* MRC Laboratory for Molecular Cell Biology, Department of Biochemistry, and § Eisai London Research Laboratories,
University College London, London WC1E 6BT, United Kingdom; and
Laboratory of Experimental Oncology, Department of
Pathology, Stanford University School of Medicine, Stanford, California 94305
The small GTPases Rho and Rac regulate
actin filament assembly and the formation of integrin
adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear. We now describe a biological
assay that allows components of the Rho and Rac signaling pathways to be identified. Permeabilization of
serum-starved Swiss 3T3 cells with digitonin in the
presence of guanosine 5-O-(3-thiotriphosphate)
(GTP
S) induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac. These responses are lost when
GTP
S is added 6 min after permeabilization, but can
be reconstituted using concentrated cytosolic extracts.
We have achieved a 10,000-fold purification of the activity present in pig brain cytosol and protein sequence
analysis shows it to contain moesin. Using recombinant proteins, we show that moesin and its close relatives
ezrin and radixin can reconstitute stress fiber assembly,
cortical actin polymerization and focal complex formation in response to activation of Rho and Rac.
MEMBERS of the Rho subfamily of small GTPases
control the adhesion, morphology, and motility
of mammalian cells, and also regulate signal
transduction pathways that affect gene transcription in the
nucleus (Hall, 1994 The biological consequences of activating Rho, Rac,
and Cdc42 have been documented in some detail in Swiss
3T3 fibroblasts. Activation of Rho using extracellular
stimuli such as lysophosphatidic acid, or microinjection of
constitutively activated forms of recombinant Rho protein, leads to the assembly of actin stress fibers and focal
adhesion complexes, whereas activation of Rac by agonists such as PDGF or insulin stimulates the formation of
lamellipodia and membrane ruffles (Ridley and Hall,
1992 There has been a great deal of interest in the signal
transduction pathways downstream of Rho, Rac, and
Cdc42 and two main approaches have been used to identify proteins that associate with these GTPases: the yeast
two-hybrid system using GTPases as bait, and protein purification techniques using a GTP-bound GTPase as an affinity ligand. The putative Rho effectors identified in this
way to date include two families of Ser/Thr kinases,
p160ROCK (p164 rho-kinase) and protein kinase N (PKN)
a protein kinase C-related kinase, and several structural
proteins with no obvious catalytic activity (Leung et al.,
1995 A growing list of targets for Rac has also emerged
and these include the Ser/Thr kinases p65PAK, mixed-lineage kinase (MLK), p70S6kinase, and p160ROCK, and
several structural proteins (Diekmann et al., 1994 Finally, there have been several reports that Rho and
Rac control the activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase thereby regulating the synthesis of
phosphatidylinositol-4,5-biphosphate (PIP2) (Chong et al.,
1994 To identify components of the Rho and Rac signal
transduction pathways, we have developed a biological assay that reconstitutes the assembly of stress fibers, lamellipodia, and focal complexes in permeabilized cells. Permeabilization has been used by others to study actin filament
assembly. In platelets, for example, Hartwig et al. (1995) We show here that stress fibers and focal adhesions, as
well as peripheral actin structures and their associated focal complexes, are induced in quiescent Swiss 3T3 permeabilized in the presence of guanosine 5 Cell Culture
Swiss 3T3 cells were cultured in DME with antibiotics (GIBCO BRL,
Gaithersburg, MD), supplemented with 10% FCS (Sigma Chemical Co.,
St. Louis, MO). Cells were seeded onto 13-mm-diam round glass coverslips (Chance Propper, West Midlands, UK) at a density of 5 × 10 cells
per ml and grown to quiescence over 5-8 d, and before use were serum
starved overnight in DME containing 2 g/liter NaHCO3.
Permeabilization
Permeabilization buffer (DK) comprised 150 mM potassium glutamate,
10 mM Hepes/KOH, 5 mM glucose, 2 mM MgCl2, 0.4 mM EGTA, pH 7.6;
this was stored as a 2× stock at Each coverslip was rinsed twice in PBS and once in DK buffer, and
then inverted on a 50-µl droplet of complete DK containing an additional
0.003% digitonin and a stimulus as stated (protocol 1). Alternatively (protocol 2), cells were permeabilized for 6 min at room temperature on a 60-µl droplet of DK buffer containing 0.003% digitonin, and then transferred
to a 40-µl droplet of complete DK (lacking digitonin), containing a stimulus as indicated. Coverslips were incubated under humidified conditions at
37°C for 20 min and then fixed for immunofluorescence.
Immunofluorescence and Microscopy
Immunofluorescence procedures are substantially as described (Nobes
and Hall, 1995 Preparation and Assay of Porcine Brain Extract
All procedures were carried out at 4°C. Pig brains were washed in PBS,
and then homogenized in a blender in 1.5 vol of DK supplemented with
100 µM DTT, 10 µM ATP, 5 µM GTP, and 1 mM PMSF, N The specific activity was determined in a semi-quantitative immunofluorescence assay, by calculating the amount of protein required to generate
cytoskeletal reorganization in 10-30% of permeabilized cells. For each
coverslip, several fields of cells were examined at random. First, cells were
counted by counting Hoechst-stained nuclei, then the cells were examined
under the rhodamine and fluorescein channels, and a second count was
made of cells showing focal adhesions and actin filament assembly. Fields
at the edge of the coverslip were not examined, nor were fields showing
evidence of damage, or fields where cells did not grow in an even monolayer. Background activity was subtracted from cells treated with stimulus
or protein alone (2-5% of response in presence of stimulus + protein).
Since this was a labor-intensive assay, duplicates were rarely performed, but assays were performed three times or more; while absolute
specific activities might vary within a tenfold range between assays, the
relative specific activities of fractions varied within twofold.
Purification of Focal Adhesion Activity from
Porcine Brain
Chromatography was carried out on a Biologic integrated chromatography system (Bio Rad Laboratories, Hercules, CA); in all cases the buffer
was DKx, where x indicates the millimolar concentration of potassium
glutamate. The data shown in Fig. 5 and Table I derive from a single representative purification, which was carried out without pause from homogenization of fresh brain to the assay of the second Q-Sepharose eluate, in 72 h. Activities were stable when snap frozen in small aliquots in
liquid nitrogen and stored at
Table I.
Purification of Bioactivity from Pig Brain
Pig brain extract was passed over a 150-ml column of Q-Sepharose
Fast-Flow (Pharmacia LKB Biotechnology Inc., Piscataway, NJ). The column was washed extensively in DK10, and proteins were eluted in DK200.
Pooled material was diluted to DK40 and loaded onto 40 ml of Cibachrome blue 3GA-Sepharose (Sigma Chemical Co.). Protein was eluted
in a gradient of DK40-600 and two peaks of activity were detected at 250 and 410 mM. These were pooled, adjusted to 600 mM K-glutamate and
passed over a 15-ml phenyl-Sepharose column; the activity was eluted at
100 mM salt in a gradient of DK600-10. The peak of activity was diluted
to 20 mM, applied to a 5-ml Q-Sepharose column, and then eluted with a
gradient of DK10-200. Activity was detected between 70 and 110 mM.
For gel filtration, a 200-µl aliquot of the activity peak from the final
Q-Sepharose column was applied to a 6-ml Bio-Silect chromatography
column (Bio Rad Laboratories: rated fractionation range 100-5 kD) and
protein was eluted in DK150, sampling 0.25-ml fractions. It should be
noted that the protein concentrations of these gel-filtration fractions were
not assessed; therefore, the activities of the fractions (unlike all other fractions and preparations in this study) were assessed in arbitrary units, based on the volume, not the absolute quantity, of protein added.
Protein Microsequencing
The peak fraction from the final Q-Sepharose column was electrophoresed on 10% SDS-PAGE gel and the proteins transferred to PVDF (Bio
Rad Laboratories) by wet-blotting in 5 mM 3-cyclohexylamino-1-propanesulfonic acid (CAPS) (Aldrich, St. Louis, MO), pH 11, 1% MeOH,
0.01% SDS at 0.75Amperes for 1.5 h. The blot was washed three times for 10 min in deionized water, and then stained with 0.1% Amido black/10% HOAc for 1 min, and destained with distilled water until the bands were
clearly visible. Bands were excised, transferred to sequencing cartridges,
and submitted to 20 rounds of NH2-terminal sequencing on a protein sequencer (GP1000A; Hewlett-Packard Co., Palo Alto, CA), using Routine
3.0 chemistry.
Western Blotting
The samples for Western blotting were derived from cells permeabilized
under equivalent conditions to those prepared for immunofluorescence assay, except that after permeabilization the supernatant was recovered
and the cell residue on the coverslip was scraped into sample buffer. (For
Fig. 9, d and e, supernatant and pellet fractions were combined.) Typically, samples for electrophoresis were equivalent to 0.25-0.5 permeabilizations. Proteins were electrophoresed on 10% or 12% SDS-PAGE gels
and transferred to PVDF membranes (Millipore Corp., Bedford, MA). The
membranes were blocked extensively in blocking buffer (1 M glycine, 5%
milk powder, 1% ovalbumin, and 5% FCS), and then immunoblotted using monoclonal antibodies against vinculin (Sigma Chemical Co.) at 1:200
dilution, and polyclonal anti-ezrin/radixin/moesin (ERM) (1:500 dilution).
HRP-conjugated secondary antibodies (Pierce) was used along with Amersham ECL system (Arlington Heights, IL) for detection of immunoreactivity.
Recombinant Proteins
Recombinant, constitutively activated RhoA (V14 or L63 mutants), C3
transferase, constitutively active (L61) and dominant negative (N17)
Rac1, RhoGAP, and human moesin were expressed as glutathione-
S-transferase (GST) fusion proteins in Escherichia coli and purified on
glutathione-Sepharose beads as described by Self and Hall (1995) Lactate Dehydrogenase
Lactate dehydrogenase activity was assayed by a method based on the
work of Kornberg (1955) Rho Assay
Rho was assayed by C3-mediated ribosylation using 32P-NAD in a method
based on that of Aktories and Just (1995) Immunodepletion
Immunodepletion was carried out using polyclonal anti-moesin antiserum,
a kind gift of Dr. Paul Mangeat (Université Montpelier, Montpelier,
France); control antisera were rabbit anti-RhoGDI or rabbit anti-rat
(Sigma Chemical Co.). Antisera were coupled to protein A-Sepharose,
and the beads repeatedly washed in DK. MonoQ eluate peak activity (10-µl aliquots) was subjected to two rounds of immunodepletion on the coupled beads, by incubation for 40 min at 4°C; afterwards, the collected immunodepletion supernatants were assayed for bioactivity as described
above (permeabilization protocol II). By Western blotting (not shown),
the supernatant of anti-moesin antibody contained no moesin, whereas
moesin was present in the supernatant of mock depletion.
Dot Blotting
Dot blots were performed essentially as described by Lamarche et al.
(1996) F-actin Overlay
66 µM G-actin purified from rabbit muscle was a gift of Dr. Laura Machesky (MRC LMCB, University College of London, UK). G-actin was
gel filtered into G buffer (2 mM Tris.HCl, pH 8.0, 50 µM CaCl2, 0.5 mM
DTT, 50 µM NaN3) to reduce the concentration of free ATP, and then incubated for 120 min at room temperature with 125 µCi [ Focal Complex and Actin Filament Assembly in
Permeabilized Swiss 3T3 Cells
Stress fibers and lamellipodia can be induced in quiescent,
confluent serum-starved Swiss 3T3 cells by addition of extracellular agonists or by microinjection of recombinant
Rho and Rac proteins, respectively. In an attempt to reconstruct these effects in vitro, we permeabilized serum-starved Swiss 3T3 cells grown on glass coverslips by exposure to isotonic buffer containing an ATP regenerating system and a low concentration (0.003%) of the nonionic
detergent digitonin. After 20 min at 37°C, the permeabilized cells were fixed and F-actin visualized by immunofluorescence. Like intact serum-starved cells, permeabilized
cells lack actin filament organization (Fig. 1 A, left) and focal adhesion complexes. However, permeabilization in the
presence of the nonhydrolyzable GTP analogue, GTP
To visualize these effects more clearly, permeabilized
cells were immunofluorescently stained for both F-actin
and the focal adhesion protein vinculin, and examined at
higher magnification. This showed that GTP To confirm this suggestion and to dissect the morphology of the GTP We then tried to stimulate the changes in actin filament
organization using recombinant, constitutively activated
Rho and Rac proteins instead of GTP
Reconstitution of Actin Filament and Integrin Complex
Assembly Using Cytosolic Extracts
Cells left in permeabilization buffer for 6 min at room
temperature, before addition of either GTP
In an attempt to reconstitute the effects of Rho, a high
speed, cytosolic supernatant was prepared from a pig brain
homogenate. Swiss 3T3 cells were permeabilized at room
temperature for 6 min and transferred to a fresh drop (40 µl) of buffer containing either Rho, concentrated cytosol
extract (1 mg/ml), or a combination of the two, and then
incubated at 37°C for another 20 min. As shown in Fig. 4,
incubation with Rho plus cytosol caused cytoskeletal reorganization in up to 50% of cells (Fig. 4, e and f), while cytosol alone (Fig. 4, c and d) or Rho alone (Fig. 4, a and b) produced no effect. Similarly, pig brain extract reconstituted the morphological effects of GTP Purification of the Activity from Pig Brain Cytosol
To purify an active component from pig brain cytosol the
permeabilization protocol was made semiquantitative.
Permeabilized cells were stimulated in the presence of
protein fractions, and then examined by immunofluorescence for both F-actin and vinculin. Several fields were assessed on each coverslip, and the proportion of "responding" cells was calculated; responding cells were defined operationally as those containing substantial bundled actin
filaments and abundant focal adhesions. A maximum of
~30% of cells was capable of responding to optimal stimuli, and the quantity of protein required to provoke response in 10% of the cells was defined as 0.1 units of activity. For assays during purification, GTP Pig brain extract (20 g) was first passed over a Q-Sepharose column, and the activity was eluted using a 200 mM salt
step (Table I). This fraction was diluted fivefold and
loaded on to a Cibachrome blue-Sepharose column and
proteins eluted using a gradient of increasing ionic strength
(Fig. 5 a and Table I). Two peaks of activity were obtained;
these were pooled and applied to a phenyl-Sepharose column and proteins eluted using a gradient of decreasing
ionic strength (Fig. 5 b and Table I). Finally, the active
fractions were applied to a Q-Sepharose column and the
activity eluted using a salt gradient. The specific activity of
the cytosolic component increased ~10,000-fold during
the purification procedure (Table I and Fig. 5 c).
200 µl of the Q-Sepharose fraction was analyzed by gel-filtration chromatography (Figs. 5 d and 6 a) and the peak
of biological activity eluted in fraction 31, although the
profile of activity was broad. A prominent protein in fraction 31, whose abundance broadly correlated with biological activity across the gel-filtration profile, migrated on
PAGE at just over 66 kD. To identify this protein, 22 µg of
the final Q-Sepharose fraction was subjected to electrophoresis on 10% polyacrylamide gels and proteins transferred to PVDF membranes. The six major bands (marked
A-F on Fig. 6 b) were excised and subjected to NH2-terminal protein microsequencing. The majority of these bands
gave no peptide sequence and were presumably NH2-terminally blocked. However, two major bands around 66 kD (bands B and C) were identified as porcine moesin
(Lankes et al., 1993 Moesin Is Required for Rho and Rac Effects on the
Actin Cytoskeleton
Antibodies recognizing moesin (polyclonal anti-ERM)
were used to probe a Western blot of the fractions obtained after gel-filtration of the final Q-Sepharose active
fraction. As seen in Fig. 6 c, moesin immunoreactivity correlated with biological activity, suggesting that this might
be the active component purified from pig brain extract.
To test this, recombinant moesin was expressed in E. coli as a GST fusion protein. In digitonin-treated cells,
cleaved recombinant moesin or GST-moesin fusion protein (at levels of 200-500 ng/ml), in the presence of GTP
As a control for the effects of full-length moesin, a construct was expressed which lacked only the COOH-terminal 22 amino acids, which constitute an F-actin binding
motif (Pestonjamasp et al., 1995 As an additional test of the specific activity of moesin
within the semipurified pig brain activity, the purified material was subjected to immunodepletion using either a
rabbit anti-moesin antiserum or an irrelevant antiserum.
When tested alongside untreated pig brain activity, moesin-immunodepleted material showed an 85% loss of bioactivity, whereas mock-depleted material had suffered only a 33% loss of activity (data not shown).
Moesin belongs to a family of closely related proteins
(ERM proteins) that also contains ezrin and radixin. To
assess whether these family members possessed the same
activity, recombinant ezrin and radixin were expressed in
E. coli in the same way as moesin, and tested for re-establishment of GTP Moesin Does Not Interact Directly with Rho or Rac
The ability of Rho or Rac to interact directly with moesin
was assessed using an in vitro bead-binding assay and a dot
blot assay. GST-moesin was incubated with [
F-actin Binding Site of Moesin Is Essential for Activity
The COOH-terminal 22 residues of moesin have been
shown to be required for interaction with F-actin (Pestonjamasp et al., 1995 Next, we established the fate of endogenous moesin during digitonin permeabilization of quiescent Swiss 3T3 cells.
Fig. 9 c shows that little moesin is lost during the 6-min
permeabilization procedure (lane 3); most remains cell associated (lane 4). This result seemed puzzling, given that
both purified and recombinant moesin were capable of reconstituting activity in these cells. To assess the ability of
Swiss 3T3-derived moesin to bind F-actin, we made total
SDS lysates from mock- and digitonin-treated cells. As
seen in Fig. 9 d, by Western blot analysis the lysates contain equal amounts of moesin (lanes 2 and 3). Fig. 9 e
shows that moesin present in quiescent Swiss cells is able
to interact with F-actin (lane 2), but after digitonin permeabilization, moesin is no longer able to bind F-actin (lane
3). Quantitation (four experiments) shows a 20-fold reduction in the actin-binding capacity of moesin during a 6-min
incubation with digitonin.
Our initial challenge was to develop a protocol for permeabilizing serum-starved Swiss 3T3 fibroblasts under sufficiently mild conditions to allow subsequent morphological
examination; this was achieved using low concentrations
of the detergent digitonin. Permeabilization in the presence of GTP Permeabilized cells rapidly lose their ability to respond
to either Rho or GTP Moesin is a member of the ERM family of proteins
(Sato et al., 1992 ERM proteins possess F-actin binding activity which, for
moesin at least, is located within its COOH-terminal 22 amino acids (Pestonjamasp et al., 1995 More recently, further links have been established between the cytoskeleton and ERM proteins, particularly
moesin, by Hirao et al. (1996) It is clear from the results reported here that both purified porcine moesin and recombinant moesin promote
GTPase-dependent cytoskeletal reorganization in permeabilized cells and both can bind F-actin. Recombinant
moesin, ezrin, and radixin preparations have equivalent
effects on cells, at least in this system, which presumably
reflects their high homology. It is not clear whether ERM
proteins are functionally redundant in vivo, though their slightly distinct localizations (Berryman et al., 1993 Curiously, although quiescent Swiss 3T3 cells contain
moesin capable of binding F-actin, during the 6-min permeabilization this activity disappears. The reasons for this
are unclear at present. One possibility is the loss of PIP2.
Hirao et al. (1996) Another potential inactivation mechanism is an alteration in the phosphorylation of moesin on threonine 558, which in platelets is phosphorylated and dephosphorylated within seconds of stimulation; inhibitor studies suggest that these changes are additionally regulated by tyrosine kinases/phosphatases. The effects of Rho on the
actin cytoskeleton are modulated by both tyrosine and
serine/threonine kinase inhibitors (Ridley and Hall, 1994 However, if either lipid dissociation or protein dephosphorylation causes moesin inactivation, the question of
how recombinant bacterial protein resupplies this loss remains. It is doubtful that moesin is either phosphorylated
or lipid associated when synthesized in bacteria; more
likely, its activity is due to small amino acid changes at the
NH2 terminus of the expression construct causing constitutive opening of an otherwise folded moesin structure.
In conclusion, although moesin does not interact directly with Rho or Rac, the experiments reported here
show that it is required for the stress fiber formation and
cortical actin polymerization. The versatility of this in vitro
assay should now allow rapid progress in characterizing
the role of moesin and other ERM family members in cytoskeletal reorganization, in both intact and permeabilized cells, and more general understanding of the Rho and Rac
signal transduction pathways.
; Machesky and Hall, 1996
). The three best-understood members of the family, Rho, Rac, and
Cdc42, share some 50% identity at the amino acid level
and like the archetypal small GTPase, Ras, act as molecular switches in intracellular signaling pathways. They cycle
between active (GTP-bound) and inactive (GDP-bound)
forms, and a large number of potential regulators of this
cycle have been identified, such as guanine nucleotide exchange factors, guanine nucleotide dissociation inhibitors
(GDIs)1, and GTPase activating proteins (GAPs) (Ueda
et al., 1990
; Cerione and Zheng, 1996
; Lamarche et al.,
1996
).
; Ridley et al., 1992
). Further analysis has revealed
that the filamentous actin found in Rac-induced lamellipodia is associated with small focal complexes which, although morphologically distinct from classical focal adhesions, share many of their constituents, including integrins, vinculin, paxillin, and focal adhesion kinase (Hotchin and
Hall, 1995
; Nobes and Hall, 1995
). Cdc42 activation in
Swiss cells (by bradykinin or by microinjection of recombinant protein) triggers the formation of filopodia (microspikes, membrane protrusions containing actin filament bundles that are associated with integrin focal
complexes (Kozma et al., 1995
; Nobes and Hall, 1995
). Activation of Cdc42 also leads to rapid activation of Rac in
these cells, making Cdc42 and Rac ideal candidates for
regulating cell movement in response to extracellular stimuli.
; Amano et al., 1996
; Ishizaki et al., 1996
; Matsui et al.,
1996
; Wanatabe et al., 1996
). One of these Rho targets is
the myosin-binding subunit of myosin light chain phosphatase, while myosin light chain (MLC) itself is a good
substrate for p160ROCK (Kimura et al., 1996
; Matsui et
al., 1996
). This suggests that increased phosphorylation of
MLC could be an important component of the Rho-dependent assembly of actin stress fibers and focal adhesions; and recently, MLC kinase inhibitors have been used
to provide evidence in support of this idea (Chrzanowska-Wodnicka and Burridge, 1996
).
; Manser
et al., 1994
; Burbelo et al., 1995
; Brill et al., 1996
; Chou
and Blenis, 1996
; Hart et al., 1996
; Lamarche et al., 1996
;
van Aelst et al., 1996
). Some progress in analyzing the
downstream roles of these putative targets has been
achieved by introducing amino acid substitutions into the
effector region of Rac that interfere selectively with target
interactions (Lamarche et al., 1996
). Substitutions at
codon 40 in Rac, for example, prevent binding to p65PAK
and MLK, but do not interfere with the induction of lamellipodia, while substitutions at codon 37, which prevent
binding to p160ROCK, block lamellipodium formation.
; Hartwig et al., 1995
; Tolias et al., 1995
). It is unclear
whether PIP 5-kinase interacts with the GTPases directly
or through an adaptor protein; however, PIP2 is known to
interact with a number of actin binding proteins and has
been suggested to be a key regulator of actin polymerization (Janmey and Stossel, 1989
; Stossel, 1993
), and it is
likely, therefore, that PIP 5-kinase is an important target
of Rho and Rac in mediating cytoskeletal reorganizations.
A recent report suggests that PIP2 also plays an essential
role in focal adhesion assembly by regulating the interaction of vinculin with talin and actin (Gilmore and Burridge, 1996
).
demonstrated that thrombin triggers actin polymerization through the Rac-dependent generation of PIP2 and the
consequent release of capping proteins, such as gelsolin,
from actin filaments (Hartwig et al., 1995
). Reorganization
of actin filaments has also been observed in permeabilized
mast cells and shown to be dependent on a trimeric G protein as well as on Rac and Rho, though the physiological
consequences of these changes are not clear in this cell
type (Norman et al., 1994
). Li et al. (1995)
have developed
an assay for actin nucleation using permeabilized yeast cells and have shown its dependence on a functional
CDC42 gene. Finally, permeabilization has been used to
look at the disassembly of focal adhesions in chick fibroblasts in response to high levels of ATP; interestingly, this
was blocked by peptides that inhibit actin-myosin interaction (Crowley and Horwitz, 1995
).
-O-(3-thiophosphate) (GTP
S), via activation of endogenous Rho and
Rac. If cells are incubated in permeabilization buffer for 6 min before nucleotide addition, the response is lost, but it
can be reconstituted using concentrated cytosolic extracts. The purified bioactivity from cytosol has been identified
as moesin by protein sequencing.
Materials and Methods
20°C. In addition, the complete buffer
contained 1 mM ATP, UTP, 5 mM creatine phosphate, 10 µg/ml creatine
phosphokinase, 100 µM DTT, GTP, and a protease inhibitor cocktail of
10 µg/ml chymostatin, leupeptin, aprotinin, antipain, pepstatin, and 1 mM
benzamidine hydrochloride.
). All solutions were prepared in PBS, cells were thoroughly
rinsed in PBS between stages, and incubations were performed at room
temperature. Cells were fixed for 10 min in 3% paraformaldehyde, and
then free aldehyde groups were quenched for 10 min in 1 mg/ml sodium
borohydride. The primary antibody, 50 µl of 1:200 dilution monoclonal
mouse anti-vinculin (Sigma Chemical Co.), was incubated with the cells
for 40 min; only in the case of unpermeabilized controls was it necessary
to include 0.05% Triton X-100 as a permeabilizing agent. Two secondary
antibodies were used sequentially: goat anti-mouse, and donkey anti-goat (both from Pierce, Rockford, IL, used at 1:200 dilution). The final antibody treatment also contained rhodamine-conjugated phalloidin and
Hoechst dye 33342 (both at 0.1 µg/ml; Sigma Chemical Co.). Finally the
coverslips were rinsed in PBS and water and mounted on slides by inversion over 5 µl Moviol mountant containing p-phenylinediamine (1 mg/ml;
Sigma Chemical Co.) as an antibleaching agent. Cells were examined using a Zeiss Axiophot microscope using Zeiss 63 × 1.4 and 100 × 1.3 oil immersion objectives (Carl Zeiss, Inc., Thorwood, NY), and photographed
on Kodak T-MAX 400ASA film (Eastman Kodak Co., Rochester, NY).
-p-Tosyl-
L-lysine chloromethyl ketone (TPCK), and N-Tosyl-L-phenylalanine chloromethyl ketone (TLCK). The homogenate was ultracentrifuged for 30 min
at 200,000 g in Beckman Ti50 rotors (Fullerton, CA), and the supernatant
filtered through Whatman No. 1 paper (Lexington, MA). Typically the
volume of supernatant obtained at this stage was equivalent to that of the
starting material.
80°C, and also to limited freeze thawing.
Gel-filtration chromatography and some biological assays were carried
out on such frozen material.
Fig. 5.
Purification of the active component from pig brain cytosol. (A) Cibachrome blue 3GA: a step-eluted fraction from
fast-flow Q-Sepharose, diluted to 50 mM salt, was passed over a
40 ml Cibachrome blue 3GA column, washed extensively, and
eluted as shown in a continuous 25-300 µS salt gradient. Protein
was monitored in line by absorbence at 280 nm, and salt concentration by conductivity, as shown. The activities of column fractions were assayed and plotted (far left ordinate axis) and the two
activity peaks were pooled. (B) Phenyl-Sepharose: pooled fractions from A were applied to a 15-ml phenyl-Sepharose column
and proteins eluted with a 300-25 µS salt gradient. (C) Q-Sepharose: pooled active fractions from B were diluted to 20 mM salt
and chromatographed on a 5-ml Q-Sepharose column. Activity
was eluted in a salt gradient to 200 µS. (D) 200 µl of the peak
fraction from C was applied to a Bio-Silect gel filtration column
with a rated separation range of 100-5 kD. The elution positions
of molecular weight standards are indicated.
[View Larger Version of this Image (33K GIF file)]
Fig. 6.
Moesin copurifies
with GTPS-dependent biological activity in permeabilized cells. (A) Silver-stained
10% SDS-PAGE using 10-µl aliquots from fractions
28-35 of the gel-filtration
column shown in Fig. 5 D. Relative activities of the gel-filtered fractions are noted
below the corresponding
lanes of the blot. (B) 22 µg of
fraction 22, the most active
from the Q-Sepharose column shown in Fig. 5 c, was
electrophoresed on 10%
SDS-PAGE and electroblotted onto PVDF, and the major protein bands A-F were excised and microsequenced. The sequences obtained are shown. (C) Proteins from a parallel gel to A were
transferred to PVDF and Western blotted with a polyclonal anti-ERM antibody.
[View Larger Version of this Image (32K GIF file)]
Fig. 9.
F-actin binding to
moesin. (a and b) F-actin
binding to recombinant and
purified pig brain moesin:
lane 1, 35 ng recombinant moesin; lane 2, 125 ng of
fraction 22 from Q-Sepharose column, estimated to
contain 30 ng moesin. (A)
immunoreactivity with monoclonal anti-moesin antibody;
(B) F-actin binding using
[32P]ATP labeled F-actin nitrocellulose overlay assay.
(C) Loss of moesin from permeabilized cells. Coverslips were incubated under the following conditions: lanes 1 and 2, mock-permeabilized for 6 min in 60 µl DK without digitonin; lanes 3 and
4, permeabilized for 6 min in 60 µl DK containing 0.003% digitonin. Cell supernatants (lanes 1 and 3) and residues (lanes 2 and
4) were prepared and proteins electrophoresed on 10% SDS-PAGE gels, transferred to PVDF, and blotted with a polyclonal
antibody against ERM proteins. (D and E) F-actin binding of
moesin in Swiss 3T3 cells. Lane 1, 75 ng recombinant moesin;
lane 2, total SDS lysate; lane 3, total SDS lysate from cells that
had been first incubated for 6 min in 60 µl DK with 0.003% digitonin. Samples equivalent to half coverslips of cells were electrophoresed on 10% SDS-PAGE gels, transferred to PVDF membranes, and then probed with (D) polyclonal anti-ERM antibody
(E) [32P]ATP-labeled F-actin.
[View Larger Version of this Image (79K GIF file)]
. Cleaved
proteins were obtained from the beads by addition of human thrombin
(Sigma Chemical Co.; 5 U/liter of culture overnight, with an additional 5 U
for 30 min before protein recovery). Contaminating thrombin was removed by incubating proteins with 10 µl p-aminobenzamidine beads
(Sigma Chemical Co.) for 30 min at 4°C. GST fusion proteins were eluted
from glutathione-Sepharose by addition of 5 mM GST. Recovered protein was dialyzed into buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.1 mM DTT), and then concentrated by ultrafiltration (Centricon 10; Amicon Corp., Danvers, MA). Protein concentration was assayed by Bradford assay (Bio Rad Laboratories) and purity assessed on
SDS-PAGE; the activity of p21 preparations was measured as the binding
of [3H]GTP in the filter-binding assay as described by Hall and Self (1986)
.
. Lysate samples equivalent to one coverslip of
cells were added to an assay mixture comprising 330 µM sodium pyruvate,
10 mM Hepes/KOH, pH 7.2, 66 µM NADH, and 0.2% Triton X-100, and
the decrease in absorbence at 340 nm was monitored over 1-3 min.
. Samples were derived from
cells permeabilized in 60-µl vol; supernatants were then removed, and cell
residue harvested by briefly washing the coverslip and then scraping off
residue into 60 µl 0.2% Triton X-100 in DK. 10-µl aliquots of this material
were incubated for 1 h at 37°C with 4 µCi 32P-NAD and 40 ng C3 transferase. The labeled proteins were boiled in SDS-PAGE sample buffer,
and 12% SDS-PAGE gels of labeled proteins were autoradiographed
overnight.
. GTPases (0.1 µg) were loaded with [
32P]GTP by incubation in 50 mM Tris, pH 7.5, 0.5% BSA, 5 mM EDTA, and 10 µCi [
32P]GTP for 10 min at 30°C, and then nucleotide exchange was halted by addition of
MgCl2 to 10 mM. Nitrocellulose was spotted with 5-µg aliquots of GST,
GST-RhoGAP, and GST-moesin in a volume of 10 µl. The nitrocellulose
was dried, blocked for 1 h in Western blocking buffer, and then incubated
for 5 min at 4°C with agitation in 3 ml buffer A containing 5 µM unlabeled
GTP, 0.5% BSA, and the labeled GTPase. Unbound protein was removed
with three brief washes in ice-cold buffer A/0.1% Tween-20, and then the
nitrocellulose was dried and autoradiographed for 120 min at
80°C with
intensifier screens.
32P]ATP (800 Ci/mmol). Actin polymerization was initiated by addition of 0.1 vol of
0.5 M KCl, 20 mM MgCl2, 0.5 mM CaCl2 (10× F buffer), and continued
for 20 min at room temperature, after which the F-actin was sedimented at
100,000 g for 30 min. An estimated 60% of [32P]ATP was incorporated
into the F-actin pellet. The F-actin was resuspended at ~20 µg/ml in F
buffer containing 5 µM phalloidin and 1 mM DTT. For blot overlays, the
protein was resuspended at 20 µg/ml in Western blocking buffer containing 5 µM phalloidin and 1 mM DTT, and incubated with preblocked
Western blots for 2 h at room temperature. The blots were washed in
TBS-T (150 mM NaCl, 10 mM Tris/HCl, pH 8.0, 0.2% Tween-20) four
times for 5 min at room temperature, and then exposed to film at
80°C
for 2-24 h.
Results
S,
provoked cytoskeletal reorganization in up to 75% of
cells, which could be detected at low magnification as an
increase in F-actin staining and sharpening of cell outline
owing to actin-mediated contraction of the cells (Fig. 1 A,
right). Permeabilization caused no loss of cells from the
substratum. Cytoskeletal reorganization was observed only
within a narrow concentration range of digitonin (0.0025-
0.004%) and could not be achieved in the absence of an ATP regenerating system, or at room temperature (20°C).
Fig. 1.
Permeabilization of
quiescent Swiss 3T3 cells in the
presence of GTPS. (A) Permeabilization (protocol 1) was performed in the absence of stimulus (left) or in the presence of 50 µM GTP
S (right). Cellular F-actin was visualized using
rhodamine-conjugated phalloidin. (B) Permeabilization (protocol 1) was performed in the
absence of stimulus (a and b), in
the presence of 50 µM GTP
S (c and d); 50 µM GTP
S with
0.1 nM C3 transferase (e and f);
50 µM GTP
S with 1 nM
N17Rac (g and h). After 20 min
at 37°C, cells were fixed and F-actin visualized with rhodamine-phalloidin (a, c, e, and g) and
vinculin visualized with a monoclonal antibody (b, d, f, and h).
In c and d, arrowheads show termini of bundled actin filaments
decorated with focal adhesions,
while arrows mark regions of
peripheral actin polymerization
decorated with linear arrays of
focal complexes. Bars: (A) 150 µm; (B) 30 µm.
[View Larger Versions of these Images (58 + 125K GIF file)]
S treatment
provoked a complex and dramatic reorganization of the
actin cytoskeleton (compare Fig. 1 B, c and d of GTP
S-treated cells with untreated cells shown in Fig. 1 B, a and
b). The cells displayed at least two types of actin structure:
first, thick, bundled filaments that traversed the cell body,
similar to actin stress fibers (Fig. 1 B, c, arrowhead); and
second, dense filamentous actin localized at the cell cortex, similar to that observed in the lamellipodia of intact
cells (Fig. 1 B, c, small arrow). These two types of structures were frequently found together in single cells, giving
rise to the complex cytoskeletal formations revealed by
F-actin staining. Moreover, cells with stress fiber-like filaments also contained typical focal adhesion complexes
(Fig. 1 B, d, arrowhead), while cells showing cortical actin
polymerization were decorated with peripheral, punctate focal complexes (Fig. 1 B, d, small arrow). The effects of
GTP
S on the actin cytoskeleton are consistent with the
activation of endogenous Rho and Rac GTPases in the
permeabilized cells.
S-treated cells, we used C3 transferase
and N17Rac, specific inhibitors of Rho and Rac function,
respectively (Paterson et al., 1990
; Ridley et al., 1992
).
Cells were permeabilized in the presence of GTP
S and
either inhibitor. C3 transferase totally blocked the assembly of stress fibers and focal adhesions and most cells now
showed clear cortical actin (Fig. 1 B, e) and peripheral focal complexes (Fig. 1 B, f). N17Rac, on the other hand,
blocked cortical actin polymerization, but stress fibers
(Fig. 1 B, g) and focal adhesions (Fig. 1 B, h) were unaffected. We therefore conclude that GTP
S activates endogenous Rho and Rac proteins in permeabilized fibroblasts.
S. Cells permeabilized in the presence of Rho (25 µg/ml) and incubated for
20 min showed dense actin bundles traversing the cell (Fig.
2 a); the bundles were decorated at their termini with large
focal adhesions (Fig. 2 b). These effects were abrogated by
inclusion of 2 ng/ml C3 transferase in the assay buffer
(data not shown). We were unable to induce lamellipodial
actin and peripheral integrin complexes using recombinant Rac protein.
Fig. 2.
Recombinant Rho stimulates formation of focal adhesions and stress fibers in permeabilized cells. Cells were permeabilized (protocol 1) in the presence of 1 µM recombinant
V14Rho. (a) Phalloidin staining for F-actin; (b) monoclonal antibody against vinculin. Bar, 30 µm.
[View Larger Version of this Image (156K GIF file)]
S or recombinant Rho (25 µg/ml, V14Rho), were no longer competent
to assemble actin filaments or integrin complexes. (Fig. 4,
a and b). It appeared, therefore, that during this incubation period one or more limiting components are either
lost from cells or inactivated. Pellets and supernatants were prepared from permeabilized cells and assayed for
lactate dehydrogenase (LDH; a marker for bulk cytosol),
Rho and focal adhesion proteins. Although 65% of the
LDH was lost from the cells during the 6-min incubation
with digitonin (Fig. 3 a), ~80% of Rho (Fig. 3 c) and essentially all cellular vinculin (Fig. 3 b), paxillin (not shown),
and RhoGDI (not shown) were retained. It seemed possible, therefore, that the cells retained sufficient cytoskeletal
components for actin filament and integrin complex assembly, but that a cytosolic component of the Rho and
Rac signal transduction pathways had been inactivated or
lost after permeabilization.
Fig. 4.
Reconstitution of Rho-induced effects after extended
permeabilization using a pig brain cytosolic extract. Cells were
permeabilized according to protocol 2 (i.e., permeabilized for 6 min in the presence of digitonin before addition of stimulus; see
Materials and Methods) in the presence of (a and b) 25 µg/ml
V14Rho, (c and d) 2 mg/ml pig brain extract, (e and f) 25 µg/ml
V14Rho plus 2 mg/ml pig brain extract. F-actin in the permeabilized cells was visualized using rhodamine-conjugated phalloidin
(a, c, and e) and focal adhesions with anti-vinculin antiserum (b,
d, and f). Bar, 30 µm.
[View Larger Version of this Image (155K GIF file)]
Fig. 3.
Retention of cytosolic and cytoskeletal markers after permeabilization.
(a) LDH activity, (b) vinculin
visualized by Western blot,
and (c) Rho visualized after
ADP ribosylation with C3
transferase and 32P-NAD. (a)
Coverslips were incubated
for 20 min at 37°C in 50 µl
vol ± 0.003% digitonin, or
for 6 min at room temperature in 60 µl vol containing
either 0.003% digitonin or
0.2% Triton X-100. Samples
were assayed for LDH in the
presence of 0.2% Triton
X-100. Data are mean ± SD from four independent determinations. (b) Coverslips were incubated for 6 min at room temperature in 60 µl buffer containing no detergent, 0.003% digitonin or
0.2% (vol/vol) Triton X-100, and supernatants and pellets were
recovered. Proteins were electrophoresed on 12% SDS-PAGE
and transferred to PVDF, and vinculin was visualized using a
monoclonal anti-vinculin antibody. (c) Cell supernatants and pellets were prepared as in b, and endogenous Rho determined as
described in Materials and Methods. Positions of molecular weight standards (kD) are marked.
[View Larger Versions of these Images (43 + 38K GIF file)]
S on permeabilized cells (data not shown). Concentrated cytosol from
growing Swiss 3T3 or Rat 1 cells, mouse brain or pig liver
could also reconstitute activity with GTP
S and Rho. It
therefore appeared that the concentrated extracts contained a factor or factors capable of reconstituting cytoskeleton reorganization in response to Rho or Rac activation.
S was used as a
stimulus.
).
S,
induced actin stress fibers and peripheral actin polymerization, both accompanied by their attendant focal complexes (Fig. 7, a and b). No cytoskeletal reorganization was seen in cells stimulated in the presence of boiled moesin,
either recombinant or purified, or in those treated with
moesin alone (data not shown). In the presence of the Rho
inhibitor C3 transferase, only cortical actin and peripheral
focal complexes were observed (Fig. 7, c and d) whereas in
the presence of N17Rac only stress fibers and focal adhesions were induced (Fig. 7, e and f). Moesin promoted
stress fiber formation in the presence of recombinant V14Rho (data not shown).
Fig. 7.
Recombinant moesin reconstitutes GTPS-
induced effects. Cells were
permeabilized according to
protocol 2, and incubated
with 50 nM (saturating) recombinant moesin and 50 µM GTP
S (a-f); additionally, in c and d 0.1 nM C3 was
present and in e and f 1 nM
N17rac. The cells shown in g
and h were incubated with
50 nM truncated moesin and
50 µM GTP
S. (a, c, e, and
g) Phalloidin staining for
F-actin; (b, d, f, and h) monoclonal antibody against vinculin. Bar, 30 µm.
[View Larger Version of this Image (140K GIF file)]
). This construct did not
support cytoskeletal rearrangement in treated cells under
any stimulus tested, at concentrations up to tenfold higher
than the full-length protein (Fig. 7, g and h; and data not
shown); indeed, the actin cytoskeletons of such cells were,
if anything, slightly disarranged or fragmented. By titration of recombinant full-length moesin and moesin purified from pig brain (amounts judged by Western blot analysis), we estimate that the E. coli-produced protein is
around 10-fold less active than the purified moesin in permeabilized cells. It is possible either that the Q-Sepharose
fraction contains additional active proteins or that the E. coli-produced protein is slightly contaminated with inhibitory proteolytic fragments, or partially incorrectly folded
or modified.
S-dependent cytoskeletal reorganization
in permeabilized cells. In parallel experiments, recombinant moesin, ezrin, and radixin exerted equivalent morphological effects at an equivalent range of concentrations
(data not shown).
32P]GTP-bound Rho or Rac for 5 min at 4°C and glutathione-
Sepharose beads added for a further 30 min. After recovery of the beads, the amount of Rho or Rac bound to
moesin was measured. GST-p160ROCK was used as a
positive control since this has been previously shown to
bind both Rho and Rac (Lamarche et al., 1996
). No significant binding of Rho or Rac to moesin was detected (data
not shown). The dot blot-binding assay was also used;
GST-moesin and GST-p50rhoGAP (which has also been
shown to bind both Rho and Rac; Lancaster et al., 1994
)
were spotted on to nitrocellulose filters and incubated with [
32P]GTP-bound Rho or Rac. As seen in Fig. 8, no
interaction with moesin could be detected.
Fig. 8.
Moesin does not interact directly with Rho or Rac. 5 µg
aliquots of GST, GST-RhoGAP,
and GST-moesin were spotted
onto nitrocellulose and the filter blocked with blocking buffer.
The filter was then incubated
with 0.1 µg of [32P]GTP-loaded
Rho or Rac and subjected to autoradiography.
[View Larger Version of this Image (78K GIF file)]
). We have compared the ability of recombinant moesin and moesin purified from pig brain cytosol to bind [
32P]ATP-labeled F-actin. As shown in Fig.
9, a and b, both preparations bind F-actin in a nitrocellulose overlay assay. To see if the F-actin binding site is required for GTP
S-stimulated actin filament assembly, a
deletion construct of moesin lacking the COOH-terminal 22 residues was used in the permeabilized cell assay. This
deletion mutant is unable to bind F-actin (Pestonjamasp et
al., 1995
) and when added to permeabilized cells along
with GTP
S, it could not promote actin filament or integrin
complex assembly (Fig. 7, g and h).
Discussion
S induced two clear types of cytoskeletal structures: (a) bundled actin filaments traversing the cells and
terminating in focal adhesions; and (b) filamentous actin
localized at the cell periphery and associated with small punctate focal complexes. By use of specific inhibitors, we
confirmed that these effects were due to activation of endogenous Rho and Rac GTPases respectively (Ridley and
Hall, 1992
; Nobes and Hall, 1995
). Recombinant, constitutively activated Rho was also able to stimulate focal adhesions and stress fibers when included in the permeabilization buffer, but recombinant Rac was unable to induce
cortical actin polymerization or formation of integrin complexes. The reason for this difference is not clear, but one possibility is that recombinant Rac is not as efficiently
posttranslationally modified in the permeabilized cells as
is Rho. It is even possible that posttranslational modification does not occur under these conditions and that for
Rho it is not essential. The use of baculovirus-expressed proteins may help to address this issue.
S, and we believed this to be caused
by the loss or inactivation of a specific activity (since Rho,
like numerous structural components of the cytoskeleton,
was not lost under these conditions). Furthermore, the response to either GTP
S or recombinant Rho could be reconstituted using concentrated cytosol prepared from pig
brain homogenates. After this observation, we undertook
the purification of a factor required for GTPase-dependent cytoskeletal reorganization, not based on direct association between Rho and a putative effector, but on a biological response to GTPase activation. A 10,000-fold purification of the activity in pig brain cytosol led to the identification
of moesin as its active component, and this was confirmed
using E. coli-derived recombinant moesin. Moesin alone
was unable to induce any cytoskeletal changes in permeabilized cells but it could cooperate with Rho to induce stress fibers and focal adhesions, and it could cooperate
with Rac to induce actin polymerization at the cell periphery
and the assembly of associated integrin complexes. Moesin
is therefore a component of both signal transduction pathways.
), which exhibit ~70-80% amino acid homology. The ERM proteins also possess significant homology in their NH2 termini with talin, the tumor suppressor
gene merlin/schwannomin (Rees et al., 1990
; Trofatter et
al., 1993
) and Band 4.1 (Arpin et al., 1994
). ERM proteins
have been reported to be localized to regions of contact between actin filaments and the plasma membrane, such
as microvilli and microspikes, cleavage furrows and sites of
cell-cell and cell-substratum attachment (Berryman et al.,
1993
; Franck et al., 1993
; Takeuchi et al., 1994
; Amieva
and Furthmayr, 1995
). Their importance as membrane-
cytoskeleton linkers was demonstrated by the severe disruption of cell adhesion and microvillus formation in cells treated with antisense oligonucleotides to ERM proteins
(Takeuchi et al., 1994
).
; Turunen et al.,
1994
). However, the regulatory mechanisms controlling
this activity in cells are not yet established. Algrain et al.
(1993)
expressed truncations of ezrin in CV1 cells and
found that the NH2-terminal portion was predominantly
membrane-associated, whereas the COOH-terminal portion colocalized with actin structures (Algrain et al., 1993
).
In Sf9 cells, expression of the COOH-terminal portion of
ezrin, but not the full-length protein, leads to the formation of long actin-rich, filopodium-like cell processes,
which is reportedly inhibited by coexpression of NH2-terminal sequences (Martin et al., 1995
). It is likely, therefore,
that the COOH-terminal F-actin binding region is masked
by NH2-terminal sequences. In addition, the F-actin binding region of all ERM proteins contains a conserved threonine (T558) and this has been shown to be phosphorylated, in moesin, during platelet activation (Nakamura et
al., 1995
).
. First, it was shown that PIP2
stabilized a high affinity interaction between moesin's
NH2-terminal portion and CD44, via PIP2 binding to the
same NH2-terminal portion of moesin; second, RhoGDI
was coimmunoprecipitated with moesin from cell lysates;
and third, the association between moesin and an insoluble fraction of BHK cells is modulated in vitro and in vivo
by regulators of Rho family GTPases. The authors proposed that Rho activation leads to synthesis of PIP2, which
then associates with moesin and alters its conformation so
as to promote membrane-cytoskeleton interaction. Since
phosphatidylinositol 4-phosphate kinases are among
known effectors of Rho and Rac, this model provides a
mechanism whereby GTPase activation may provoke local
effects at cell-cell and cell-substratum adhesions, where
moesin has been shown to reside. In addition, a potential
link between moesin and Rho GTPases was established in
the shape of RhoGDI (Ueda et al., 1990
). This protein is
supposed to sequester guanosinediphosphate (GDP)-
bound (inactive) GTPases in the cytosol, but if the association between the Rho-RhoGDI complex and moesin is regulatable, then RhoGDI may assist the movement of the
GTPase to its site of action. It should be noted, however,
that GDI was not found by Western blotting in our purified bioactivity from porcine brain (data not shown).
; Franck et al., 1993
) suggest that there is some specialization in
their activities.
showed that the affinity of moesin for
CD44 is modulated by PIP2; however, they found constitutively high affinity association between CD44 and an NH2-terminal moesin construct. Similarly, the effects of isolated
NH2- and COOH-terminal ezrin constructs on cells were regulated by coexpression of the reciprocal portions of the
protein (Martin et al., 1995
). It is reasonable to speculate
that PIP2 binding either provokes or accompanies a gross
conformational change, or unclasping, in moesin, which
renders the protein competent to bind both CD44 and actin. A precedent for a PIP2-regulated conformational
change exists in the focal adhesion protein vinculin. Digitonin, a cholesterol-chelating detergent (Elias et al., 1978
), recently has been shown to specifically extract moesin
from BHK cell membranes along with a small number of
other cytoskeletal proteins (Harder et al., 1997
). In permeabilized cells, digitonin may rapidly extract PIP2 directly
from moesin, or disrupt phospholipid turnover, thereby
preventing the PIP2-dependent interaction of moesin with
its normal ligands.
;
Nobes and Hall, 1995
), and kinases are among the putative
Rho effectors (Kimura et al., 1996
; Wanatabe et al., 1996
);
moesin may be a direct or indirect target of one of these
kinases.
Received for publication 4 February 1997 and in revised form 6 June 1997.
Please address all correspondence to Alan Hall, MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK. Tel.: 44-171-380-7909. Fax: 44-171-380-7909. e-mail: Alan.Hall{at}ucl.ac.uk
GAP, GTPase activating protein;
GDI, guanine nucleotide dissociation inhibitor;
GST, glutathione-S-transferase;
GTPS, guanosine 5
-O-(3-thiotriphosphate);
MLC, myosin light chain;
PIP2, phosphatidylinositol-4,5-bisphosphate;
PVDF, polyvinylidene difluoride;
ROCK, Rho-kinase.
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