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