The actin-cytoskeleton linker protein ezrin is regulated during
osteosarcoma metastasis by PKC
L Ren1, SH Hong1, J Cassavaugh1, T Osborne1, AJ Chou2, SY Kim1, R Gorlick3, SM Hewitt4
and C Khanna1
1Tumor and Metastasis Biology Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA;2Department
of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA;3Department of Pediatrics, Albert Einstein College
of Medicine, The Children’s Hospital at Montefiore, Bronx, NY, USA and4Tissue Array Research Program, Laboratory of
Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
Ezrin is a member of the ERM (ezrin, radixin, moesin)
protein family and links F-actin to the cell membrane
following phosphorylation. Ezrin has been associated with
tumor progression and metastasis in several cancers
including the pediatric solid tumors, osteosarcoma and
rhabdomyosarcoma. In this study, we were surprised to
find that ezrin was not constitutively phosphorylated but
rather was dynamically regulated during metastatic
progression in osteosarcoma. Metastatic osteosarcoma
cells expressed phosphorylated ERM early after their
arrival in the lung, and then late in progression, only at the
invasive front of larger metastatic lesions. To pursue
mechanisms for this regulation, we found that inhibitors of
PKC (protein kinase C) blocked phosphorylation of ezrin,
and that ezrin coimmunoprecipitated in cells with PKCa,
PKCi and PKCc. Furthermore, phosphorylated forms of
ezrin and PKC had identical expression patterns at the
invasive front of pulmonary metastatic lesions in murine
and human patient samples. Finally, we showed that the
promigratory effects of PKC were linked to ezrin
phosphorylation. These data are the first to suggest a
dynamic regulation of ezrin phosphorylation during
metastasis and to connect the PKC family members with
Oncogene (2009) 28, 792–802; doi:10.1038/onc.2008.437;
published online 8 December 2008
Keywords: ezrin; ERM; PKC; tumor metastasis
Metastatic disease continues to be the most common
cause of death for patients with cancer. We have shown
that ezrin is required for metastasis in two pediatric solid
tumors of mesenchymal origin, osteosarcoma and
rhabdomyosarcoma (Khanna et al., 2004; Yu et al.,
2004). The expression of ezrin has been linked to poor
survival in several cancers including carcinomas of the
breast, colon, endometrium and ovary, cutaneous and
uveal melanomas, brain tumors and soft tissue sarcomas
(Ohtani et al., 1999; Khanna et al., 2004; Yu et al., 2004;
Elliott et al., 2005; Ilmonen et al., 2005; Weng et al.,
2005; Kobel et al., 2006; Bruce et al., 2007).
Ezrin is a member of the ERM (ezrin,radixin, moesin)
protein family (Berryman et al., 1993; Bretscher et al.,
2000). ERM proteins provide a physical link from
F-actin to membrane-associated proteins on the surface
of cells (Tsukita et al., 1994; Reczek et al., 1997). This
linker function makes ezrin essential for many funda-
mental cellular processes, including the determination of
cell shape, polarity and surface structure, cell adhesion,
motility, cytokinesis, phagocytosis and integration of
membrane transport with signaling pathways (Serrador
et al., 1999; Ng et al., 2001; Bretscher et al., 2002; Wu
et al., 2004). Ezrin is expressed in many normal tissues
and has been demonstrated to be important during
embryogenesis (Dard et al., 2004; Polesello and Payre,
2004; Saotome et al., 2004). Nonetheless, the redun-
dancy that exists between ezrin and the other ERM
proteins may be sufficient to compensate for the loss of
ezrin in some physiological processes. Indeed, ezrin
knockout mice survive for 21 days after birth, suggesting
some ERM functional redundancy during embryogen-
esis. Interestingly, the lethal phenotypes in these mice
are restricted to the intestinal villi, a site in which other
ERM proteins are not expressed (Kivela et al., 2000;
Saotome et al., 2004). Death in these mice is believed to
result from the intestinal villous malformations. In our
studies of ezrin’s role in cancer, a functional redundancy
of ERM proteins may be suggested. Following suppres-
sion of ezrin in murine osteosarcoma cells, there were no
changes in in vitro viability, proliferation or primary
tumor growth in vivo (Khanna et al., 2004). Despite
expression of other ERM proteins, the suppression of
ezrin in several murine and human cancer models
resulted in the inhibition of metastasis (Makitie et al.,
2001; Khanna et al., 2004; Pang et al., 2004; Yu et al.,
2004; Ilmonen et al., 2005; Weng et al., 2005). This
suggested that ezrin, rather than other ERM proteins,
Received 25 March 2008; revised 3 October 2008; accepted 1 November
2008; published online 8 December 2008
Correspondence: Dr C Khanna, Tumor and Metastasis Biology
Section, Pediatric Oncology Branch, Center for Cancer Research,
National Cancer Institute and Comparative Oncology Program,
37 Convent Drive, Rm 2144, Bethesda, MD 20892, USA.
Oncogene (2009) 28, 792–802
& 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00
contributed a unique and necessary function to cells
Phosphorylation of the C-terminal threonine of ERM
proteins is important for their activation. ERM proteins
exist in inactive forms in which the C-terminal tail binds
to, and masks, the N-terminal FERM domain (band
4.1, ezrin, radixin, moesin homology domains) (Pearson
et al., 2000). The activation of ERM proteins is
mediated by both C-terminal threonine phosphorylation
(T567 in ezrin, T564 in radixin, T558 in moesin) (Matsui
et al., 1999; Gautreau et al., 2000; Pearson et al., 2000)
and exposure to polyphosphoinositides (Fievet et al.,
2004). It is most likely that phosphorylation of other
residues in ERM proteins are needed to maintain an
open activated conformation and to direct specific
effects in cells (Krieg and Hunter, 1992; Srivastava
et al., 2005). Several protein kinases have been found to
phosphorylate the C-terminal threonine residue of ERM
proteins. Examples include protein kinase Ca (PKCa)
(Ng et al., 2001; Chuan et al., 2006), PKCy (Pietromo-
naco et al., 1998), PKCi (Wald et al., 2008), Rho
kinases/ROCK (Matsui et al., 1998; Oshiro et al., 1998),
G protein-coupled receptor kinase 2 (GRK2) (Cant and
Pitcher, 2005) and myotonic dystrophy kinase-related
Cdc42-binding kinase (MRCK) (Nakamura et al.,
2000). Most studies that have investigated the kinase
responsible for ezrin T567 phosphorylation have been
conducted in cell-free systems or non-cancer cell lines.
In this study, we were surprised to find that ezrin
phosphorylation was dynamically regulated during the
metastatic process in osteosarcoma. On the basis of our
previous data, we had assumed that the phosphorylated,
‘activated’, form of ezrin would be constitutively
expressed during metastasis. By following the progres-
sion of metastasis in highly metastatic murine and
human osteosarcoma cells, high expression of phos-
phorylated ezrin was observed early after cells arrived in
the lung. Surprisingly, at later points in the metastatic
process there was a loss of phosphorylated ezrin, most
notable several days after metastatic cells arrived in the
lung, and most evident as metastatic lesions progressed
in size, particularly within the central portions of large
metastases. Re-expression of phosphorylated ezrin was
then found at the invasive front of larger metastatic
lesions. Using pharmacological inhibitors, to uncover
the kinase responsible for this regulation, we found the
phosphorylation of ezrin at T567 to be dependent on
PKC family members (BIM, Ro31-8220, Go ¨ 6976) but
not by inhibition of Rho-kinase (Y27632) or PI3 kinase
(LY294002). Furthermore, ezrin and several PKC iso-
forms were found to coimmunoprecipitate in osteosa-
rcoma cells. In support of this in vitro data, staining of
lung metastases in mice showed that phospho-PKCa
and phosphorylated ERM were both expressed in cells
found at the leading front of metastatic lesions. The
connection between PKC and ezrin expression was
further supported by similar decreases in osteosarcoma
cell migration following either PKC or ezrin inhibition.
Lastly, cells overexpressing an activated phospho-
mimetic ezrin mutant (T567D) were less responsive to
the PKC inhibitors suggesting that PKC-induced
motility was in part ezrin dependent. These data are
the first to describe a dynamic regulation of ezrin
phosphorylation during metastatic progression. This
regulation is linked with PKC activation of ezrin and
reciprocally suggests a role for ezrin in mediating some
PKC prometastatic effects. Targeting PKC activation of
ezrin during specific times in metastatic progression may
be considered as a means to improve outcome for
patients with metastasis.
Phosphorylated ERM proteins are regulated during
Human and murine osteosarcoma cells were injected
intravenously into mice. Lung samples were harvested at
various time points to assess the expression of ezrin and
phosphorylated ERM (Figure 1 and Supplementary
Figure S1). At present, an antibody that is specific for
phosphorylated ezrin does not exist. Early (5 days) after
K7M2 osteosarcoma cells arrived in the lung, both ezrin
and phosphorylated ERM expression were uniformly
high (Figure 1). It is important to note that most ezrin in
K7M2 cells cultured in vitro is not phosphorylated
(Supplementary Figure S2). At this time point, the
metastatic cells were identified as single cells or in small
clusters of 5–20 cells, with both total ezrin and
phosphorylated ERM expression distributed through-
out the cytosol and cell membrane. Fifteen days after
arrival of the cells in the lung, small metastatic lesions
(0.1–0.5mm) were observed throughout the lung. These
metastatic foci continued to express total ezrin uni-
formly in the cytoplasm; however, phosphorylated
ERM was dramatically decreased. With further progres-
sion of the metastatic lesions, the majority of the cells,
particularly those in the center of the mass expressed
very low phosphorylated ERM. Interestingly, as these
larger metastatic lesions progressed, phosphorylated
ERM began to be re-expressed but only at the periphery
of the expanding metastatic lesions. This result was seen
consistently following both experimental (Figure 1g)
and spontaneous metastasis assays (data not shown)
using K7M2 murine osteosarcoma cells in mice, using
human MNNG/HOS osteosarcoma
(Figure 1h) and in six of six human metastatic
osteosarcoma patient samples (Figure 1i). Observations
of single metastatic cells and the expression of ezrin and
phosphorylated ERM in these cells were limited to the
use of the experimental metastasis assay (tail vein
injection of tumor cells). Subsequent events during
metastatic progression were assessed and found con-
cordant following both experimental metastasis and
spontaneous metastasis that developed from orthotopic
primary appendicular osteosarcoma tumors. These data
suggested that phosphorylation of ERM proteins was
dynamically regulated during metastatic progression,
and furthermore that the expression of phosphorylated
ERM was not necessary throughout the metastatic
process as we had expected. Phosphorylated ERM
Regulation of ezrin by PKC
L Ren et al
expression appeared to be important early after arrival
of metastatic cells in the lung and during further
progression at the invasive front of larger metastatic
PKC is responsible for phosphorylation (activation)
of ezrin on Thr 567 in osteosarcoma
To determine the kinase involved in the regulation of
ezrin phosphorylation, the mouse ezrin protein sequence
was included in a MotifScan (http://scansite.mit.edu)
search for kinase candidate(s). As shown in Table 1, six
kinases were identified in this scan as potential kinases
for ezrin phosphorylation at threonine 567, four of
which were PKC isoforms (classical PKCa, b, g and z).
For unknown reasons, although structurally similar to
PKCz the motif scan did not identify PKCi. The scan
scores start at 0.000 if the sequence optimally matches a
given motif, and the scores increase for sequences as
they diverge from the optimal match. These data
suggested the hypothesis that PKC family kinases are
responsible forthe phosphorylationofezrin on
Day 5Day 15
MNNG/HOS-xenograft Human OS patient sample
Day 24 K7M2-mouse
nodules were evaluated by immunohistochemistry for expression of phosphorylated ERM and total ezrin. Samples were derived after
tail vein injection of K7M2 murine osteosarcoma cells (a–g and j) or MNNG/HOS human osteosarcoma cells (h and k), and from
human patient tissue samples (i and l). K7M2 lung metastases were examined at days 5, 15, 20 and 24 after injection (a–g and j).
A similar pattern of immunoreactivity for ezrin and phosphorylated ERM were seen in K7M2 pulmonary metastases derived from
spontaneous lung metastasis from an appendicular primary tumor in mice (data not shown). Total ezrin and phosphorylated ERM
were stained on adjacent lung sections. a and d, Bar¼40mm; b, c, e and f, bar¼100mm; g–l, bar¼200mm.
Phosphorylation (activation) of ERM proteins is dynamically regulated during metastatic progression. Metastatic lung
Table 1MotifScan results for ezrin T567 site
PKCa, b, g
14-3-3 Mode 1
aPredicted Ser/Thr kinases for ezrin T567 phosphorylation site.
bThe scan scores start at 0.000 if the sequence optimally matches a
given motif and the scores increase for sequences as they diverge from
the optimal match. Lower scores in the output are thus better matches.
cThe percentile ranking of ezrin T567 phosphorylation site in respect to
all potential motifs in vertebrate proteins in Swiss-Prot.
Regulation of ezrin by PKC
L Ren et al
Thr 567 (activation) in osteosarcoma. Outside the
setting of cancer, several kinases (PKC, ROCK,
GRK2, p38, MRCK and so on) have been previously
shown to contribute to the phosphorylation of ezrin
(Matsui et al., 1998; Oshiro et al., 1998; Pietromonaco
et al., 1998; Ng et al., 2001; Weng et al., 2005; Wald
et al., 2008). Very recently, PKC phosphorylation of
ezrin in androgen-primed prostate cancer cells was
reported, lending further support to our hypothesis in
metastasis (Chuan et al., 2006).
To test the hypothesis that PKC family kinases are
active in phosphorylation of ezrin at threonine 567 in
selected kinases were exposed to K7M2 murine oste-
osarcoma cells. Following exposure, cell lysates were
collected and analysed for phosphorylation of ERM
proteins at the C-terminal threonine. As shown in
Figure 2a, BIM (inhibits PKC and other kinases)
suppressed threonine phosphorylation of ERM proteins
in a dose-dependent manner. Exposure of cells to
Y27632 (inhibits Rho kinase), LY294002 (inhibits PI3
kinase) and U0126 (inhibits MEK1/2) did not change
phosphorylated ERM at the C-terminal threonine
(Figure 2a). The specific modulation of either AKT
(LY294002) or MAPK-44/42 (U0126) confirmed each
agent’s biological activity and the presence of the
pathway in K7M2 cells (Figure 2a). To further
demonstrate that the inhibition of phosphorylated
ERM was mediated by PKC, isoform selective PKC
inhibitors were evaluated in both murine K7M2 and
human MNNG/HOS cells as shown in Figure 2b. In
both cell lines, treatment with Ro31-8220 (inhibits PKC)
and Go ¨ 6976 (inhibits PKC isoforms; PKCa, bI, bII
and g) resulted in decreased phosphorylated ERM levels
in a time- and concentration-dependent manner. In situ
evaluation demonstrated that phosphorylated ERM
proteins were mainly localized on the cell membrane,
largely concentrated in the membrane protrusions
005 1020 0.51246
0 1 5 10 20 1 5 10 20
0 5 10 20 5 1020
with various concentrations of PKC inhibitor BIM, Rho kinase inhibitor Y27632, PI3 kinase inhibitor LY294002 and MEK inhibitor
U0126. Phospho-ERM expression is shown by western blot analysis (phosphorylated ERM is comprised of a phospho-ezrin/radixin
band * and a phospho-moesin band **). The blots were probed for b-actin as the loading control, and phospho-Akt (Y374) or
phospho-MAPK 42/44. (b) K7M2 and MNNG/HOS cells were treated with PKC inhibitors BIM, Ro 31-8220 or Go ¨ 6976 at various
concentrations for 60min or for different time periods as indicated. The level of phospho-ERM was analysed by western blotting.
(c) Imuunofluorescent detection of phospho-ERM in untreated K7M2 cells or in K7M2 cells treated with 5mM of Ro 31-8220 for
60min. Bar¼20mm in low magnification photographs and 5mm in high magnification photographs.
Ezrin (T567) phosphorylation is dependent on protein kinase C (PKC). (a) Murine osteosarcoma K7M2 cells were incubated
Regulation of ezrin by PKC
L Ren et al
(microspikes) (Figure 2c). Following the treatment of
cells with the PKC inhibitor Ro31-8220, this membra-
nous and microspike expression of phosphorylated
ERM was markedly diminished (Figure 2c). It should
be noted that PKC inhibitors were active at concentra-
tions as low as 1mM within 10min of exposure. The
rapid effects of PKC inhibitors on ERM phosphoryla-
tion may be explained by the rapid intrinsic turnover of
T567 phosphorylation (Zhu et al., 2007) or through the
activation of an ERM-specific phosphatase following
PKC inhibition (Forte et al., 2008). The physiological
relevance of these exposures is not known. However, it is
important to note that significantly greater exposures to
other kinase inhibitors had no effect on phosphorylated
ERM expression in these cells. Nonetheless, the lack of
complete specificity of pharmacological inhibitors of
kinases, including the isoform-specific PKC inhibitors
used in our studies, should be considered when
interpreting these data.
PKC isoforms a, g and i complex with ezrin in vivo
To further explore the connection of ezrin’s phospho-
rylation by PKC isoforms, we screened human and
murine osteosarcoma cells for the expression of all PKC
isoforms by immunoblot analysis. PKCa, g, e, z, i and d
were expressed in murine K7M2, K12 and human
MNNG/HOS osteosarcoma cell lines (Figure 3a). How-
ever, PKCb, y and Z were not detectable. In addition,
ezrin and the PKC isoforms a, g and i coimmunopre-
cipitated in the osteosarcoma cells (Figure 3b). We could
not demonstrate PKCd, PKCe and PKCz coimmuno-
precipitation with ezrin. On the basis of PKC coimmu-
noprecipitation results, we next used small interfering
RNAs (siRNAs) to knockdown the expression of
PKCa, g and i in the K7M2 osteosarcoma cells. The
individual knockdown of PKCg and i was nearly 100%;
however, no changes were seen in expression of
phosphorylated ERM (data not shown). Consistent
with reports in the literature, despite repeated attempts
with several siRNAs and siRNA pools, we could not
knock PKCa by more than 50% (data not shown).
PKCa expression was not further modulated when pools
of PKCa, g and i siRNAs were combined. Accordingly,
the siRNA studies were unable to definitively answer if
PKCa, PKCg and PKCi are responsible for the
regulation of ezrin phosphorylation in ostoesarcoma
cells (Supplementary Figure S3).
Phospho-PKCa and phospho-ERM show identical
expression patterns in osteosarcoma lung metastases
To study the association between ezrin and PKC in vivo,
we performed immunohistochemistry using phospho-
PKCa (Ser657) and phospho-ERM in lung nodules of
K7M2 tumor-bearing mice. The phosphorylation of
PKC on serine 657 controls accumulation of the active
enzyme and contributes to the maintenance of the
phosphatase-resistant conformation (Bornancin and
Parker, 1997) of PKC. As shown in Figure 4, intense
staining of phospho-PKCa (membranous, cytoplasmic
and nuclear) was seen at the leading edges of metasta-
static lesions (Figures 4a and b). At these same locations
within the metastatic lesions, phospho-ERM was also
highly expressed and localized predominantly on the
tumor cell membrane (Figures 4d and e). Conversely,
both active PKCa expression and phosphorylated ERM
expression were low in the central portions of these
lesions (Figures 4c and f). Interestingly, low phospho-
PKCa expression in the central portions of the meta-
static lesions was largely localized to the nucleus with no
membranous or cytoplasmic staining.
PKC-mediated tumor cell migration is in part mediated
As previously reported, the suppression of total ezrin
protein, using full-length antisense constructs (K7M2
As1.46) resulted in an inhibition of metastasis in mice
without any measurable changes in primary tumor
growth (Khanna et al., 2004). To extend these findings
and explore the biological significance of PKC regula-
tion on ERM phosphorylation, we independently
examined the effects of ezrin and PKC on cell motility
using wound healing and transwell migration assays.
The cells with knockdown of ezrin (K7M2 As1.46 cells;
low ezrin, poorly metastatic) had a significant delay in
migration in the wound-healing migration assay com-
pared with the parental K7M2 cells (high ezrin, highly
metastatic; Figure 5a). Exposure of cells to the PKC
inhibitor Go ¨ 6976 at 1mM also blocked cell migration
through the wounded area (Figure 5a). Treatment of
cells with Go ¨ 6976 at 1mM had no effect on proliferation
in K7M2 cells, supporting the antimigratory role.
Similar results were also seen with the PKC inhibitor
Ro31-8220 (data not shown). To confirm the antimi-
gratory phenotype associated with downregulation of
ezrin and by PKC inhibition, we performed a modified
Boyden chamber transwell assay. These results demon-
strated significant inhibition of cell motility following
ezrin suppression (antisense Ezrin-As1.46; 5?104cells,
P¼0.004; 2.5?104cells, P¼0.01) or following either
treatment with the PKC inhibitor Go ¨ 6976 (0.1mM,
P¼0.05; 1mM, P¼0.03) (Figure 5b).
To link the similar anti-migratory results obtained
following either ezrin knockdown or PKC inhibition, we
performed similar experiments using an ezrin mutant
that has been previously described to mimic the
activated ‘open’ confirmation of ezrin (Pearson et al.,
2000). The Thr 567 to Asp (EzrinT567D-GFP, acti-
vated-ezrin-GFP) mutation was detected by western blot
analysis by the virtue of its larger size at 110kDa
(Figure 6a). As predicted, the expression of the active
ezrin (EzrinT567D-GFP) was localized to the cell
membrane (Figure 6b). Treatment of both parental
K7M2/GFP cells with the PKC inhibitor Go ¨
resulted in decreased migration as expected. However,
K7M2/EzrinT567D-GFPcells were more able to
migrate in the presence of a PKC inhibitor (Figure 6c)
compared with parental and GFP-expressing K7M2
cells. These data provide a link between PKC-mediated
activation of ezrin and the metastatic phenotype of
enhanced cell motility.
Regulation of ezrin by PKC
L Ren et al
To study the mechanisms associated with ezrin’s role in
metastasis, we followed the expression of ezrin and its
phosphorylation serially during the metastatic cascade.
Our previous studies have confirmed a unique depen-
dence of metastatic cells on ezrin rather than the other
ERM proteins (Khanna et al., 2004; Yu et al., 2004;
of the PKC isoforms in K12, K7M2 and MNNG/HOS osteosarcoma cell lines. PKCb, Z and y isoforms are not expressed in
osteosarcoma cells, although they are expressed in Jurkat cells or lung tissue. (b) Immunoprecipitated ezrin from K7M2 or MNNG/
HOS cell extracts were probed for pan-PKC, PKCa, g, d, i, e, z and ezrin. Ezrin interactions were detected with PKC isoforms a, g and i.
Normal rabbit serum was used as the negative control.
Ezrin forms protein–protein complexes with PKCa, i and g in osteosarcoma cells. (a) Western blot analysis shows expression
of phosphorylated ERM and phospho-PKCa in K7M2 murine osteosarcoma lung nodules (harvested 24 days after cell injection to
mice). Adjacent lung sections were labeled with antibodies against either phospho-PKCa (Ser 657) (a–c) or phospho-ERM (d–f).
Phospho-PKCa (a and b) and phospho-ERM (d and e) immunoreactivity was evident at the periphery of metastatic lesions. Both
active PKCa expression and phosphorylated ERM expression was low in the central portions of the metastatic lesions (Figures 4c and
f). Bar¼200mm in low magnification photographs and 50mm in high magnification photographs.
Matching expression patterns of phospho-ERM and phospho-PKCa in metastatic lesions. Immunohistochemistry detection
Regulation of ezrin by PKC
L Ren et al
Krishnan et al., 2006). As expected, we found high levels
of total ezrin expressed in all lesions at all time points
during metastasis. Our expectation was that the expres-
sion of phosphorylated ezrin would be equally and
continuously high. We were surprised to find that
phosphorylated ERM was not expressed throughout
all stages of progression. ERM was phosphorylated
early after metastatic cells arrived in the lung. As these
multicellular lesions progressed, they lost the expression
of phosphorylated ERM. However, as these lesions grew
even larger, phosphorylated ERM was again expressed,
but only at the leading edge, or invasive front, of the
lesions. These data, confirmed in human osteosarcoma
metastases, suggested that phosphorylated ezrin was not
needed throughout metastatic progression and that the
phosphorylation of ezrin was regulated in metastatic
The high expression of phosphorylated ERM in the
early stages of metastasis is consistent with an existing
hypothesis that ezrin contributes to the survival of
metastatic cancer cells following their arrival in the lung.
ERM proteins are regulated by an intramolecular
association of the N-terminal and C-terminal domains
that masks their protein–protein binding sites (Bretscher
et al., 2002). Unfolding of the molecule into an active
conformation occurs following binding to phosphoino-
sitides and phosphorylation of the C-terminal threonine
(T558 in moesin, T567 in ezrin, T564 in radixin) (Fievet
et al., 2004). The open molecules bind with various
membrane proteins (Tsukita et al., 1994; Bretscher et al.,
1997; Reczek et al., 1997; Simons et al., 1998; Yonemura
et al., 1998) at the N-terminal region and F-actin
through the C-terminal domain (Bretscher et al., 1997;
Hishiya et al., 1999; Pearson et al., 2000). As osteo-
sarcoma cells in culture express high levels of ezrin and
phosphorylated ERM, it was possible that the high
levels of phosphorylated ERM seen in single cells that
arrived in the lung following tail vein injection was
merely a residual effect of their growth in vitro.
However, it was not expected that the residual ex-
pression of phosphorylated ERM would persist for
5 days. The loss of ERM phosphorylation later in the
course of metastatic progression was a consistent finding
that suggested two possibilities. Namely, that phospho-
rylation of ERM proteins was not required during this
stage of metastatic growth or that dephosphorylation of
ERM proteins was necessary for progression. Depho-
sphorylation of C-terminal threonine of moesin is a
Cell number (x1000)Go 6976
25 0 um 0.1 um1 um
(K7M2) or antisense mediated ezrin knockdown cells (As 1.46) were ‘wounded’ using a P-200 pipette, and images of the denuded area
were taken at 0 and 24h. Migration of cells with suppression of ezrin (As1.46) was markedly decreased at 24h compared to K7M2
control cells (high ezrin). K7M2 cells that had been treated with 1mM PKC inhibitor Go ¨ 6976 also showed a significant reduction in
migration. (b) Ezrin knock down (As1.46) and control K7M2 cells were seeded on transwell plates in complete medium (left panel).
After an 18-h incubation, cells migrating to the lower chamber were stained with Calcein AM and the fluorescent intensity was
quantitated. Ezrin knockdown cells (As1.46) had a marked decrease in migration compared to control cells (K7M2) using either 5?104
cells (P¼0.004) or 2.5?104cells (P¼0.01) as starting material. Similarly, K7M2 cells were seeded on transwell plates with 0.1%
dimethylsulfoxide or PKC inhibitor Go ¨ 6976 (right panel). Cell migration was inhibited in the presence of Go ¨ 6976 at 0.1mM (P¼0.05)
and at 1mM (P¼0.03).
Suppression of ezrin or inhibition of PKC decreases osteosarcoma cell migration. (a) Nearly confluent wild-type ezrin
Regulation of ezrin by PKC
L Ren et al
crucial step for transendothelial migration of lympho-
cytes (Brown et al., 2003). Accordingly, it is interesting
to speculate that ezrin dephosphorylation is an active
and necessary step, required for the development of cell–
cell contacts needed during progression of metastatic
cells to multicellular clusters. Again, consistent with our
hypothesis that ezrin is needed for the survival of
metastatic cells, especially those encountering the
foreign microenvironment of a secondary metastatic
site, we observed the re-expression of phosphorylated
ERM at the invasive front of metastatic lesions. At this
time, we do not have data to causally associate the
expression of phosphorylated ERM in single metastatic
cells, either early after their arrival in the lung or at the
periphery of established grossly detectable lesions, with
metastatic success. Nonetheless, these data suggest a
dynamic regulation of ERM during cancer progression
rather than our initial expectation that constitutive
activation of ezrin would be a requirement for metas-
tases; furthermore, it appears that the phosphorylated
and dephosphorylated states of ezrin are dynamically
regulated during the process of metastatic progression.
Several kinases have been implicated in the regulation
of ERM protein function. PKCa has been shown to
interact with ezrin, both in vitro and in vivo, and can
phosphorylate ezrin at T567 in vitro (Ng et al., 2001).
Using a MotifScan program, we were able to identify six
kinases that had the potential to phosphorylate ezrin at
T567, three of which were classical (or conventional)
PKCs and one was PKCz. A screen using a panel of
kinase inhibitors showed that only PKC inhibitors could
decrease phosphorylation of ERM in osteosarcoma
cells. Immunoprecipitation experiments then verified
that PKCa, PKCg and PKCi interact with ezrin.
SiRNA-mediated knockdown PKCg, and PKCi failed
to decrease phosphorylation of ERM in osteosarcoma
cells. SiRNA knockdown of PKCa was incomplete
following the exposure of cells to siRNAs for PKCa
alone, or siRNAs for PKCa, PKCg and PKCi used in
combination, as such were unable to ask if PKCa alone
or if the combination of PKCa, PKCg and PKCi
regulated ezrin phosphorylation in osteosarcoma cells.
The use of selective pharmacological inhibitors or more
effective genetic knockdown strategies for specific PKC
0 hr no treat
Gö 6976 0.1 µM
mutant (T567D-GFP) in K7M2 osteosarcoma cells allowed association between PKC, ezrin and cell migration to be assessed.
(a) Expression of endogenous ezrin and GFP-tagged ezrin mutant proteins in K7M2 parental cells were resolved by western blot
analysis and with an anti-ezrin antibody. Lane 1, GFP transfectants; lanes 2–4, three different clones of EzrinT567D-GFP
transfectants. (b) Localization of GFP and EzrinT567D-GFP in K7M2 cells was assessed by GFP fluorescence. Whereas GFP was
distributed throughout the cytoplasm and nucleus, EzrinT567D-GFP (active ezrin) was discretely localized to the cell membrane with
F-actin. (c) Cell migration (wound healing) assay using K7M2 control, GFP control, and three different clones of EzrinT567D-GFP
cells (T567D1, T567D3 and T567D2.4) before and after 24h of treatment with either 0.1% dimethylsulfoxide or 0.1mM Go ¨ 6976. The
constitutive activation of ezrin in the EzrinT567D-GFP prevented PKC inhibitors from suppressing osteosarcoma cell migration.
PKC-mediated cell migration is in part dependent on ezrin. Expression of constitutively activated phospho-mimetic ezrin
Regulation of ezrin by PKC
L Ren et al
isoforms are required to better understand which PKC
isoforms are individually or collectively involved in the
dynamic regulation of ezrin phosphorylation during
Protein kinase C isoforms have been connected with
several aspects of cancer biology including carcinoge-
nesis, progression and chemotherapy resistance (Blobe
et al., 1994). Several of the steps associated with
metastatic progression have been linked to PKC,
including resistance to apoptosis, migration and inva-
sion (Herbert, 1993; Musashi et al., 2000; Sullivan et al.,
2000; Koivunen et al., 2004). To further connect our
growing understanding of ezrin’s role in the metastatic
phenotype, with our finding that PKC isoforms regulate
ezrin phosphorylation, we hypothesized that some of the
previously established effects of PKC on metastasis-
specific functions, (that is, motility) were mediated
through ezrin. To test this hypothesis, we mutated the
C-terminal threonine of ezrin to aspartic acid. The result
of this site-directed mutation was a constitutively
T567D). Using motility as an example of a PKC-related
function, we found osteosarcoma cells with wild-type
expression of ezrin to have significantly reduced migra-
tion following exposure to PKC inhibitors; whereas,
cells expressing the phospho-mimetic ezrin (active) were
not affected by PKC inhibitors. These data suggest that
part of the PKC-mediated effects on cell migration are
mediated through ezrin phosphorylation and may
suggest that other recognized effects of PKC on
metastasis are also mediated through ezrin.
In summary, our data suggest that the phosphoryla-
tion of ERM proteins, including ezrin, is regulated
during the metastasis process in osteosarcoma. PKC is
responsible for the phosphorylation of ezrin C-terminal
threonine both early after cells arrive at secondary sites
and during the invasion of the metastatic lesion into the
surrounding parenchyma. Efforts are underway to
develop isoform-specific PKC inhibitors as therapeutic
drugs to treat cancer. The connection between ezrin and
PKC suggest that cancers that are dependent on ezrin
may be responsive to PKC inhibitors and that these
inhibitors may be especially active in patients at high
risk for metastasis.
Materials and methods
K7M2 and K12 murine osteosarcoma cell lines have been
described previously (Khanna et al., 2001). MNNG/HOS
human osteosarcoma cell lines were obtained from ATCC
(Manassas, VA, USA) and grown in DMEM (Invitrogen,
Carlsbad, CA, USA) containing 10% fetal bovine serum.
Geneticin (0.8mg/ml) (Invitrogen) was added to the medium
of K7M2 cells that had been transfected with antisense ezrin or
ezrin mutant constructs.
In vivo studies and immunohistochemistry
The experimental and spontaneous metastasis assays were
performed as previously described (Khanna et al., 2000). For
the experimental metastasis assay, the entire lung was
harvested from mice on days 5, 15, 20 and 24 after injection.
For spontaneous metastasis assay, the lungs were collected
from days 0 to 50 after the removing of primary tumor. Lungs
were inflated by tracheobronchial injection of 1.0ml neutral
buffered 10% formalin (Fisher, Newark, DE, USA). All
tissues were fixed in formalin immediately after collection for
24h, and then transferred to 80% ethanol. All tissues were
embedded in paraffin, sectioned at 5mm thickness and
mounted on glass slides. Slides were deparaffinized and
rehydrated as previously described (Khanna et al., 2001).
Slides were incubated in preheated target retrieval solution
(Dako, Carpinteria, CA, USA), pH6, in a steam cooker for
20min. Anti-ezrin antibody (Sigma, St Louis, MO, USA) was
used at 1:500 dilution, anti-phospho-ezrin(Thr567)/radix-
in(Thr564)/moesin(Thr558) (phosphorylated ERM) antibody
(Cell Signaling Technology Inc., Danvers, MA, USA) and
anti-phospho-PKCa (Ser657) antibody (Upstate, Swampscott,
MA, USA) at 1:100 dilution. The samples were counter-
stained with hematoxylin (Dako) for 30s and mounted.
Immunoprecipitation and western blotting
Cells were lysed in RIPA buffer (150mM NaCl, 50mM Tris,
pH 8.0, 0.1% SDS, 0.25% deoxycholate, 1% NP-40) with
protease inhibitor cocktail (Roche Diagnostics, Indianapolis,
IN, USA) and 1mM calyculin A (Alexis Biochemicals, Lausen,
Switzerland). Lysates containing 1–2mg protein were pre-
cleared with A/G agarose beads (Pierce Biotechnology Inc.,
Rockford, IL, USA) and incubated overnight at 41C with 7ml
of anti-ezrin serum (kindly provided by Dr Anthony Bretscher,
Cornell University) or normal rabbit serum. Proteins were
resolved on a 6% SDS–polyacrylamide gel electrophoresis and
transferred to nitrocellulose membrane. To detect the expres-
sion of PKC isoforms, OS cells were lysed in SDS sample
loading buffer. Proteins were resolved on a 4–12% SDS–
polyacrylamide gel electrophoresis and transferred to nitro-
cellulose membrane. Western blotting was performed with
anti-ezrin (1:4000 dilution) (Sigma), anti-PKCa (1:1000 dilu-
tion) (Upstate), anti-PKCg (1:1000 dilution) (BD Biosciences,
Palo Alto, CA, USA), anti-PKCd (1:500 dilution) (BD
Biosciences), anti-PKCi (1:250 dilution) (BD Biosciences),
anti-PKCe (1:500 dilution) (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA, USA), anti-PKCz (1:500 dilution) (Santa
Cruz Biotechnology Inc.), anti-PKCZ (1:500 dilution) (Santa
Cruz Biotechnology Inc.), anti-PKCb (1:250 dilution) (BD
Biosciences), anti-PKCy (1:200 dilution) (Santa Cruz Biotech-
nology Inc.) and anti-panPKC (1:500 dilution) (Santa Cruz
Biotechnology Inc.) antibodies.
Cells were fixed with 3% paraformaldehyde/phosphate-
buffered saline, and permeablized with 0.2% Triton-X 100.
After blocking with 0.2% bovine serum albumin for 30min,
cells were incubated with anti-phosphorylated ERM antibody
(Cell Signaling Technology Inc.) at 1:100 dilution for 30–
60min, followed by FITC-labeled secondary antibody (Mole-
cular Probe, Eugene, OR, USA). Stained cells were mounted
with VectaShield (Vector Laboratories, Burlingame, CA,
USA) mounting medium and visualized using a Leica DMIRB
fluorescent microscope at 10? or 40? magnification.
Kinase inhibitor treatments
Equal number of cells were plated in six-well tissue culture
plates, grown to 70% confluence and then treated with the
pharmacological inhibitors for PKC (BIM, Ro31-8220, Go ¨
6976) (Alexis Biochemicals), Rho-kinase (Y27632) (Santa
Cruz), PI3 kinase (LY294002) (Cell Signaling Technology
Regulation of ezrin by PKC
L Ren et al
Inc.) or MEK1/2 (U0126) (Sigma). Dose titration (0, 0.1, 1, 5,
10, 20mM) at 60min and time course (0, 10, 30, 60, 120min)
studies at optimal doses were performed. Dimethylsulfoxide
was used as the control for the treatments. The treated cells
were lysed in 200ml of 1? Laemmli’s buffer. Western blot
analysis was performed using anti-phosphorylated ERM
antibody (1:1000 dilution). The phosphorylation of Akt and
ERK1/2 (p44/42) confirmed the inhibition of PI3 kinase and
K7M2 cells were plated in six-well tissue culture plates and
grown to 70% confluence in complete medium. A ‘wound’ was
made by scraping with a P200 pipette tip in the middle of the
cell monolayer. Floating cells were removed by washing with
phosphate-buffered saline and fresh complete medium con-
taining dimethylsulfoxide or 1mM Go ¨ 6976 was added. Cells
were incubated at 371C for 24h. Phase contrast images were
then taken using a Leica DMIRB inverted microscope.
Cell migration assay
In vitro tumor cell migration was assessed using a 24-Multiwell
Insert System (HTS FluoroBlok, BD Biosciences) containing
an 8-mm pore size polyethylene terephthalate membrane.
Briefly, 0.5ml of tumor cells (5?104or 2.5?104cells per
ml) resuspended in DMEM medium containing 5% fetal
bovine serum was added to the upper chamber (in triplicate).
DMEM medium containing 10% fetal bovine serum was
added to the lower chamber. The PKC inhibitor (Ro31-8220
or Go ¨ 6976) was added in both upper and lower chambers. The
cells were incubated for 18h at 371C and 5% CO2. Migrated
cells were then stained with 5mM Calcein AM (Molecular
Probe) in HBSS buffer for 1h at 371C. To quantitate tumor
cell migration, fluorescently labeled cells were detected at an
excitation wavelength of 485nm and emission wavelength of
530nm using a Wallac Victor 3 microplate reader (Perkin-
Elmer, Wellesley, MA, USA). Migration experiments were
repeated at least three times. Unpaired t-test with Welch’s
correction was used for the migration studies. Statistical
analyses were performed using GraphPad Prism version 4.0b
for the Macintosh (GraphPad Software).
Transfection and expression of ezrin mutant
K7M2 cells in six-well tissue culture plates were grown in
DMEM with 10% fetal bovine serum to 70% confluence.
Transfection of the mutant ezrin construct (pEGFP-N1-
ezrinT567D-GFP) was performed with Trans IT-LT1 (Mirus
Bio Co. Madison, WI, USA) transfection reagent following the
manufacturer’s protocol. Two days after transfection, selection
was started using media containing 0.8mg/ml G418. Stable
transfected single cell clones (T567D1, T567D3 and T567D2.4)
were utilized for all experiments.
K7M2 cells expressing GFP or ezrin-T567D-GFP were fixed
with 3% paraformaldehyde/phosphate-buffered saline, and
permeabilized with 0.2% Triton-X 100. Cells were incubated
with rhodamine phalloidin (Invitrogen) at 1:200 dilution for
20min and then washed with phosphate-buffered saline.
Confocal images were obtained in Zeiss confocal microscope
LSM510 using Zeiss LSM imagine browser software (Carl
Zeiss, Oberkochen, Germany).
We would like to thank Drs Joseph Briggs and Luowei Li for
experimental advice and their critical review of this manu-
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
Regulation of ezrin by PKC
L Ren et al