1 Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road,
Glasgow, G61 1BD Scotland
2 The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121,
1066 CX Amsterdam, The Netherlands
* Author for correspondence (e-mail: g.stapleton{at}beatson.gla.ac.uk )
Accepted 23 April 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: CD44, Ezrin, Protein Kinase C
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two such gene products upregulated in these libraries are CD44 and the
CD44-interacting protein, Ezrin. CD44 is a transmembrane glycoprotein and the
major cell surface receptor for the ECM component, hyaluronan. Extensive
alternative splicing of the CD44 gene results in a large number of
CD44 isoforms that participate in functions including lymphocyte homing, cell
adhesion and migration (Aruffo et al.,
1990; Jalkanen et al.,
1987
; Thomas et al.,
1992
). CD44 is also known to be upregulated in a variety of
tumours with metastatic or invasive properties
(Gunthert et al., 1991
;
Sy et al., 1997
). Similarly,
ezrin was found to be upregulated in a number of screens designed to identify
genes involved in metastasis and invasion
(Jooss and Muller, 1995
;
Khanna et al., 2001
;
Nestl et al., 2001
;
Ozanne et al., 2000
). Ezrin is
a member of the Ezrin-Radixin-Moesin family
of proteins (ERM) that serve as molecular crosslinkers between the actin
cytoskeleton and the plasma membrane
(Mangeat et al., 1999
). In
this capacity, ERM proteins participate in a number of cell processes
including the maintenance of cell morphology, cell adhesion and motility
(Bretscher et al., 2000
).
CD44 expression is required for invasion by both v-fos-transformed
and EGF-transformed fibroblasts; in these cells it localises to the tips of
pseudopodial cell extensions (Lamb et al.,
1997a). CD44 variant isoforms localise to the tips of
`invadapodia' in Met1 cells, where they function to localise
matrixmetalloproteases for directional degradation of the ECM
(Bourguignon et al., 1998
;
Yu and Stamenkovic, 1999
;
Yu and Stamenkovic, 2000
).
Ezrin is the only ERM protein to be upregulated by the v-fos
oncogene, being required for the formation of the extending pseudopod
(Lamb et al., 1997b
).
Both CD44 and ezrin are regulated by phosphorylation. CD44 is
phosphorylated exclusively on serine residues
(Neame and Isacke, 1992), and
the major phosphorylation site on the CD44 cytoplasmic tail is located at
serine 325, and there is an additional less well characterised Protein Kinase
C (PKC) consensus site at serine 291. The significance of CD44 phosphorylation
is not fully understood, although CD44 phosphorylation regulates cell
migration on a hyaluronan substrate (Peck
and Isacke, 1996
) and may regulate association with the
cytoskeleton in macrophages (Camp et al.,
1991
). Phosphorylation of a conserved threonine at the C-terminus
of ERM proteins, together with phospholipid interaction, is required for
plasma membrane localisation (Barret et
al., 2000
; Nakamura et al.,
1999
; Niggli et al.,
1995
). Ezrin is also tyrosine phosphorylated by the EGF receptor
and the HGF receptor (Crepaldi et al.,
1997
; Krieg and Hunter,
1992
), and while tyrosine phosphorylation is required for motility
and morphogenesis in an epithelial cell line, it has no effect on cellular
localisation of ezrin (Crepaldi et al.,
1997
). Amongst the various kinases that have been shown to be
capable of phosphorylating ezrin and CD44 is PKC. Purified CD44 is
phosphorylated by PKC in vitro (Kalomiris
and Bourguignon, 1989
). The PKC
isoform phosphorylates
moesin and ezrin in vitro (Pietromonaco et
al., 1998
; Simons et al.,
1998
), and more recently PKC
was shown to phosphorylate
ezrin in vivo (Ng et al.,
2001
).
PKC is a family of serine-threonine kinases that are differentially
regulated by lipid and calcium (Mellor and
Parker, 1998). Conventional isoforms of PKC (
, ßI,
ßII,
) require phosphatidylserine and diacylglycerol together with
calcium for activation. The novel isoforms (
,
,
, µ)
are calcium independent, and the atypical isoforms (
/
,
)
require neither lipid or calcium for activation. A number of reports support a
role for PKC in cell motility, invasion and regulation of the cytoskeleton.
PKC
overexpression resulted in increased motility and adhesion of a
non-metastatic mammary epithelial cell line
(Sun and Rotenberg, 1999
),
whereas a constitutively active form of PKC
increased invasion of
intestinal cells (Batlle et al.,
1998
). The novel PKC
isoform is involved in the regulation
of cell adhesion and spreading (Berrier et
al., 2000
; Chun et al.,
1996
), whereas PKC
regulates migration of endothelial cells
(Tang et al., 1997
). Atypical
PKC isoforms are involved in regulating the organisation of the actin
cytoskeleton (Laudanna et al.,
1998
; Uberall et al.,
1999
).
AP-1 activity is required for invasion by the
squamous-cell-carcinoma-derived cell line A431 in response to EGF
(Malliri et al., 1998). A431
cells overexpress the EGF receptor: treatment with EGF results in the rapid
rearrangement of the actin cytoskeleton including Rac-dependent membrane
ruffling and Rho-dependent cortical actin polymerisation and cell rounding,
leading to increased cell motility and invasion. Downregulation of AP-1
activity in A431 cells by expression of dominant-negative c-Jun (TAM67)
results in cells that are no longer motile, invasive or capable of Rac- and
Rho-dependent reorganisation of the actin cytoskeleton in response to EGF
(Malliri et al., 1998
).
Although it is clear that a new profile of gene expression is required to
implement the invasion programme, it is also necessary that the regulatory
signals required for the function of each new gene product in the context of
invasion are also in place. Here we investigate the regulation of CD44 and
ezrin localisation in A431 cells after EGF treatment to initiate cell
invasion, compared with invasion-defective A431 cells expressing TAM67 (TA
cells). We find that correct EGF-dependent colocalisation of both CD44 and
ezrin is defective in TA cells and provide evidence that associated
upregulation of PKC contributes to disruption of the invasion
programme.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines, culture and transfection
A431 cells, A431 cells stably expressing an empty neomycinencoding vector
(NA cells) and A431 cells expressing both a neomycin-encoding plasmid and an
expression construct encoding TAM67 (TA cells) have been described previously
(Malliri et al., 1998).
PKC
-expressing cell lines were established by transfecting a PKC
expression plasmid using Fugene6 (Roche) and selecting colonies grown in the
presence of G418. Cells were maintained in DMEM (Sigma) containing 10% FCS
(Harlan SeraLab) and, in the case of NA, TA and A431-
cells, 500
µg/ml G418, at 37°C, 5% CO2. For EGF or TPA treatment, cells
were plated either on tissue culture plates or on glass coverslips, allowed to
attach overnight, then transferred to medium without FCS for 2 days. 10 ng/ml
EGF or 100 ng/ml TPA was used to treat cells.
Wound-healing assays
A431 and A431- cells were grown as a confluent monolayer, serum
starved for 2 days, then wounded using a disposable pipette tip. 10 ng/ml EGF
was added to the medium and the extent of wound closure was determined after
36 hours.
Immunofluorescence
Cells were seeded onto glass coverslips, allowed to attach overnight, then
transferred to serum-free medium for 2 days. Following various treatments,
cells were fixed in 4% formaldehyde in PBS for 15 minutes and permeabilised in
PBS containing 0.1% Triton X-100. Cells were blocked in 10% FCS, 0.5% BSA in
PBS for 1 hour, followed by the addition of antibody diluted in blocking
buffer. The antibody dilutions used were: CD44, neat; ezrin, 1:500. Cells were
washed in blocking buffer then incubated in either goat-anti-mouse IgG TRITC
conjugate (Sigma) (for CD44) or goat-anti-rabbit IgG FITC conjugate (Sigma)
(for ezrin), both diluted in blocking buffer at 1:60, together with 500 ng/ml
TRITC- or FITC-conjugated phalloidin. Cells were washed in blocking buffer
followed by a final wash in PBS containing 0.1% Triton X-100. Coverslips were
mounted onto glass slides using Vectashield (Vector Laboratories), followed by
confocal microscopic analysis using a Biorad MRC 600 confocal illumination
unit attached to a Nikon Diaphot inverted microscope. Cells were treated with
PKC inhibitors for 30 minutes at 3 µM, followed by EGF treatment and
fixation.
Western analysis
Cell lysates were prepared by washing cells twice in ice-cold PBS, followed
by scraping into lysis buffer (20 mM Hepes pH 7.4, 5 mM EDTA, 10 mM EGTA, 5 mM
Na fluoride, 1 mM KCl, 0.4% Triton X-100, 10% glycerol, 1 mM benzamidine, 5
µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 1 mM Na vanadate).
Cleared supernatants were collected and quantified using the copper
sulphate/bicinchoninic acid method (Sigma). 50 µg of protein lysate was
electrophoresed on SDS-PAGE gels (no reducing agent was used for CD44
analyses). Proteins were transferred to a polyvinylidene difluoride membrane
(Immobilon-P, Millipore) using a semi-dry blotter. Filters were blocked in 5%
semi-skimmed milk powder in TBST (20 mM Tris pH 7.6, 137 mM NaCl, 0.1%
Tween-20) for 1 hour prior to incubation with CD44 antibody (1:10 dilution),
ezrin antibody (1:10,000 dilution) or PKC antibodies (at dilutions recommended
by the vendor). Filters were washed in 5% semi-skimmed milk powder in TBST
then incubated with a horseradish-peroxidase-conjugated sheep anti-mouse or
anti-rabbit Ig (Amersham, dilution 1:5000) for 1 hour. Filters were washed in
TBST followed by ECL (Amersham) to detect positive signals.
Cell fractionation
Soluble and particulate fractions were prepared as previously described
(Goodnight et al., 1995).
Cells were washed in ice-cold PBS, scraped into 20 mM Tris pH 7.5, 2 mM EDTA,
2 mM EGTA, 1 mM PMSF, 20 µg/ml leupeptin, 80 µg/ml aprotinin and 0.1%
2-mercaptoethanol then sonicated for 10 seconds. The soluble supernatant was
collected, and the pellet was resuspended in 1xSDS PAGE sample buffer
and sonicated for 10 seconds.
Northern analysis
Total RNA was prepared using RNazol B (Biogenesis Ltd) according to the
manufacturer's instructions. Electrophoresis of 20 µg of total RNA in a 1%
agarose, 200 mM MOPS, 7% formaldehyde gel was followed by capillary transfer
to a Hybond-N nylon filter (Amersham) and UV crosslinking using a UV
Stratalinker 1800 (Stratagene). Radiolabelled PKC and 7S ribosomal cDNA
probes were prepared by random priming using the AmershamPharmacia DNA
labelling kit and
-32P-dCTP (Amersham). Hybridisation was
carried out at 68°C overnight in 0.25 M NaPO4 pH 7.2, 7% SDS, 1
mM EDTA and 1% BSA. Filters were washed three times in 20 mM NaPO4
pH 7.2, 1% SDS, 1 mM EDTA, 20 minutes, 68°C and exposed for
autoradiography.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
After a 5 minute exposure to EGF, a proportion of CD44 swiftly localises to membrane ruffles and also concentrates in newly formed apical microvilli (Fig. 1B,a,b). We found identical, localisation of the standard 85 kDa form of CD44 (CD44s)-GFP fusion protein in A431 cells before and after EGF treatment (data not shown). Similarly, EGF results in an identical relocalisation of ezrin to membrane ruffles and microvilli (Fig. 1Bc,d) (Bretscher, 1989), precisely overlapping CD44 localisation (Fig. 1Be,f). Thus, signals from the EGF receptor result in the assembly of functionally interacting proteins at locomotory structures in a similar manner to that observed in FBR cells, where the activity of each is required for cell invasion.
Expression of dominant-negative c-Jun (TAM67) results in CD44 and
ezrin mislocalisation
Previously we have shown that A431 cells require AP-1 activity for
EGF-induced invasion, motility and associated reorganisation of the actin
cytoskeleton (Malliri et al.,
1998). We therefore examined whether the localisation of two gene
products required for invasion, CD44 and ezrin, was affected in A431 cells
expressing TAM67 (TA cells) (Malliri et
al., 1998
). CD44 localisation is completely disorganised in TA
cells (Fig. 2A, compare c with
a). CD44 is mostly absent from sites of cell-cell contact and the intensity of
staining is greatly reduced compared with NA cells (A431 cells expressing an
empty neomycin expression vector). Upon EGF treatment of TA cells, CD44
localisation remains generally disorganised and has a lower signal intensity
(Fig. 2A, compare d with
b).
|
The immunolocalisation data suggested either that CD44 was now uniformly
distributed over the cell surface of TA cells or that CD44 expression levels
were greatly reduced in these cells. Western analysis showed that the
expression levels and isoform profile of CD44 is unaffected in TA cells
(Fig. 2B). A431 and TA cells
both express the standard 85 kDa form (CD44s), the 145 kDa epithelial isoform
(CD44E) and a >200 kDa form that most probably corresponds to CD44v3 8-10,
which is expressed in A431 cells (Grimme
et al., 1999). Thus CD44 localisation, rather than isoform
expression levels, is affected by downregulated AP-1 activity. Although it is
known that CD44 is an AP-1 target gene, our results are consistent with other
reports showing that dominant-negative versions of c-Jun
(Johnston et al., 2000
;
Young et al., 1999
) and JunD
(Ui et al., 2000
) do not
affect expression of all AP-1 target genes.
Likewise, ezrin fails to relocalise to the plasma membrane or microvilli in response to EGF in TA cells, remaining in the cytosol of both resting and EGF-treated TA cells (Fig. 3A). Ezrin protein levels are also equal in NA and TA cells (Fig. 3B). Invasion-defective TA cells, therefore, are incapable of assembling two proteins necessary for cell motility and invasion into locomotory structures. AP-1 activity is required for expression of the gene(s) necessary to signal correct ezrin and CD44 localisation from the EGF receptor.
|
Protein Kinase C activity is required for ezrin and CD44
localisation
PKC is capable of phosphorylating both CD44 and ezrin, and the localisation
of ERM proteins to the membrane is regulated in part by phosphorylation of a
conserved C-terminal threonine (Nakamura
et al., 1999). To address whether PKC activity is required for
CD44 and ezrin localisation in A431/NA cells, we inhibited PKC activity using
the broad spectrum PKC inhibitor, Ro-31-8220 (identical results were obtained
using another PKC inhibitor, GF109203X) (data not shown).
Serum-starved cells were pretreated with 3 µM Ro-31-8220 for 30 minutes before EGF treatment and fixation. In both EGF-treated and untreated cells, CD44 localisation is affected (Fig. 4A). After inhibitor pre-treatment, serum-starved cells have a disrupted actin cytoskeleton, and the cells appear to be detaching from each other. CD44 staining is reduced in cell-cell contacts and appears as a uniform signal over the cell surface (Fig. 4A). However, when the same inhibitor-treated cells were stained for E-cadherin, a marker for the integrity of cell-cell contacts, the same disrupted staining pattern resulted (data not shown). This suggests that the effect of the PKC inhibitor was an indirect effect caused by disruption of cell-cell contacts, and we conclude that the PKC inhibitor has not directly affected CD44 localisation in untreated cells.
|
Upon EGF treatment, however, Ro-31-8220-treated A431 cells still reorganise the actin cytoskeleton, producing large membrane lamellipodia and cortical actin polymerisation, yet CD44 remains localised to cell-cell contacts and does not mobilise to the newly formed membrane structures (Fig. 4A). EGF-induced CD44 localisation is therefore directly dependent on PKC activity and is not an indirect consequence of effects on the actin cytoskeleton. Ezrin distribution in A431 cells is again affected by the presence of the PKC inhibitor (Fig. 4B). In Ro-31-8220-treated serum-starved cells, ezrin remains cytosolic; however, upon EGF treatment, ezrin fails to efficiently relocalise to the plasma membrane even though lamellipodia formation and cortical actin polymerisation have occurred (Fig. 4B). We conclude that CD44 and ezrin localisation requires PKC activity and suggest that the failure of CD44 and ezrin to localise in TA cells may be caused by aberrant PKC signalling in the cells.
Phorbol ester activation of conventional and novel Protein Kinase C
is insufficient for relocalisation of ezrin and CD44
We next established whether activation of PKC alone is sufficient to
relocalise both proteins and whether direct activation of PKC in TA cells
would result in ezrin or CD44 relocalisation. This was achieved by determining
the distribution of CD44 and F-actin (Fig.
5A,B) and ezrin (Fig.
5C,D) in NA and TA cells before and after treatment with phorbol
ester (TPA), a potent activator of conventional and novel PKC isoforms.
Treatment of NA cells results in actin concentrating in sites of cell-cell
contact and an increase in actin cables, with little or no membrane ruffling
observed (Fig. 5A). This is
consistent with a previous report describing the effect of phorbol ester on
the actin cytoskeleton in A431 cells
(Vaaraniemi et al., 1999).
Interestingly, TPA treatment of TA cells results in the same reorganisation of
the actin cytoskeleton (Fig.
5B). Although TPA- and EGF-induced reorganisation of the actin
cytoskeleton is different (compare Fig.
4A,B upper panels with Fig.
5A), it is clear that PKC can be activated in TA cells and can
transduce signals to effector molecules required for reorganisation of the
actin cytoskeleton.
|
TPA treatment however has no effect on CD44 distribution in either NA or TA cells. CD44 remains tightly associated with cell junctions and basal surface plaques in NA cells (Fig. 5A), and no change in organisation or intensity of CD44 staining was seen in TA cells treated with TPA (Fig. 5B). Similarly, ezrin distribution is not affected by TPA treatment of either NA or TA cells (Fig. 5C,D). Ezrin remains cytosolic in the presence of TPA, even though PKC activation has resulted in actin cytoskeletal rearrangements. Therefore, PKC is necessary for CD44 and ezrin localisation to the plasma membrane of A431 cells; however, additional signal(s) from the EGF receptor are required, and these signals are not delivered in TA cells. These conclusions, however, do not extend to atypical PKC isoforms that are not activated by phorbol ester.
Aberrant Protein Kinase C isoform expression in AP-1-deficient TA
cells
In light of the effects seen using PKC inhibitors on CD44 and ezrin
localisation, we next investigated whether expression of TAM67 resulted in
changes to PKC isoform expression. Liu et al. have previously shown that a
single representative isoform from each PKC subgroup is expressed in A431
cells (conventional, PKC; novel, PKC
; atypical, PKC
) (Liu
et al., 1994). While PKC
and PKC
expression levels are unchanged
in TA cells, PKC
expression is reduced three-fold in TA cells
(Fig. 6A). More significantly,
however, we found that an extra novel PKC isoform, PKC
, is expressed in
TA cells (Fig. 6A). The 3.4 kb
PKC
transcript is also upregulated in TA cells and absent from NA
cells, indicating transcriptional upregulation of PKC
(Fig. 6B). We have confirmed
PKC
upregulation by western analysis of a number of independent TA cell
lines, finding that all cell lines expressing TAM67 also express PKC
(data not shown). Together, these data establish that PKC expression levels
and isoform profile are altered in TA cells by the addition of an extra novel
isoform and suggest that signalling pathways through PKC may too be
affected.
|
PKC translocates to the cellular particulate fraction upon EGF
treatment
In order to determine whether PKC is activated in response to EGF
and whether the increased expression of an extra isoform might interfere with
the activation of endogenous PKC isoforms, we examined translocation of PKC
isoforms from the cytosol to the plasma membrane after treatment with EGF or
TPA. Soluble and insoluble protein fractions were prepared from serum-starved,
EGF-treated and TPA-treated NA cells and the same for TA cells, followed by
western analysis to determine the relative distribution of each PKC isoform
(Fig. 7A).
|
Phorbol ester treatment of A431 cells results in the expected shift of
PKC from the cytosol to the membrane fraction
(Fig. 7A). TA cells treated
with TPA are also capable of translocating PKC
, indicating that
signalling events independent of the EGF receptor function equivalently in
A431 and TA cells. There is no detectable PKC
in the membrane fraction
after EGF treatment of NA or TA cells. It may be that only a very small,
undetectable fraction of PKC
translocates after EGF treatment or that
EGF does not activate PKC
. Nevertheless, there is no difference in the
cellular partitioning of PKC
between NA and TA cells.
In unstimulated NA cells, PKC can be detected in both the cytosolic
and membrane fractions. Treatment with TPA results in a small increase in
PKC
in the particulate fraction; however EGF treatment does not result
in any significant change in the amount of PKC
in either fraction
(Fig. 7A). The distribution of
PKC
is equivalent in NA and TA cells. These data suggest either that
PKC
is not being activated in response to EGF or that the sensitivity
of this assay is such that a small change in cellular localisation will not be
detected. Nevertheless, there is no difference in PKC
fractionation in
response to EGF by either cell type. Atypical PKC
distribution remains
largely cytosolic, with only a small amount detected in the membrane fraction
(Fig. 7A). This partitioning
pattern is conserved in the TA cells and does not change after TPA or EGF
treatment. This is consistent with the inability of PKC
to bind phorbol
ester and has been seen also in fibroblasts overexpressing PKC
(Goodnight et al., 1995
).
We next examined the distribution of PKC in TA cells to determine
whether it was activated by EGF. In unstimulated cells, PKC
is detected
in both the cytosolic and membrane fractions; however after either TPA or EGF
treatment, there is a large shift of PKC
to the membrane fraction
(Fig. 7A), indicating
PKC
activation in TA cells treated with EGF. In summary, the
fractionation of PKC after growth factor treatment shows that there is no
gross effect on endogenous PKC membrane translocation owing to the expression
of PKC
. PKC
, however, is clearly sent to the membrane of
EGF-treated TA cells, indicating activation of the kinase in response to
growth factors.
EGF-induced phosphorylation of endogenous PKC and
are
equivalent in NA and TA cells
A parallel approach to confirm the activation state of endogenous PKC in NA
and TA cells utilised phosphospecific PKC antibodies. A phosphoPKC (pan)
antibody capable of detecting a subset of phosphorylated PKC isoforms was used
in western analysis of EGF- and TPA-treated NA and TA cell lysates
(Fig. 7B). The antibody
efficiently recognised phosphorylated PKC in response to both EGF and
TPA in both cell types. This is inconsistent with the fractionation data where
EGF fails to relocalise PKC
to the membrane; however it may be that the
sensitivities of each assay differ such that only a small undetectable amount
of PKC
fractionates to the membrane. Neither TPA or EGF resulted in an
increased phosphorylation of PKC
in either NA or TA cells, suggesting
that PKC
is not efficiently activated in A431 cells at the timepoint
tested. Nevertheless, it is evident that the activation profile of PKC
and PKC
by EGF is equivalent in both NA and TA cells, and therefore the
expression of PKC
in TA cells does not appear to affect activation of
endogenous conventional or novel PKC isoforms.
Expression of PKC in A431 cells mimics aspects of the TA
phenotype
To determine whether any of the effects described in TA cells were
attributable to PKC, we established stable A431 cell lines expressing
PKC
(A431-
) (Fig.
8A). Because TA cells are non-motile and non-invasive, we
determined firstly whether motility had been similarly affected by PKC
expression. EGF induces the closure of a wound made in a monolayer of
serum-starved A431 cells after 36 hours; however, A431-
cell lines are
unable to migrate into the wound (Fig.
8B), indicating a defect in cell motility. Expression of
PKC
is therefore sufficient to disrupt EGF-receptor-directed signalling
to implement the invasion programme.
|
We next examined the effect of PKC expression on EGF-induced ezrin
(Fig. 9) and CD44
(Fig. 10) localisation,
together with actin cytoskeletal rearrangements, by immunofluorescence. While
the distribution of actin, ezrin and CD44 appears unaffected in serum-starved
A431-
cells (Fig. 9A,
Fig. 10A), confocal sections
perpendicular to the substratum reveal that A431-
cells are
significantly flatter than A431 cells (Fig.
9B, Fig. 10B).
Upon EGF treatment, A431-
cells do round up and increase their height
in response to EGF; however this is still reduced compared with EGF-treated
A431 cells. EGF-treated TA cells similarly do not round or increase in height
compared with A431 cells. Cell rounding in EGF-treated A431 cells is a
consequence of F-actin cortical polymerisation and cell contraction.
PKC
expression in A431-
cells similarly has effects on actin
cytoskeletal reorganisation. F-actin cortical polymerisation and cell
contraction are reduced in A431-
cells. More significantly, very little
membrane ruffling is produced in response to EGF; however A431-
cells
still form apical microvilli (Figs
9 and
10). this is in contrast to TA
cells where neither structure is formed. PKC-
therefore has partially
mimicked TAM67 expression by affecting a subset of cytoskeletal events but
still results in non-motile cells.
|
|
Ezrin and CD44 localisation is partially affected in EGF-treated
A431 cells. There is reduced membrane localisation by both proteins in
response to EGF (Fig. 9A,
Fig. 10A); however, CD44 and
ezrin can localise to apical microvilli
(Fig. 9B,
Fig. 10B). In both cases, the
absence of membrane ruffles accounts for the lack of either protein at the
membrane. Again this partially mimics the effect of TAM67 expression, where
extension of membrane ruffles does not occur, and consequently the
mislocalisation of CD44 and ezrin in TA cells. PKC
expression therefore
has disrupted ezrin and CD44 localisation to membrane ruffles, and this may be
an indirect effect resulting from effects on the actin cytoskeleton. When
membrane structures, such as microvilli, are formed, ezrin and CD44 are able
to localise. We conclude that PKC
expression in A431 cells contributes
to the disruption of cell invasion by TAM67, most probably through effects on
the actin cytoskeleton, which in turn, affects membrane ruffling and therefore
localisation of CD44 and ezrin to these structures and consequently cell
motility.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Constitutive expression of dominant-negative Jun (TAM67) results in changes
in gene expression, some of which will be through directly affecting AP-1
activity, whereas others may be the result of interfering with the expression
and activity of other transcription factors (e.g.
Li et al., 2000). CD44 is a
known AP-1 target gene, and downregulation of its expression might have been
expected in TA cells. However, we have found that expression of TAM67 is
incapable of downregulating 14% of genes that are upregulated in
v-fos expressing fibroblasts
(Johnston et al., 2000
). Our
findings are also consistent with others where expression of dominant-negative
versions of c-jun (Young et al.,
1999
) or JunD (Ui et al.,
2000
) does not affect expression of all AP-1 targets. It is worth
noting nevertheless that when used in an inducible system TAM67 was able to
revert expression of two AP-1-dependent genes
(Li et al., 2000
). The future
use of an inducible TAM67 in A431 cells could extend our findings, revealing
the timing of PKC
expression and indeed whether PKC
upregulation
is a direct consequence of downregulated AP-1.
Inhibiting AP-1 activity by TAM67 in A431 cells results in the failure of
the EGF receptor to colocalise CD44 and ezrin to the plasma membrane. Here we
show that PKC activity is required for CD44 and ezrin translocation. Further,
TAM67 expression is associated with abnormal PKC expression and function. We
have previously shown that downregulation of AP-1 activity in A431 cells
similarly results in the uncoupling of signalling events from the EGF receptor
to the reorganisation of the actin cytoskeleton
(Malliri et al., 1998). These
events were shown to be dependent on Rac and Rho GTPase activities, and these
signalling pathways failed to correctly signal to the cytoskeleton in TA
cells. We have subsequently shown that Rac and Rho GTPases are required for
CD44 and ezrin localisation, suggesting that an intact cytoskeleton is
required for their localisation (G.S., unpublished). Nevertheless, the
requirement for PKC activity for CD44 and ezrin localisation is independent of
actin cytoskeletal rearrangements, as CD44 and ezrin both fail to localise to
the plasma membrane in the presence of PKC inhibitors, even though actin
polymerisation and lamellipodial extensions are produced
(Fig. 4A,B). Thus two
signalling pathways downstream of the EGF receptor are perturbed in
TAM67-expressing cells, each presumably contributing to the non-invasive state
of the cells.
There are several mechanism(s) through which the introduction of an extra
PKC isoform in TA cells could affect endogenous PKC function. Firstly,
PKC might block the function of endogenous isoforms either by competing
for upstream activators or by binding to PKC-binding partners and substrates.
PKC
is capable of phosphorylating the conserved C-terminal threonine of
the ERM family member, moesin and ezrin
(Simons et al., 1998
).
Although it seems contradictory that ezrin should not localise in TA cells if
PKC
is now expressed, it is known that other events, such as
phosphatidyl-inositol 4,5-bisphosphate
[PtdIns(4,5)P2]-binding is required for ezrin localisation
(Barret et al., 2000
), and it
may be that additional signals, including those to localise the kinase, are
lacking in TA cells. Nevertheless, it cannot be predicted how PKC
will
behave in epithelial cells in response to EGF receptor signalling compared to
its function in T cells.
Secondly, PKC could phosphorylate a new set of substrates in the
cell, independently of endogenous PKC function, which somehow interferes with
the correct localisation of CD44 and ezrin. Thirdly, PKC
may interfere
with signalling from the EGF receptor by interacting with signalling molecules
that are not normally associated with endogenous PKC in A431 cells. To this
end, PKC
is known to associate with a number of signalling molecules,
including Vav1, a GDP-exchange factor for Rac
(Moller et al., 2001
), and the
Src family tyrosine kinase, Lck (Liu et
al., 2000
).
The data presented suggest that both ezrin and CD44 receive some common
regulatory signal(s) to determine their cellular localisation and subsequent
function; however it may be that signalling to one protein may suffice to
localise the other. The phosphorylation of CD44 appears to be complex, and a
number of kinases have been described as capable of phosphorylating CD44.
ROK is another candidate kinase for both CD44 and ezrin phosphorylation
(Bourguignon et al., 1999
;
Matsui et al., 1998
); however,
we have found no differences in ROK
expression levels in TA and NA
cells (G.S., unpublished). Ezrin phosphorylation at threonine 567 by
PKC
has been demonstrated in a breast cancer cell line
(Ng et al., 2001
). Although
PKC
phosphorylation is evident in A431 cells we do not see an
accompanying shift to the insoluble fraction even though phorbol ester induces
efficient translocation. It seems likely that EGF is an inefficient activator
of PKC
and that only a small amount is translocated.
The inhibition of cell motility, membrane ruffling and associated
localisation of CD44 and ezrin to these structures by PKC expression
supports its contribution to the effects attributed to TAM67. Indeed, the
inhibition of membrane ruffling suggests that PKC
may interfere with
signalling through Rac GTPase. Further investigation of the mechanism of
inhibition by PKC
will reveal not only the endogenous signalling
pathways required for invasion that PKC
has affected but will also shed
light on the potential of PKC
as a novel suppressor of cell
invasion.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B. and Seed, B. (1990). CD44 is the principal cell surface receptor for hyaluronate. Cell 61,1303 -1313.[Medline]
Barret, C., Roy, C., Montcourrier, P., Mangeat, P. and Niggli,
V. (2000). Mutagenesis of the phosphatidylinositol
4,5-bisphosphate (PIP(2)) binding site in the NH(2)-terminal domain of ezrin
correlates with its altered cellular distribution. J. Cell
Biol. 151,1067
-1080.
Batlle, E., Verdu, J., Dominguez, D., del Mont Llosas, M., Diaz,
V., Loukili, N., Paciucci, R., Alameda, F. and de Herreros, A. G.
(1998). Protein kinase C-alpha activity inversely modulates
invasion and growth of intestinal cells. J. Biol.
Chem. 273,15091
-15098.
Berrier, A. L., Mastrangelo, A. M., Downward, J., Ginsberg, M.
and LaFlamme, S. E. (2000). Activated R-ras, Rac1, PI
3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated
integrin beta1 cytoplasmic domains. J. Cell Biol.
151,1549
-1560.
Bourguignon, L. Y., Gunja-Smith, Z., Iida, N., Zhu, H. B., Young, L. J., Muller, W. J. and Cardiff, R. D. (1998). CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells. J. Cell Physiol. 176,206 -215.[Medline]
Bourguignon, L. Y., Zhu, H., Shao, L., Zhu, D. and Chen, Y. W. (1999). Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells. Cell Motil. Cytoskeleton 43,269 -287.[Medline]
Bretscher, A., Chambers, D., Nguyen, R. and Reczek, D. (2000). ERM-Merlin and EBP50 protein families in plasma membrane organization and function. Annu. Rev. Cell Dev. Biol. 16,113 -143.[Medline]
Camp, R. L., Kraus, T. A. and Pure, E. (1991). Variations in the cytoskeletal interaction and posttranslational modification of the CD44 homing receptor in macrophages. J. Cell Biol. 115,1283 -1292.[Abstract]
Chun, J. S., Ha, M. J. and Jacobson, B. S.
(1996). Differential translocation of protein kinase C epsilon
during HeLa cell adhesion to a gelatin substratum. J. Biol.
Chem. 271,13008
-13012.
Crepaldi, T., Gautreau, A., Comoglio, P. M., Louvard, D. and
Arpin, M. (1997). Ezrin is an effector of hepatocyte growth
factor-mediated migration and morphogenesis in epithelial cells. J.
Cell Biol. 138,423
-434.
Goodnight, J. A., Mischak, H., Kolch, W. and Mushinski, J.
F. (1995). Immunocytochemical localization of eight protein
kinase C isozymes overexpressed in NIH 3T3 fibroblasts. Isoform-specific
association with microfilaments, Golgi, endoplasmic reticulum, and nuclear and
cell membranes. J. Biol. Chem.
270,9991
-10001.
Grimme, H. U., Termeer, C. C., Bennett, K. L., Weiss, J. M., Schopf, E., Aruffo, A. and Simon, J. C. (1999). Colocalization of basic fibroblast growth factor and CD44 isoforms containing the variably spliced exon v3 (CD44v3) in normal skin and in epidermal skin cancers. Br. J. Dermatol. 141,824 -832.[Medline]
Gunthert, U., Hofmann, M., Rudy, W., Reber, S., Zoller, M., Haussmann, I., Matzku, S., Wenzel, A., Ponta, H. and Herrlich, P. (1991). A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65, 13-24.[Medline]
Hennigan, R. F., Hawker, K. L. and Ozanne, B. W. (1994). Fostransformation activates genes associated with invasion. Oncogene 9,3591 -3600.[Medline]
Jalkanen, S., Bargatze, R. F., de los Toyos, J. and Butcher, E. C. (1987). Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells. J. Cell Biol. 105,983 -990.[Abstract]
Johnston, I. M., Spence, H. J., Winnie, J. N., McGarry, L., Vass, J. K., Meagher, L., Stapleton, G. and Ozanne, B. W. (2000). Regulation of a multigenic invasion programme by the transcription factor, AP-1: re-expression of a down-regulated gene, TSC-36, inhibits invasion. Oncogene 19,5348 -5358.[Medline]
Jooss, K. U. and Muller, R. (1995). Deregulation of genes encoding microfilament-associated proteins during Fos-induced morphological transformation. Oncogene 10,603 -608.[Medline]
Kalomiris, E. L. and Bourguignon, L. Y. (1989).
Lymphoma protein kinase C is associated with the transmembrane glycoprotein,
GP85, and may function in GP85-ankyrin binding. J. Biol.
Chem. 264,8113
-8119.
Khanna, C., Khan, J., Nguyen, P., Prehn, J., Caylor, J., Yeung,
C., Trepel, J., Meltzer, P. and Helman, L. (2001).
Metastasis-associated differences in gene expression in a murine model of
osteosarcoma. Cancer Res.
61,3750
-3759.
Krieg, J. and Hunter, T. (1992). Identification
of the two major epidermal growth factor-induced tyrosine phosphorylation
sites in the microvillar core protein ezrin. J. Biol.
Chem. 267,19258
-19265.
Lamb, R. F., Hennigan, R. F., Turnbull, K., Katsanakis, K. D., MacKenzie, E. D., Birnie, G. D. and Ozanne, B. W. (1997a). AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44. Mol. Cell Biol. 17,963 -976.[Abstract]
Lamb, R. F., Ozanne, B. W., Roy, C., McGarry, L., Stipp, C., Mangeat, P. and Jay, D. G. (1997b). Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts. Curr. Biol. 7, 682-688.[Medline]
Laudanna, C., Mochly-Rosen, D., Liron, T., Constantin, G. and
Butcher, E. C. (1998). Evidence of zeta protein kinase C
involvement in polymorphonuclear neutrophil integrin-dependent adhesion and
chemotaxis. J. Biol. Chem.
273,30306
-13035.
Li, J. J., Cao, Y., Young, M. R. and Colburn, N. H. (2000). Induced expression of dominant-negative c-jun downregulates NFkappaB and AP-1 target genes and suppresses tumor phenotype in human keratinocytes. Mol. Carcinog. 29,159 -169.[Medline]
Liotta, L. A., Steeg, P. S. and Stetler-Stevenson, W. G. (1991). Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64,327 -336.[Medline]
Liu, Y., Witte, S., Liu, Y. C., Doyle, M., Elly, C. and Altman,
A. (2000). Regulation of protein kinase Ctheta function
during T cell activation by Lck-mediated tyrosine phosphorylation.
J. Biol. Chem. 275,3603
-3609.
Malliri, A., Symons, M., Hennigan, R. F., Hurlstone, A. F.,
Lamb, R. F., Wheeler, T. and Ozanne, B. W. (1998). The
transcription factor AP-1 is required for EGF-induced activation of rho-like
GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431
cells. J. Cell Biol.
143,1087
-1099.
Mangeat, P., Roy, C. and Martin, M. (1999). ERM proteins in cell adhesion and membrane dynamics. Trends Cell Biol. 9,187 -192.[Medline]
Marchisio, P. C., Cirillo, D., Teti, A., Zambonin-Zallone, A. and Tarone, G. (1987). Rous sarcoma virus-transformed fibroblasts and cells of monocytic origin display a peculiar dot-like organization of cytoskeletal proteins involved in microfilament-membrane interactions. Exp. Cell Res. 169,202 -214.[Medline]
Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M.,
Kaibuchi, K. and Tsukita, S. (1998). Rho-kinase
phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins
and regulates their head-to-tail association. J. Cell
Biol. 140,647
-657.
Mellor, H. and Parker, P. J. (1998). The extended protein kinase C superfamily. Biochem. J. 332,281 -292.[Medline]
Moller, A., Dienz, O., Hehner, S. P., Droge, W. and Schmitz, M.
L. (2001). Protein kinase C theta cooperates with Vav1 to
induce JNK activity in T-cells. J. Biol. Chem.
276,20022
-20028.
Nakamura, F., Huang, L., Pestonjamasp, K., Luna, E. J. and
Furthmayr, H. (1999). Regulation of F-actin binding to
platelet moesin in vitro by both phsophorylation of threonine 558 and
polyphosphatidylinositides. Mol. Biol. Cell
10,2669
-26685.
Neame, S. J. and Isacke, C. M. (1992). Phosphorylation of CD44 in vivo requires both Ser323 and Ser325, but does not regulate membrane localization or cytoskeletal interaction in epithelial cells. EMBO J. 11,4733 -4738.[Abstract]
Neame, S. J. and Isacke, C. M. (1993). The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts. J. Cell Biol. 121,1299 -12310.[Abstract]
Nestl, A., von Stein, O. D., Zatloukal, K., Thies, W. G.,
Herrlich, P., Hofmann, M. and Sleeman, J. P. (2001). Gene
expression patterns associated with the metastatic phenotype in rodent and
human tumors. Cancer Res.
61,1569
-1577.
Ng, T., Parsons, M., Hughes, W. E., Monypenny, J., Zicha, D.,
Gautreau, A., Arpin, M., Gschmeissner, S., Verveer, P. J., Bastiaens, P. I. et
al. (2001). Ezrin is a downstream effector of trafficking
PKC-integrin complexes involved in the control of cell motility.
EMBO J. 20,2723
-2741.
Niggli, V., Andreoli, C., Roy, C. and Mangeat, P. (1995). Identification of a phosphatidylinositol-4,5-bisphosphate-binding domain in the N-terminal region of ezrin. FEBS Lett. 376,172 -176.[Medline]
Ozanne, B. W., McGarry, L., Spence, H. J., Johnston, I., Winnie, J., Meagher, L. and Stapleton, G. (2000). Transcriptional regulation of cell invasion: AP-1 regulation of a multigenic invasion programme. Eur. J. Cancer 36,1640 -1648.[Medline]
Peck, D. and Isacke, C. M. (1996). CD44 phosphorylation regulates melanoma cell and fibroblast migration on, but not attachment to, a hyaluronan substratum. Curr. Biol. 6, 884-890.[Medline]
Pietromonaco, S. F., Simons, P. C., Altman, A. and Elias, L.
(1998). Protein kinase C-theta phosphorylation of moesin in the
actin-binding sequence. J. Biol. Chem.
273,7594
-7603.
Saez, E., Rutberg, S. E., Mueller, E., Oppenheim, H., Smoluk, J., Yuspa, S. H. and Spiegelman, B. M. (1995). c-fos is required for malignant progression of skin tumors. Cell 82,721 -732.[Medline]
Simons, P. C., Pietromonaco, S. F., Reczek, D., Bretscher, A. and Elias, L. (1998). C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton. Biochem. Biophys. Res. Commun. 253,561 -565.[Medline]
Sun, X. G. and Rotenberg, S. A. (1999).
Overexpression of protein kinase Calpha in MCF-10A human breast cells
engenders dramatic alterations in morphology, proliferation, and motility.
Cell Growth Differ. 10,343
-352.
Sy, M. S., Mori, H. and Liu, D. (1997). CD44 as a marker in human cancers. Curr. Opin. Oncol. 9, 108-112.[Medline]
Tang, S., Morgan, K. G., Parker, C. and Ware, J. A.
(1997). Requirement for protein kinase C theta for cell cycle
progression and formation of actin stress fibers and filopodia in vascular
endothelial cells. J. Biol. Chem.
272,28704
-28711.
Thomas, L., Byers, H. R., Vink, J. and Stamenkovic, I. (1992). CD44H regulates tumor cell migration on hyaluronate-coated substrate. J. Cell Biol. 118,971 -977.[Abstract]
Uberall, F., Hellbert, K., Kampfer, S., Maly, K., Villunger, A.,
Spitaler, M., Mwanjewe, J., Baier-Bitterlich, G., Baier, G. and Grunicke, H.
H. (1999). Evidence that atypical protein kinase C-lambda and
atypical protein kinase C-zeta participate in Ras-mediated reorganization of
the F-actin cytoskeleton. J. Cell Biol.
144,413
-425.
Ui, M., Mizutani, T., Takada, M., Arai, T., Ito, T., Murakami, M., Koike, C., Watanabe, T., Yoshimatsu, K. and Iba, H. (2000). Endogenous AP-1 levels necessary for oncogenic activity are higher than those sufficient to support normal growth. Biochem. Biophys. Res. Commun. 278,97 -105.[Medline]
Vaaraniemi, J., Palovuori, R., Lehto, V. P. and Eskelinen, S. (1999). Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity, the confluence, and transformation state of kidney epithelial cell lines. J. Cell Physiol. 181,83 -95.[Medline]
Young, M. R., Li, J. J., Rincon, M., Flavell, R. A.,
Sathyanarayana, B. K., Hunziker, R. and Colburn, N. (1999).
Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is
required for tumor promotion. Proc. Natl. Acad. Sci.
USA 96,9827
-9832.
Yu, Q. and Stamenkovic, I. (1999). Localization
of matrix metalloproteinase 9 to the cell surface provides a mechanism for
CD44-mediated tumor invasion. Genes Dev.
13, 35-48.
Yu, Q. and Stamenkovic, I. (2000). Cell
surface-localized matrix metalloproteinase-9 proteolytically activates
TGF-beta and promotes tumor invasion and angiogenesis. Genes
Dev. 14,163
-176.