
rescue experiments revealed that overexpression of palladin is
able to reverse the migratory effect induced by palladin shRNA,
further demonstrating the specificity of the approach and con-
firming the inhibitory function of palladin phosphorylation on
invasive migration of breast cancer cells.
phorylation modulates its actin regulatory activity. We show that
phosphorylation of palladin promotes actin bundling, consistent
with the observation that the Akt motif at Ser507 resides in
a domain that is critical for crosslinking actin (Dixon et al.,
2008). We propose that by promoting the formation of actin
bundles and reorganizing the actin cytoskeleton, palladin phos-
phorylation inhibits cell migration. It is also worth noting that
a link between actin filaments and integrin receptors plays a
crucial role in dictating the organization and stability of adhe-
sions during cell migration (Vicente-Manzanares et al., 2009).
Polymerization of actin fibers regulates clustering of activated b1
integrins at the leading edge of migrating fibroblasts (Galbraith
et al., 2007). Interestingly, palladin can stabilize b1 integrins in
fibroblasts (Liu et al., 2007). Since palladin phosphorylation
modulates the organization of actin cytoskeleton, it is possible
that Ser507 phosphorylation by Akt1 could regulate b1 integrin
signaling to the migration phenotype.
Coordinated upregulation of both the stimulatory and inhibi-
tory branch of actin motility machinery has been observed
in invasive carcinoma cells (Wang et al., 2004). Accordingly,
expression of palladin is increased in human breast tumor tis-
sues (Goicoechea et al., 2009) and invasive mammary tumors
in rats (Wang et al., 2004). Since our data show that palladin
phosphorylation functions to inhibit invasive migration, it will be
interesting to determine the relative phosphorylation levels of
palladin in breast tumor tissues. This will be possible only with
the use of specific phospho-Ser507 palladin antibodies.
In summary, the present study defines a mechanism that
links palladin to Akt1-mediated inhibition of breast cancer cell
migration. We show that Akt1, but not Akt2, phosphorylates pal-
ladin at Ser507. In turn, this induces reorganization of the actin
Figure 6. Regulation of Breast Cancer Cell Random Migration by Palladin Phosphorylation
(A) MDA-MB-231 cells were infected with palladin shRNA lentiviral vector or empty vector for 48 hr. Cells were then transfected with shRNA-resistant (*) HA-pal-
ladin WT*, HA-palladin Ser507Alamutant*,or controlvector. Twenty-fourhours after transfection,cell migrationon NIH3T3 cell-secretedECMwas monitored by
time-lapse microscopy for 1 hr. Representative phase-contrast images from the movies are shown.
(B) Quantification of the velocity of random migration in (A). Data are represented as mean ±SEM. See also Movies S1–S4.
Palladin Phosphorylation Regulates Cell Migration
340 Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc.
cytoskeleton through actin-bundling activity and ultimatelyleads
to inhibition of migration of breast cancer cells. Undoubtedly,
more isoform-specific substrates that confer functional selec-
tivity remain to be identified. Given the fact that Akt isoforms
astherapeuticagents, thesefindings underscoretheimportance
of dissecting the precise mechanisms by which the PI3K and
Akt pathway regulates breast cancer invasive migration leading
to metastatic dissemination.
HEK293T, HeLa, MCF7, MDA-MB-231, and MDA-MB-468 cells were main-
tained in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro; Manassas,
VA) supplemented with 10% fetal bovine serum (FBS) (HyClone; Waltham,
MA). SKBR3 cells were cultured in McCoy’s 5A medium (Cambrex; East Ruth-
erford, NJ) supplemented with 10% FBS. BT-549 cells were grown in RPMI
1640 medium supplemented with 10% FBS. SUM-159-PT cells were cultured
in Ham’s F12 medium (Cellgro) supplemented with 5% FBS, 1 mg ml?1hydro-
cortisone (Sigma-Aldrich; St. Louis), and 5 mg ml?1insulin (Sigma-Aldrich).
Figure 7. Phosphorylation of Palladin Regulates Actin Organiza-
tion and Bundling
(A) shRNA-resistant (*) HA-palladin WT* or HA-palladin Ser507Ala mutant*
was overexpressed in MDA-MB-231 cells infected with palladin shRNA
lentiviral vector or empty vector. Twenty-four hours after transfection,
cells were fixed and stained with anti-palladin and Alexa Fluor 488-conju-
(B) MDA-MB-231 cells were infected and transfected as described in (A).
Cell lysates were incubated with F-actin, and F-actin binding (left panel)
and bundling (right panel) was determined by differential centrifugation
at 150,000 3 g for 1.5 hr and 10,000 3 g for 1 hr, respectively. Pellet (P)
and supernatant (S) fractions were immunoblotted with anti-Palladin to
visualize palladin and stained with Ponceau S to reveal total F-actin
protein. All results are representative of three independent experiments.
See also Figure S5.
MCF10A were grown in DMEM/Ham’s F12 medium supplemented with
5% equine serum (GIBCO-brl; Carlsbad, CA), 10 mg ml?1insulin, 500 ng
ml?1hydrocortisone (Sigma-Aldrich), 20 ng ml?1EGF (R&D Systems;
Minneapolis, MN), and 100 ng ml?1cholera toxin (List Biological Labs;
Growth Factors and Inhibitors
Cells were stimulated with recombinant human IGF-1 (R&D Systems) at
in Figure 5B). Recombinant human EGF was added to cells at 20 ng ml?1
for 10 min. Wortmannin (Sigma-Aldrich) and SN30978 (referred to Akti-1/2
in DeFeo-Jones et al., 2005; gift from Peter Shepherd [Symansis; Timaru,
New Zealand and University of Auckland, New Zealand]) were added to
cells 15 min prior to growth factor stimulation at final concentrations of
100 nM and 5 mM, respectively. Rapamycin (Sigma-Aldrich) was added
to cells 15 min prior to stimulation at 100 nM. LY294002 (Alexis Biochem-
icals; Farmingdale, NY) was added to cells 15 min prior to growth factor
stimulation at a final concentration of 10 mM.
Anti-Akt1 monoclonal antibody, anti-Akt2 polyclonal antibody, anti-phos-
pho-Akt Ser473 (pAkt) monoclonal antibody, anti-phospho-Akt substrate
(pAkt-motif) monoclonal antibody, anti-phospho-GSK3b monoclonal anti-
body, anti-GSK3b monoclonal antibody, anti-p110a monoclonal anti-
body, and anti-phospho-S6K1 polyclonal antibody were obtained from Cell
Signaling Technology (Danvers, MA). Anti-Akt polyclonal antibody and anti-
GFP monoclonal antibody were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-palladin polyclonal antibody was from ProteinTech
Group (Chicago). Horseradish peroxidase-conjugated anti-mouse and anti-
rabbit immunoglobulin G (IgG) antibodies were purchased from Chemicon
(Billerica, MA). Cy3-conjugated anti-rabbit IgG antibody was from Jackson
Laboratory (Bar Harbor, ME). Anti-b-actin monoclonal antibody was pur-
chased from Sigma-Aldrich. Anti-Akt1 polyclonal antibody was raised against
a synthetic peptide (VDSERRPHFPQFSYSASGTA) and generated in house.
Anti-HA monoclonal antibody was purified from the 12CA5 hybridoma.
HA-Myr-Akt1(Yoeli-Lerner et al., 2005) and HA-GSK3b (Ding et al., 2000) plas-
mids have been described previously. HA-Myr-Akt2, HA-p110a/pBABE-puro,
HA-p110a H1047R/pBABE-puro, and HA-p110a E545K/pBABE-puro plas-
mids were obtained from Addgene (Cambridge, MA). Retroviral vectors
expressing Akt chimeras 1122, 2211, and 2111 were gifts from Jeffrey Field
(Zhou et al., 2006) (University of Pennsylvania). HA-Palladin and GFP-Palladin
plasmids were gifts from Mikko Ro ¨nty (Ro ¨nty et al., 2007) (University of
Helsinki, Finland). HA-Palladin Ser507Ala was constructed by site-directed
mutagenesis with the following primers: sense, 50-AGGCCTCGTTCTAGAG
CAAGGGACAGTGGAG-30; antisense, 50-CTCCACTGTCCCTTGCTCTAGAA
CGAGGCCT-30. ShRNA-resistant variants of palladin were constructed by
Palladin Phosphorylation Regulates Cell Migration
Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc. 341
site-directed mutagenesis with the following primers: sense, 50-CCTCCGATG
CATCGGAGG-30. All sequences were verified by DNA sequencing.
For shRNA-mediated knockdown of Akt isoforms, a set of single-stranded
oligonucleotides encoding the Akt1 or Akt2 target shRNA and its complement
were synthesized. The hairpin sequences have been validated previously
(Irie et al., 2005): Akt1, sense, 50-CCGGGAGTTTGAGTACCTGAAGCTGCTC
GAGCAGCTTCAGGTACTCAAACTCTTTTTG-30; Akt1, antisense, 50-AATTCA
TC-30; Akt2, sense, 50-CCGGGCGTGGTGAATACATCAAGACCTCGAGGTCT
TGATGTATTCACCACGCTTTTTG-30; Akt2, antisense, 50-AATTCAAAAAGCG
oligonucleotide pair was annealed and inserted into pLKO. To produce lentivi-
ral supernatants, 293T cells were cotransfected with control or Akt1 or Akt2
shRNA-containing pLKO vectors, VSVG, and psPAX2 for 48 hr. Palladin
shRNA sequence (sense, 50-CCGGGGCACAAAGGATGCTGTTATTCTCGAG
AATAACAGCATCCTTTGTGCCTTTTTG-30; antisense, 50-AATTCAAAAAGG
been validated previously (Ro ¨nty et al., 2007) and was cloned into the pLKO
lentiviral expression system as described above. For siRNA-mediated knock-
down of Akt isoforms, Akt1 siRNA oligos (sense, 50-GAGUUUGAGUACCU
GAAGCUGUU-30; antisense, 50-CAGCUUCAGGUACUCAAACUCUU-30) and
Akt2 siRNA oligos (sense, 50-GCGUGGUGAAUACAUCAAGACUU-30; anti-
sense, 50-GUCUUGAUGUAUUCACCACGCUU-30) were purchased from
Dharmacon (Lafayette, CO). Cells were transfected with Akt1, Akt2, or control
luciferase GL2 siRNA (Dharmacon) using Lipofectamine 2000 (Invitrogen;
Carlsbad, CA) according to the manufacturer’s protocol.
In Vitro Kinase Assays
hr before harvesting. Palladin was immunoprecipitated from cell extracts and
incubated with 500 ng recombinant Akt1 (Cell Signaling Technology) or Akt2
(Cell Signaling Technology) in the presence of 250 mM cold ATP in a kinase
buffer for 1 hr at 30?C. The kinase reaction was stopped by the addition of
SDS-PAGE loading buffer, and the samples were assayed by immunoblotting.
Transwell Migration and Invasion Assays
Transwell filters (8 mm pore size; Corning; Corning, NY) were left uncoated for
migration assays or coated with 1–10 mg Matrigel (BD Biosciences; San Jose,
CA) for invasion assays. Cells (3 3 105) in serum-free medium containing 0.1%
BSA were added to the upper chambers in triplicate. NIH 3T3-cell-conditioned
medium was added to the lower chambers. After 2–22 hr incubation at 37?C,
nonmigrated cells at the top of the filters were removed. Cells that had
migrated to the bottom of the filters were fixed and stained using the Hema-
3 stain set (Protocol; Fisher Scientific; Pittsburgh) or X-gal.
Cells were plated on tissue culture dishes containing NIH 3T3-cell-secreted
ECM for 1 hr before time-lapse microscopy. Phase-contrast images of
live cells were taken every 2.5 min for 1 hr with an inverted microscope
(Eclipse2000; Nikon; Melville, NY) equipped with a heated stage and digital
image analysis software (IPLab; Scanalytics; Fairfax, VA). Velocity of random
migration was quantified using NIS-Elements AR software (Nikon).
3D culture assay was performed as previously described (Debnath et al.,
2003). Briefly, chamber slides were coated with a 50:50 mixture of growth-
factor-reduced Matrigel (BD Biosciences) and bovine dermal collagen I (Matri-
gel/collagen; Vitrogen Cohesion Technologies; Palo Alto, CA) and allowed to
solidify for 30 min. MCF10A cells (2000–4000) in assay medium (DMEM/
Ham’s F12 medium supplemented with 2% equine serum, 10 mg ml?1insulin,
500 ng ml?1hydrocortisone, 5 ng ml?1EGF, 100 ng ml?1cholera toxin, 2 mg
ml?1puromycin, and 2% Matrigel/collagen) were seeded to the coated
chamber slides. The assay medium was replaced every 4 days.
Actin Binding and Bundling Assays
Cells were lysed 24 hr posttransfection in ice-cold lysis buffer (1% NP-40,
150 mM NaCl, 10 mM KCl, 20 mM Tris-HCl [pH 7.5], 0.1% SDC, 0.1% SDS,
Proteinase inhibitor cocktail [Sigma-Aldrich], 50 nM calyculin [Sigma-Aldrich],
1 mM sodium pyrophosphate, 20 mM sodium fluoride). Actin binding and
bundling assays were performed according to the manufacturer’s protocol
(Cytoskeleton; Denver). Briefly, actin filaments (F-actin) were prepared by
incubation of human nonmuscle monomeric G-actin in polymerization buffer
(50 mM KCl, 2 mM MgCl2, 1 mM ATP, 0.18 mM CaCl2, 5 mM Tris [pH 8]) for
1 hr at room temperature. Lysates were precleared by centrifugation at
150,000 3 g for 1 hr at 4?C. Precleared lysates were then incubated with
10 mM purified F-actin for 30 min at room temperature. The reaction mixtures
were centrifuged at 150,000 3 g for 1.5 hr and 10,000 3 g for 1 hr to sediment
actin filaments and bundles, respectively. The amount of palladin and F-actin
in both the supernatant and precipitant were detected by immunoblotting and
Ponceau S staining.
Cells plated on coverslips were fixed with 2% paraformaldehyde for 10 min
and permeabilized with 0.5% Triton X-100 for 1 min. Cells were then blocked
with 1% BSA in 20 mM Tris-HCl (pH 7.5) for 20 min and incubated with anti-
palladin polyclonal antibody for 1 hr. After washing twice with phosphate-buff-
ered saline (PBS), cells were incubated with Cy3-conjugated anti-rabbit IgG
antibody for 1 hr.F-actin was visualized with Alexa Fluor 488-conjugated phal-
loidin (Invitrogen). Cells were then rinsed twice with PBS and mounted with
Prolong Gold antifade reagent/4,6-diamidino-2-phenylindole (DAPI) (Pierce;
Rockford, IL). Images of cells were acquired using a fluorescence micro-
scope (Eclipse TE300; Nikon) and digital image analysis software (IPLab;
Immunoblots and Immunoprecipitation
Cells were washed with ice-cold PBS and lysed in EBC buffer (0.5% NP-40,
120 mM NaCl, 50 mM Tris-HCl [pH 7.4], Proteinase inhibitor cocktail, 50 nM
calyculin, 1 mM sodium pyrophosphate, 20 mM sodium fluoride, 2 mM
EDTA, 2 mM EGTA) for 25 min on ice. Cell extracts were precleared by centri-
fugation at 13,000 rpm for 10 min at 4?C, and protein concentration was
measured with the Bio-Rad protein assay reagent using a Beckman Coulter
DU-800 machine. Lysates were then resolved on 8% acrylamide gels by
SDS-PAGE and transferred electrophoretically to nitrocellulose membrane
(Bio-Rad) at 160 mA for 80 min. The blots were blocked in TBST buffer
(10 mM Tris-HCl [pH 8], 150 mM NaCl, 0.2% Tween 20) containing 5% (w/v)
diluted in blocking buffer at 4?C overnight. Membranes were washed three
times in TBST and incubated with horseradish peroxidase-conjugated sec-
ondary antibody for 1 hr at room temperature. Membranes were washed three
times and developed using enhanced chemiluminescence substrate (Pierce).
For immunoprecipitation, lysates were incubated with 1–2 mg antibody for
2–4 hr at 4?C followed by incubation with 15 ml protein A/G Sepharose beads
(Amersham Biosciences; Pittsburgh) for another 2 hr. Immune complexes
were washed four times with NETN buffer (0.5% NP-40, 1 mM EDTA, 20 mM
Tris-HCl [pH 8], 100 mM NaCl) and once with PBS. Precipitates were resolved
by SDS-PAGE, and the separated proteins were analyzed by western blot.
Supplemental Information includes Supplemental References, five figures,
and four movies and can be found with this article online at doi:10.1016/j.
We thank Mikko Ro ¨nty, Jeffrey Field, Peter Shepherd, and Symansis for
providing reagents; Isaac Rabinovitz for his technical support with time-lapse
Palladin Phosphorylation Regulates Cell Migration
342 Molecular Cell 38, 333–344, May 14, 2010 ª2010 Elsevier Inc.
microscopy; Shiva Kazerounian for assistance with 3D cultures; and members
of the Toker laboratory for discussions. This study was supported in part by
grants from the National Institutes of Health (A.T., CA122099) and the Susan
G. Komen Breast Cancer Foundation (Y.R.C., PDF0706963).
Received: October 12, 2009
Revised: January 15, 2010
Accepted: February 5, 2010
Published: May 13, 2010
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