1 Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan
2 Laboratory for Cellular Morphogenesis, RIKEN Center for Developmental Biology,
Kobe 650-0047, Japan
3 College of Medical Technology, Kyoto University, Sakyo-ku, Kyoto 606,
Japan
* Author for correspondence (e-mail: yonemura{at}cdb.riken.go.jp )
Accepted 11 April 2002
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Summary |
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Key words: ERM protein, Microvilli, Rho, PtdIns(4,5)P2, Neomycin
![]() |
Introduction |
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Soluble ERM proteins in the cytoplasm are `dormant' in terms of their
crosslinking activity through intramolecular association between FERM and
C-terminal tail domains (Gary and
Bretscher, 1995); this interaction was recently confirmed by
crystal structure analysis (Pearson et
al., 2000
). When these dormant ERM proteins are activated, both
domains are exposed and allowed to interact with membrane proteins (for the
FERM domain) and actin filaments (for the C-terminal tail)
(Berryman et al., 1995
;
Bretscher et al., 1995
;
Gary and Bretscher, 1995
;
Hirao et al., 1996
;
Matsui et al., 1998
). These
activated ERM proteins together with ERM-binding membrane proteins are
directly involved in the organization of microvilli
(Yonemura et al., 1999
).
Evidence explaining the mechanism of ERM protein activation is
accumulating. Polyphosphoinositides such as phosphatidylinositol
(4,5)-bisphosphate [PtdIns(4,5)P2] bind to ERM proteins
and appear to open closed ERM proteins, enhancing their binding to membrane
proteins and actin filaments (Niggli et
al., 1995; Hirao et al.,
1996
; Heiska et al.,
1998
; Huang et al.,
1999
; Nakamura et al.,
1999
; Barret et al.,
2000
; Niggli,
2001
). In support of this, recent crystal structure analysis
showed that the N-terminal half of radixin has a basic cleft that can bind to
the headgroup of PtdIns(4,5)P2
(Hamada et al., 2000
).
Phosphorylated ERM proteins, especially ERM proteins phosphorylated at the
C-terminal threonine residue (C-terminal threonine-phosphorylated ERM
proteins; CPERMs), are also considered to be closely involved in ERM protein
activation. The amount of moesin phosphorylated at the C-terminal threonine
residue increases in platelets upon activation by thrombin
(Nakamura et al., 1995). At
the initial stage of anoxic injury and apoptosis, most microvilli disappear
from the cell surface, and in both cases ERM proteins are translocated from
the microvilli to the cytoplasm with concomitant dephosphorylation
(Chen et al., 1995
;
Kondo et al., 1997
;
Hayashi et al., 1999
). Moesin
phosphorylated in vitro and phosphorylated moesin purified from platelets
showed enhanced binding both to actin filaments and to EBP50
(Simons et al., 1998
;
Nakamura et al., 1999
).
Hayashi et al. showed that CPERMs represent an active form of ERM proteins in
vivo in terms of their exclusive localization at the plasma membrane
(Hayashi et al., 1999
). This
was experimentally confirmed using ERM proteins in which the C-terminal
threonine residue was mutated to aspartic acid
(Oshiro et al., 1998
;
Huang et al., 1999
;
Yonemura et al., 1999
).
However, this phosphorylation may not be required for activating ERM proteins
but for stabilizing activated ERM proteins, as phosphorylation of the
C-terminal half of radixin does not enhance its actin-filament-binding ability
but rather inhibits its interaction with the FERM domain
(Matsui et al., 1998
).
A small GTPase, called Rho, which organizes stress fibers
(Tapon and Hall, 1997), is
also thought to be involved in ERM protein activation. Interestingly, ERM
proteins may regulate Rho activity and vice versa (for reviews, see
Bretscher, 1999
;
Mangeat et al., 1999
;
Tsukita and Yonemura, 1999
).
The in vitro association of ERM proteins with the plasma membranes of cultured
cells was dependent on Rho activation
(Hirao et al., 1996
). In
several lines of cultured cells, activation of RhoA but not Rac1 or Cdc42
produced CPERMs, forming microvilli with a concomitant accumulation of ERM
proteins in microvilli (Shaw et al.,
1998
; Matsui et al.,
1998
; Matsui et al.,
1999
). One of the direct effectors of Rho, Rho-kinase, can
phosphorylate the C-terminal threonine of ERM proteins in vitro
(Matsui et al., 1998
) and
appears to do so also in vivo (Oshiro et
al., 1998
). However, Matsui et al.
(Matsui et al., 1999
) showed
that Rho-dependent production of CPERMs was not suppressed by Y-27632, a
specific inhibitor of ROCK kinases, including Rho-kinase
(Uehata et al., 1997
).
Overexpression of another direct effector of Rho, phosphatidylinositol
4-phospate 5-kinase type I
(PtdIns4P 5-kinase
), which
produces PtdIns(4,5)P2, increased the level of CPERMs and
induced microvillar formation (Matsui et
al., 1999
). Furthermore, Barret et al. showed that mutagenesis of
the PtdIns(4,5)P2-binding site in the N-terminal domain of
ezrin alters its membrane localization
(Barret et al., 2000
),
suggesting that PtdIns(4,5)P2 is important in ERM protein
function at the plasma membrane.
Thus, it was postulated that PtdIns(4,5)P2, which is
produced in a Rho-dependent manner, directly activates ERM proteins and that
activated ERM proteins are phosphorylated at their C-terminal threonine
residue for stability (Matsui et al.,
1999). To evaluate this hypothesis, we further examined the roles
of Rho, CPERMs and PtdIns(4,5)P2 in various types of
cultured cells. During the course of these studies we found, unexpectedly,
that in certain cell lines that neither the activation of Rho nor the
phosphorylation of ERM proteins was required in the activation of ERM proteins
or for their stabilization. In this study, we examined the molecular mechanism
behind the Rho-independent activation of ERM proteins, paying special
attention to the role of PtdIns(4,5)P2.
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Materials and Methods |
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EGF treatment of A431 cells
Subconfluent A431 cells grown on coverslips were cultured in DMEM without
FCS for 6-24 hours. Recombinant human epidermal growth factor (EGF) (GIBCO
BRL) was added at a final concentration of 100 ng/ml. Cells were incubated for
30 seconds to 30 minutes and quickly fixed and processed for
immunofluorescence microscopy.
Microinjection
Several reagents were introduced into cells by microinjection using a set
of manipulators (MN-188 and MO-189; Narishige, Tokyo, Japan) connected to an
Eppendorf microinjector 5242 (Eppendorf, Inc., Hamburg, Germany) and a Zeiss
Axiovert 135 microscope. As markers for microinjected cells
fluorescein-conjugated 70,000 MW anionic, lysine-fixable dextran (F-dextran)
or tetramethylrhodamine-conjugated 70,000 MW, lysine-fixable dextran
(Rh-dextran) (Molecular Probes, Inc.) was used. C3 transferase (1µg/µl)
or antibiotics such as neomycin sulfate (1-10 mM) were microinjected with 1%
F- or Rh-dextran in PBS into the cytoplasm of cultured cells on coverslips.
For transfection, expression vectors pEF-BOS-HAx3-V14RhoA for constitutively
active RhoA, pA/mEz-VSVG for VSVG-tagged ezrin
(Yonemura et al., 1999),
pA/mEz-T/A-VSVG where the C-terminal threonine residue of ezrin was mutated to
alanine (Yonemura et al.,
1999
) or pEF-BOS-HAx3-PI4P5K for PI4P5K
(Matsui et al., 1999
) at
0.2-0.6 µg/µl in a solution containing 1 mM EDTA and 10 mM Tris-HCl (pH
8.0) was injected into the nuclei of cells. Cells were examined 6-8 hours
after injection.
Immunofluorescence microscopy
In most cases, cells were fixed with ice-cold 10% trichloroacetic acid
(TCA) for 15 minutes (Hayashi et al.,
1999). When F-dextran or Rh-dextran was microinjected into cells,
cells were fixed with ice-cold 10% TCA plus 1% paraformaldehyde to ensure the
immobilization of F- or Rh-dextran within cells. The presence of
paraformaldehyde in 10% TCA solution did not affect the staining of ERM
proteins or CPERMs. To visualize actin filaments with rhodamine phalloidin or
FITC phalloidin (Molecular Probes, Inc.), cells were fixed with 4%
paraformaldehyde in 0.1 M Hepes buffer (pH 7.5) for 15 minutes. After three
washes with PBS containing 30 mM glycine (G-PBS), cells were treated with 0.2%
Triton X-100 in G-PBS for 10 minutes and washed with G-PBS. Cells were soaked
in blocking solution (G-PBS containing 4% normal donkey serum) for 5 minutes
and incubated with primary antibodies diluted with the blocking solution for
30 minutes. Cells were then washed three times with G-PBS and incubated with
secondary antibodies for 30 minutes. FITC- or Cy3-conjugated donkey anti-mouse
IgG antibody, FITC- and Cy3-conjugated donkey anti-rat IgG antibody and FITC-,
Cy3- and Cy5-conjugated donkey anti-rabbit IgG antibody were from Jackson
ImmunoResearch Laboratories, Inc. Cy5-conjugated goat anti-mouse IgG antibody
was from Amersham International. Cells were washed three times then mounted in
90% glycerol-PBS containing 0.1% para-phenylendiamine and 1% n-propylgalate.
Specimens were observed using a Zeiss Axiophot photomicroscope or Olympus IX70
with appropriate combinations of filters and mirrors. Images were recorded
with a cooled CCD camera (SenSys 0400, 768x512 pixels; Photometrics)
controlled by a Power Macintosh 7600/132 and the software package IPLab
Spectrum V3.1 (Scanalytics Inc.).
Electron microscopy
L cells were cultured on CELLocate coverslips (Eppendorf) and microinjected
with C3/F-dextran or neomycin/F-dextran. They were kept in a CO2
incubator for 30 minutes and fixed with 2.5% glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.4) for 2 hours at room temperature. L cells treated
with staurosporine (10 nM) for 1 hour were also fixed under the same
condition. Microinjected cells were identified under a fluorescence
microscope, and their phase contrast as well as fluorescence images were
recorded together with their location information printed on CELLocate
coverslips. Samples were then processed conventionally and examined under a
scanning electron microscope (S-3500N, Hitachi Co.). Microinjected cells were
identified on the basis of the recorded images.
For transmission electron microscopy, MDCK II cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hours at room temperature and then post-fixed with 1% OsO4 in the same buffer for 2 hours on ice. The samples were rinsed with distilled water, stained with 0.5% aqueous uranyl acetate for 2 hours at room temperature, dehydrated with ethanol and embedded in Polybed 812 (Polyscience). Ultra-thin sections were cut, doubly stained with uranyl acetate and lead citrate and viewed with a JEM 1010 transmission electron microscope (JEOL).
Soluble and insoluble ERM proteins
Subconfluent L cells cultured on a 35 mm dish were treated with 10 nM
staurosporine for 1 hour then washed twice with ice-cold PBS. After removal of
PBS, 500 µl of an ice-cold sonication buffer (150 mM NaCl, 1 mM EGTA, 1 mM
DTT, 10 µg/ml leupeptin, 10 mM Hepes buffer, pH 7.5) containing 10 nM
staurosporine was added. Cell were scraped off and collected into 1.5 ml
tubes. 500 µl of the cell suspension was transferred to a new tube and
sonicated. The homogenates were centrifuged at 10,000 g for 10
minutes at 4°C to recover the soluble and insoluble fractions. Equivalent
amounts of supernatant and pellet were applied to SDS-polyacrylamide gels
followed by immunoblotting with pAb TK89.
To examine the effects of neomycin on the solubility of ERM proteins, subconfluent L cells or MDCK II cells cultured on a 35 mm dish were processed as described above, except that 1 mM neomycin sulfate was added to the sonication buffer instead of staurosporine. After sonication, the homogenates were kept on ice for 30 minutes. Then soluble and insoluble fractions were recovered and analyzed as described above.
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Results |
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|
In serum-starved A431 cells, EGF treatment causes rapid and transient
microvillar elongation and membrane ruffling with concomitant ERM protein
translocation to these structures and CPERM production
(Chinkers et al., 1979;
Bretscher, 1989
;
Yonemura et al., 1999
). C3
blocked this EGF-induced ERM protein activation and microvillar elongation in
A431 cells (Fig. 2A-C). In
serum-starved A431 cells where the amount of CPERM is very low, expression of
a constitutively active form of Rho (V14RhoA) resulted in a dramatic increase
in the amount of CPERM localized at microvilli
(Fig. 2D,E). Thus, Rho activity
is indispensable for ERM protein activation in several types of cells.
|
Dephosphorylation of CPERMs can result in microvillar breakdown, but
CPERM production is not necessary for EGF-induced microvillar formation
We next addressed whether phosphorylation of ERM proteins at the C-terminal
threonine is required for ERM protein activation. Since staurosporine, a
potent protein kinase inhibitor with a broad specificity, suppresses
production of CPERMs in platelets
(Nakamura et al., 1995), L
cells were cultured in the presence of staurosporine at 10 nM for 1 hour.
Almost all CPERMs were dephosphorylated, and ERM proteins were translocated
from microvilli to the cytoplasm (Fig.
3Aa-d). Scanning electron microscopy confirmed the disappearance
of microvilli from the surface of staurosporine-treated L cells
(Fig. 3Ae). Biochemical
findings were consistent with these morphological observations
(Fig. 3B): in the homogenate
obtained from staurosporine-treated L cells, ERM proteins were mostly
recovered in the soluble fraction after centrifugation, whereas from control L
cells a considerable amount of ERM protein was associated with the insoluble
fraction. Similar effects of staurosporine were observed in epithelial MTD-1A,
A431, fibroblastic NIH3T3, CV1 and myeloma P3 cells (data not shown). These
results indicate that phosphorylation at the C-terminal threonine is required
for ERM proteins to function as crosslinkers in these cells. CPERM production
may be required for activating ERM proteins, or it may be required only for
maintaining their active state. In other words, without phosphorylation,
activated ERM proteins may be quickly inactivated by their intramolecular
association. To solve this problem, we tried to activate ERM proteins within
cells experimentally and examined the role of CPERM production. We chose A431
cells for this purpose because activation of ERM proteins in an inactive state
is clearly observed, especially following EGF stimulation after
serum-starvation; this was not observed in other cells. Interestingly, when
serum-starved staurosporine-treated A431 cells were stimulated with EGF,
elongation of microvilli-like structures and recruitment of ERM proteins to
these structures occurred without CPERM production
(Fig. 4Aa-d). These structures
were confirmed to contain actin filaments (such as microvilli and ruffling
membranes) (Fig. 4Ae,f),
although staurosporine treatment affected the shape of these structures to
some extent. These ERM proteins returned to the cytoplasm faster than those in
control cells stimulated with EGF (Fig.
4Ba-j). The C-terminally VSVG-tagged ezrin (Ez-VSVG) was
previously shown to behave in a similar manner to endogenous ezrin
(Algrain et al., 1993
;
Crepaldi et al., 1997
). When
the C-terminal threonine residue of ezrin (T567) was mutated to alanine
(Ez-T/A-VSVG; non-phosphorylatable ezrin), the expressed mutant ezrin in
serum-starved A431 cells was recruited to microvilli and ruffling membrane
after EGF stimulation, as Ez-VSVG was (Fig.
5), supporting the theory that ERM proteins can be activated
without phosphorylation. We could not confirm this rapid and transient
translocation of ERM proteins by the biochemical analysis described above,
probably because not only activating but also inactivating pathways are
working after EGF stimulation, rendering activated ERM protein unstable during
cell fractionation (data not shown). These findings show that production of
CPERMs is not required for ERM protein activity itself and confirm the role of
phosphorylation in maintaining the active state of ERM proteins in vivo.
|
|
|
Rho-independent activation of ERM proteins
In contrast, in kidney-derived cells such as MDCK II cells, microinjection
of C3 transferase did not dephosphorylate CPERMs
(Fig. 6Aa) and did not affect
their subcellular localization, although stress fibers were apparently
disrupted (Fig. 6Ab). Similar
findings were obtained with other kidney-derived cells: MDCK I, LLC-PK1 and
MDBK cells (data not shown). These findings were consistent with a previous
report showing no effects of C3 on the distribution of ERM proteins in MDCK
cells (Kotani et al., 1997).
Interestingly, staurosporine also induced dephosphorylation of CPERMs in these
cells, but microvilli-like structures on apical membranes remained intact or
occasionally elongated where dephosphorylated ERM proteins were concentrated
(Fig. 6Ac,d). Transmission
electron microscopy revealed that these structures are normal microvilli
(Fig. 6Ae,f). In the homogenate
obtained from staurosporine-treated MDCK cells, ERM proteins were recovered
both in the soluble and the insoluble fraction after centrifugation; the same
result was obtained for control MDCK II cells
(Fig. 6B). These findings
indicate that in these cells ERM proteins are activated in a Rho-independent
manner and that their activated forms are stabilized by mechanisms other than
phosphorylation of their C-terminal threonine residue.
|
Involvement of PtdIns(4,5)P2 both in
Rho-dependent and -independent activation of ERM proteins
PtdIns(4,5)P2 activates ERM proteins as crosslinkers in
vitro, and this activation had nothing to do with phosphorylation
(Hirao et al., 1996).
Furthermore, PtdIns(4,5)P2 was suggested to play a crucial
role in a Rho-dependent activation of ERM proteins in vivo
(Matsui et al., 1999
). We thus
examined the effects of increasing PtdIns(4,5)P2
concentration on the ERM activity within cells in more detail. Firstly,
PtdIns4P 5-kinase
, which produces
PtdIns(4,5)P2
(Ishihara et al., 1996
), was
overexpressed in serum-starved A431 cells in which Rho was not activated and
CPERMs were undetectable. As shown in Fig.
7A,B, overexpressed PtdIns4P 5-kinase
was
occasionally associated with vesicular structures in the cytoplasm to which
considerable amounts of ERM proteins were recruited. Immunofluorescence
microscopy showed that these recruited ERM proteins were phosphorylated at
their C-terminal threonine residue (Fig.
7C). Actin filaments were also concentrated at these vesicles
(data not shown). Thus, we concluded that the local production of
PtdIns(4,5)P2 by PtdIns4P 5-kinase
on the
vesicles recruited and activated the ERM proteins and phosphorylated them
without Rho activity.
|
We next examined the effects of depleting intracellular
PtdIns(4,5)P2. Neomycin, an aminoglycoside antibiotic that
binds to polyphosphoinositides, especially PtdIns(4,5)P2,
with high affinity was used (Wang et al.,
1984). As neomycin could not cross plasma membranes effectively,
we microinjected neomycin into the cytoplasm to interfere with
PtdIns(4,5)P2 and
PtdIns(4,5)P2-binding proteins. As shown in
Fig. 8Aa-c, microinjection of
10 mM neomycin induced rapid dephosphorylation of CPERMs and translocation of
ERM proteins from microvilli to the cytoplasm within 10 minutes in L cells.
Scanning electron microscopy showed that neomycin caused microvillar breakdown
(Fig. 8Ad). One of the
aminoglycosides, spectinomycin, which has a lower affinity for
PtdIns(4,5)P2 than neomycin
(Wang et al., 1984
), had no
effect on ERM proteins at a concentration of 10 mM (data not shown).
Ampicillin, which is from a different group of antibiotics, had no effect at
10 mM (data not shown). Supporting these morphological observations, when L
cell homogenate was incubated in the presence of 1 mM neomycin for 30 minutes
followed by centrifugation, ERM proteins were mostly recovered in the soluble
fraction, whereas in the absence of neomycin, considerable amounts of ERM
proteins were associated with the insoluble fraction
(Fig. 8B). Similar findings
were obtained for other types of cells in which ERM proteins were activated in
a Rho-dependent manner as described above.
|
The question of whether PtdIns(4,5)P2 is also involved in the Rho-independent activation of ERM proteins naturally arose. Interestingly, when 10 mM neomycin was microinjected into MDCK I, MDCK II, LLC-PK1 or MDBK cells, ERM proteins were translocated from microvilli to the cytoplasm (Fig. 8Cb). As shown above, in these cells the C-terminal threonine is not necessarily phosphorylated for activated ERM proteins to be stabilized, but CPERMs disappeared by microinjection of neomycin (Fig. 8Ca). Neomycin microinjection inhibited EGF-induced ERM protein activation in serum-starved A431 cells (data not shown). This finding indicates the importance of PtdIns(4,5)P2 in ERM protein activation common in a variety of cells.
We used an ezrin-, moesin-specific mAb or a pan-ERM pAb (M11, M22 and TK89,
respectively) for immunofluorescence microscopy to see whether activation
mechanisms of ERM proteins differ from each other. In all cases described in
this study, ezrin, moesin and total ERM proteins behaved similarly (data not
shown). In many cases, the expression level of radixin was too low for
detection by immunofluorescence microscopy. Considering that all ERM proteins
behaved similarly in cell fractionation experiments and that cellular
distributions of ERM proteins are quite similar to each other in many types of
cultured cells (Franck et al.,
1993; Takeuchi et al.,
1994
), each ERM protein appears to use the same activation
mechanism.
![]() |
Discussion |
---|
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---|
One of the CPERMs, moesin, and a moesin mutant, in which aspartate was
substituted for threonine 558 to mimic a CPERM, bind to actin filaments more
strongly than non-phosphorylated wild-type moesin in vitro
(Huang et al., 1999;
Nakamura et al., 1999
).
However, this study indicates that the production of CPERM is not essential
for ERM proteins to work as membrane-cytoskeleton crosslinkers in vivo.
Next, we were able to clarify the role of Rho activity in the activation of
ERM proteins. We confirmed that ERM protein activation is dependent on Rho
activity in a group of cultured cells, including L, MTD-1A, PtK2, 3Y1 and A431
cells. Because the transient activation of ERM proteins in A431 cells via EGF
stimulation, which is observed even in the presence of staurosporine, was not
observed after microinjection of C3, Rho appears to play an important role in
the activation of ERM proteins rather than in the maintenance of the active
state, that is, phosphorylation. We clearly visualized here that increasing
Rho activity activates ERM proteins in serum-starved A431 cells. In this group
of cells, the activity of PtdIns4P 5-kinase may be
functionally Rho-dependent, and therefore C3 transferase may have decreased
the amount of PtdIns(4,5)P2 required for ERM protein
activation.
We further found that ERM protein activation with concomitant CPERM
production is independent of Rho activity in a group of cells including MDCK
cells. Since dephosphorylation of CPERMs by staurosporine did not result in
ERM protein inactivation in this group of cells, ERM proteins may be
stimulated for activation very frequently. Conversely, this group of cells may
have systems such as oligomerization or adduct formation of ERM proteins other
than the production of CPERMs to maintain activated ERM proteins
(Berryman et al., 1995). If
PtdIns(4,5)P2 is important for ERM protein activation
generally, then PtdIns4P 5-kinase in this group of cells may be
functionally Rho independent. Actually, the activity of PtdIns4P
5-kinase has been reported to be dependent on Rac
(Hartwig et al., 1995
) or ARF6
(Honda et al., 1999
); however,
unknown systems for PtdIns4P 5-kinase synthesis may be dominant in
these cells.
Lastly, we presented several findings showing that polyphosphoinositides, especially PtdIns(4,5)P2, are essential for ERM protein activation regardless of Rho activity. In serum-starved, PtdIns4P 5-kinase-overexpressed A431 cells, ERM proteins were translocated to the surface of cytoplasmic vesicles where PtdIns4P 5-kinase was localized, resulting in the production of CPERMs and in further recruitment of actin filaments. This supports the idea that local accumulation of PtdIns(4,5)P2 causes local activation of ERM proteins. On the other hand, microinjection of neomycin into L cells to disrupt the interaction between PtdIns(4,5)P2 and ERM proteins induced inactivation of ERM proteins, as seen by dephosphorylation of CPERMs, translocation of ERM proteins from microvilli to the cytoplasm and microvillar breakdown. Similar results were obtained with all cultured cells tested. Translocation of ERM proteins from the insoluble fraction to the soluble fraction was demonstrated biochemically by adding neomycin to cell homogenates. These findings show that one polyphosphoinositide, PtdIns(4,5)P2, is the most likely candidate for a direct activator of ERM proteins in vivo at present.
Barret et al. mapped the PtdIns(4,5)P2-binding site in
ezrin and generated ezrin mutants that were unable to bind to
PtdIns(4,5)P2 in vitro
(Barret et al., 2000).
Furthermore, one of these ezrin mutants was unable to associate with the
plasma membrane. These results suggested that
PtdIns(4,5)P2 has an important role in ERM protein
activation and membrane targeting. Our results with
PtdIns(4,5)P2 confirm and extend their data and
interpretation.
Neomycin binds to polyphosphoinositides including
PtdIns(4,5)P2 with high affinity
(Schacht, 1978;
Wang et al., 1984
;
Gabev et al., 1989
) and has
been used for inhibition of binding between PtdIns(4,5)P2
and PtdIns(4,5)P2-binding proteins in permeabilized cells
or in vitro (Cockcroft et al.,
1987
; Liscovitch et al.,
1991
). When neomycin was microinjected into sea urchin eggs
(Swann et al., 1992
) or
Xenopus oocytes (Carnero and
Lacal, 1995
), several events at fertilization and oocyte
maturation were blocked by neomycin in the cytoplasm at a concentration of
5-10 mM. In this study, 10 mM neomycin was microinjected, and its
intracellular concentration colud be roughly estimated as 1 mM, a reasonable
concentration for inhibiting binding between PtdIns(4,5)P2
and PtdIns(4,5)P2-binding proteins in the cytoplasm.
Although microinjection of neomycin into cultured cells appears to be rarely
performed, this technique can be applied widely to analyze the roles of
PtdIns(4,5)P2 and
PtdIns(4,5)P2-binding proteins.
Another issue that we should discuss here is the relationship between ERM
proteins and the toxicity of aminoglycosides. Aminoglycosides were reported to
accumulate specifically in renal proximal tubular cells and hair cells of the
inner ear, where they cause cell injury
(Tulkens, 1989;
Lim, 1986
). Interestingly,
microvillar breakdown was observed in these cells
(Wersäll et al., 1973
;
Jones and Elliott, 1987
).
Therefore, it is interesting to speculate that the suppression of the
Rho-independent activation of ERM proteins by accumulated neomycin is directly
involved in the nephrotoxicity (and probably also ototoxicity) of
aminoglycosides.
A conformational change in an ERM protein upon activation, which was
proposed as a model (Gary and Bretscher,
1995), is being evaluated by crystal structure analysis
(Pearson et al., 2000
).
However, several important points have not been clarified yet. What is the
kinase(s) responsible for the production of CPERMs in vivo that maintains the
active state of ERM proteins? If PtdIns(4,5)P2 is the
major activator for ERM proteins, what are the spatial and temporal changes in
PtdIns(4,5)P2 concentration within cells? What is the role
of ERM proteins in the regulation of Rho activity in vivo? Furthermore, the
specific role of each ERM proteins or their synergistic roles needs to be
elucidated. Although moesin-null mice appeared normal
(Doi et al., 1999
), they
should be examined in more detail. Radixin- or ezrin-null mice may serve as a
model to demonstrate the basic functions of ERM proteins in the body.
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Acknowledgments |
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![]() |
References |
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Barret, C., Roy, C., Montcourrier, P., Mangeat, P. and Niggli,
V. (2000). Mutagenesis of the phosphatidylinositol
4,5-bisphosphate (PIP2) binding site in the NH2-terminal
domain of ezrin correlates with its altered cellular distribution.
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