From the Molecular Mechanisms of Disease Laboratories, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324
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ABSTRACT |
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Point and deletion mutants of moesin were
examined for F-actin binding by blot overlay and co-sedimentation, and
for intra- and intermolecular interactions with N- and C-terminal
domains with yeast two-hybrid and in vitro binding assays.
Wild-type moesin molecules interact poorly with F-actin and each other,
and bind neither C- nor N-terminal fragments. Interaction with F-actin is strongly enhanced by replacement of Thr558 with
aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by
deletion of the entire N-terminal membrane-binding domain of both wild
type and T558D mutant molecules, and by exposure to
phosphatidylinositol 4,5-diphosphate. Activation of F-actin binding is
accompanied by changes in inter- and intramolecular domain
interactions. The T558D mutation renders moesin capable of binding wild
type but not mutated (T558D) C-terminal or wild type N-terminal
fragments. The interaction between the latter two is prevented. DelN11
truncation enables binding of wild type N and C domain fragments. These
changes suggest that the T558D mutation, mimicking phosphorylation of
Thr558, promotes F-actin binding by disruption of
interdomain interactions between N and C domains and exposure of the
high affinity F-actin binding site in the C-terminal domain.
Oscillation between activated and resting state could thus provide the
structural basis for transient interactions between moesin and the
actin cytoskeleton in protruding and retracting microextensions.
Moesin is one of several closely related and widely expressed
proteins (1-3). They include ezrin and radixin and share ~75% overall sequence identity and regions, denoted A-H, of even higher structural conservation (4). A common secondary structure is predicted
to consist of a 320-residue globular N-terminal domain, a central
~200-residue predominantly Moesin and related proteins are required for the formation of membrane
microextensions (5, 7, 8, 24) and for the GTPase-mediated formation of
stress fibers, focal adhesion complexes, and microextensions (25-27).
Interactions of proteins in the cell cortex underlie a variety of
cellular responses, such as changes in cell shape, adhesion, movement,
and signal transduction. The function of moesin in these dynamic
processes at the membrane/cytoskeletal interface very likely depends on
regulation of its binding activities (28, 29).
Several proposals have been made, but it has not been firmly
established as yet how the linkage function of moesin operates and how
it is regulated in cells. In vitro experiments have
demonstrated homotypic and heterotypic associations between ezrin and
moesin and small amounts of dimeric molecules have been isolated from cell lysates by immunoprecipitation (30, 31). This, combined with the
fact that isolated N and C domains of these proteins interact, has led
to the suggestion that oligomers are functionally important and that
they are formed by disruption of an intramolecular interaction between
N-terminal and C-terminal domains (30, 32, 33). Consistent with this
idea is that the binding sites mediating this head-to-tail association
are masked in the full-length protein, when tested for interaction of
full-length molecules with either C or N domains (30).
A second proposal has been that phosphorylation activates binding
functions of moesin. In human platelets, for instance, thrombin activation leads to a rapid, but transient, increase in the
phosphorylation of a single threonine, Thr558,
in the C-terminal domain and near the presumptive location of the
F-actin binding site (20, 28). This residue is conserved in moesin,
ezrin, and radixin, and it is modified also in RAW macrophages (34),
Swiss3T3 cells (29),1 NIH3T3
cells,2 and RB2H3 mast
cells.3 Phosphorylation at
this site is regulated by the activity of Rho, and Rho-associated
kinase phosphorylates two residues in the C-terminal region, one of
which is Thr558 (29). Thr558 can also be
phosphorylated by Phosphorylation of Thr558 of moesin is of significance in
human platelets, because it is modulated by physiological activation, it can be manipulated by protein kinase and phosphatase inhibitors, and
it is accompanied by changes in moesin distribution and platelet morphology (28). We believe that phosphorylation of Thr558
can be mimicked by replacement of threonine with aspartate, and the
present study was undertaken to test the hypothesis that this modification activates the F-actin binding function of moesin. We show
that Thr558 substitution greatly enhances F-actin binding
activity of moesin by comparing the actin filament binding properties
of wild type moesin and a series of mutants by blot overlay and
co-sedimentation assays and by examining in detail interdomain and
intermolecular interactions by in vitro assays and with the
yeast two-hybrid system in vivo. Furthermore, we present
several lines of strong evidence that this activation step is linked to
the disruption of interactions between the N- and C-terminal domains.
This in turn changes the overall conformation of the protein and
exposes the high affinity binding site in the C-terminal domain.
GST-Moesin Mutants--
Mutations of moesin Thr558
within the sequence KYKT558L were introduced into moesin
cDNA by PCR4 using
oligonucleotides as primers that specify both the desired mutation and
carry a convenient restriction site to facilitate subcloning and
isolation of mutant clones. The following oligonucleotides were
synthesized: A, GEX5'(+) (5'-CCAAAATCGGATCTGGTTCCG-3'); B, MSN T558D
Bgl2(
To substitute Thr558 with Asp (D), two PCR reactions were
performed using pGhuMo (pGEX-KG-human moesin, or GST-MSN (20)) plasmid DNA as template with oligonucleotides A and B as primers in
reaction 1, and C and D in reaction 2. The product of reaction 1 was
digested with BglII yielding a 1.1-kb fragment, and the
product of reaction 2 gave a 0.5-kb fragment by digestion with
BglII and AatII. The two fragments were then
subcloned by three-way ligation into pGhuMo that had been predigested
with BglII and AatII to delete the C-terminal portion of moesin. This resulted in pKG-MSNT558D (GST-MSNT558D).
To substitute Thr558 with Ala, two PCRs were performed. In
reaction 1, pGhuMo served as template, and oligonucleotides A and E
served as primers. In reaction 2, UIII (Ref. 3; human moesin in
pBSSK+) served as template, and F and G served as primers.
Reaction 1 yielded a 1.1-kb BglII-HindIII
fragment, and reaction 2 yielded a 0.5-kb
HindIII-SalI fragment. These two fragments were
subcloned by three-way ligation into
BglII/SalI-predigested pGhuMo to delete the
C-terminal portion of moesin and to generate pKG-MSNT558A (GST-MSNT558A).
To delete KYKT558L, two PCRs were performed with
oligonucleotides A and H as primers in reaction 1 and I and D in
reaction 2 using pGhuMo as template. The first reaction yielded a
1.1-kb BglII-PvuI fragment, and the second
yielded a 0.5-kb PvuI-AatII fragment. The two
fragments were subcloned by three-way ligation into
BglII/AatII-predigested pGhuMo to result in
pKG-MSN
pKG-MSNT558A was digested with HindIII to delete the
C-terminal 19 codons and recircularized to generate pKG-MSN
pKG-MSNT558D was digested with BglII to delete encoding
residues 198-558 and recircularized to generate pKG-MSN
pKG-MSNc (GST-moesin C-terminal region residues 404-577) was
constructed by inserting the EcoRI-XhoI fragment
from MSNc/pCR3 (see below) into pGEX-KG.
pKG-MSNn (GST-moesin N-terminal region residues 1-310) was generated
by subcloning the EcoRI-SalI fragment from
pAS1-MSNn (see below) into pGEX-KG.
pKG-EZR (GST-EZR1-586(P4T,N6S)) and pKG-EZRn (GST-EZR1-310(P4T,N6S))
were constructed by replacing the moesin
NcoI-NcoI or NcoI-SalI
fragment in pKG-MSN with the human ezrin
NcoI-NcoI or NcoI-SalI
(from pAS1-EZRn)2 fragments, respectively. pG3X-EZRc
(GST-EZR280-586) was made by inserting the ezrin C-terminal
BamHI-BamHI fragment into pGEX-3X vector.
pKG-RDX (GST-RDX1-583) was as described by Pestonjamasp et
al. (20). pKG-RDXn (GST-RDX1-449) was generated by deleting the pig radixin C-terminal HindIII-HindIII fragment
from pKG-RDX and recircularizing the plasmid. pKG-RDXc
(GST-RDX373-583) was constructed by subcloning the C-terminal
XbaI-XhoI fragment from pKG-RDX into pGEX-KG.
PGEX-NF2 (GST-merlin) and pGEX-NFc (GST-merlin C-terminal region,
residues 254-595) were as described by Huang et al.
(23).
To confirm the correct sequence, all PCR-derived mutants were sequenced
using the ABI PRISM DYE Terminator cycle sequencing system
(Perkin-Elmer).
Untagged Moesin Mutants in pET Vectors--
Wild type and
mutated moesin cDNAs were subcloned into derivatives of pET vectors
(Stratagene) carrying a T7 promoter for expression of untagged moesin
proteins in Escherichia coli and in vitro (41).
The NdeI-HindIII fragment from pGhuMo,
pKG-MSNT558D, and pKG-MSN Untagged Moesin Mutants in pCR3 Vector--
The
EcoRI-XhoI fragment encoding full-length moesin
from pGhuMo was subcloned into pCR3 (InVitrogen) to generate MSN/pCR3. The C-terminal PstI-XhoI fragment from pGhuMo
was subcloned into pCR3 to make MSNc/pCR3 encoding residues 421-577
with codon 421 as the initiation methionine. Likewise, the
PstI-XhoI fragment from pKG-MSNT558D was
subcloned into pCR3 to generate MSNcT558D/pCR3. MSNn/pCR3 encoding
moesin's N-terminal region residues 1-310 was generated by digesting
MSNn-GFP/pCR32 with SmaI and EcoRV
(to delete GFP) and recircularization.
GAL4-Moesin Hybrid Plasmids--
The
NcoI-NcoI fragment from UIII was inserted into
pAS1 and pACT2 to create pAS1-MSN
pAS1-MSN
pACT2-MSN (G4AD-HA-moesin) and pACT2-MSNT558D (G4AD-HA-moesin T558D
mutant) were constructed by subcloning into pACT2 the EcoRI-XhoI fragment from pGhuMo and
pKG-MSNT558D, respectively.
pACT2-MSNc (G4AD-HA-moesin C-(404-577)) and pACT2-MSNcT558D
(G4AD-HA-moesin C-(404-577/T558D)) were generated by subcloning the
EcoRI-XhoI fragment from MSNc/pCR3 and
MSNcT558D/pCR3 into pACT2.
pACT2-MSNc
pAS1-MSN (G4BD-moesin) was constructed by subcloning the
BamHI-XhoI fragment, excised from pACT2-MSN by
partial digest with BamHI and XhoI into pAS1-NF2
(23), and predigested with BamHI and SalI (to
delete NF2).
pAS1-MSNT558D (G4BD-moesin T558D mutant) was created by replacing the
NdeI-SalI fragment in pAS1-MSN with the
NdeI-SalI fragment from pKG-MSNT558D.
pAS1-MSNc (G4BD-moesin C-(404-577)) was made by subcloning the
BamHI-XhoI fragment from pACT2-MSNc into
pAS1-NF2 predigested with BamHI and SalI (to
delete NF2).
pACT2-MSNn (G4AD-HA-moesinN1-310) was generated by subcloning the
EcoRI-SalI fragment from pAS1-MSNn into pACT2
cut with EcoRI and XhoI. pAS1-MSNn
(G4BD-HA-moesin N-terminal region 1-310) was constructed by subcloning
the SmaI-SmaI fragment from pACT2-MSN into pAS1.
It was not suitable for the study because of high background signal in
yeast two-hybrid assays.
Recombinant Proteins, Antibodies, and Western
Blot--
GST-moesin fusions were expressed in E. coli
transformed with corresponding plasmids according to the instructions
(Pharmacia Biotech Biotech). Untagged moesin proteins were expressed in
E. coli strain BL21(DE3) transformed with pET plasmids
carrying moesin inserts according to instructions (Stratagene, Palo
Alto, CA). The recombinant moesin proteins were analyzed by SDS-PAGE,
Coomassie staining, and Western blot as described previously using
rabbit polyclonal antibody pAs90-7 (8) to verify the expression of desired proteins. Before use for F-actin blot overlay, the recombinant proteins were quantitated by Western blot followed by densitometry and
using known amounts of moesin to construct a standard curve.
F-actin Blot Overlay Assays--
Protein samples were resolved
by SDS-PAGE under reducing conditions and electrotransferred to
nitrocellulose membranes. The blots were incubated in TTBS (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween
20) containing 0.005% sodium azide overnight at 4 °C and in
blocking buffer (TTBS containing 5% dry milk and 0.005% sodium azide)
overnight at 4 °C. After washing with TTBS, the blots were probed
with [ F-actin Co-sedimentation Assays--
Co-sedimentation of
in vitro translated, 35S-labeled moesin with
polymerized F-actin was based on reported methods (42-44) and done
with modifications. Phosphatidylinositol (PI) or phosphatidylinositol 4,5-diphosphate (PIP2) (Sigma) was dissolved in distilled
water to a final concentration of 1 mg/ml and sonicated three times each for 10 s. In vitro translated proteins were
incubated with or without 50 µg/ml PI or PIP2 in
polymerization buffer (100 mM KCl, 2 mM
MgCl2, 0.2 mM CaCl2, 0.2 mM ATP, 1 mM dithiothreitol, 0.01%
NaN3, 5 mM Tris-HCl, pH 8.0) containing 0.75%
Triton X-100 for 10 min and centrifuged at 150,000 × g
for 30 min in a Beckman TL-100 ultracentrifuge using Ultra-Clear
centrifuge tubes (5 × 41 mm) and a TLA-100.3 rotor to clear
protein aggregates. The supernatants were incubated with or without 0.5 mg/ml F-actin in polymerization buffer containing 0.75% Triton X-100
for 30 min, transferred into centrifuge tubes with or without
underlying 20% sucrose in polymerization buffer, and centrifuged again
as above. Aliquots of the supernatants and pellets that had been rinsed
with polymerization buffer were solubilized by boiling in SDS sample
buffer, analyzed by SDS-PAGE and autoradiography, and the
35S-labeled protein bands were quantitated by densitometry.
Identical results were obtained with or without the 20% sucrose
cushion, and those without are presented.
In Vitro Translation and Protein Binding Assay--
pET or pCR3
plasmids carrying moesin inserts were transcribed and translated
in vitro using a TNT coupled rabbit reticulocyte lysate
system (Promega, Madison, WI) in the presence of T7 RNA polymerase and
L-[35S]methionine according to the
manufacturer's instructions. In vitro translation products
with or without 50 µg/ml PI or PIP2 were incubated with
GST fusion proteins, which had been immobilized on
glutathione-Sepharose beads and washed four times with
phosphate-buffered saline, in binding buffer (200 mM NaCl,
20 mM Tris-HCl, pH 8, 0.2% Triton X-100) at 4 °C. After
1.5 h, the beads were washed six times in binding buffer. Bound
proteins were boiled in SDS sample buffer, resolved by SDS-PAGE, and
subjected to autoradiography.
Yeast Two-hybrid Assays--
Pairs of hybrid plasmids were
transformed (45) into the yeast reporter strain Y190 (46), and
co-transformants were selected on SC media lacking Trp and Leu at
30 °C for 4 days. Single colonies were patched onto selective
plates, incubated at 30 °C for 3 days, and subjected to a filter
lift assay for F-Actin Binding of Moesin Mutants: Enhancement by T558D
Mutation--
As a first step, a series of mutations of
Thr558 and flanking sequences were introduced into moesin
by polymerase chain reaction, and the mutants were subcloned to yield
GST fusion proteins. Mutant and wild-type GST-moesin were then
expressed in E. coli, and effects of the mutation on F-actin
binding were examined by F-actin blot overlay with 32P-
(27) or 125I-labeled F-actin (20).
As shown in Fig. 1, control wild type
(WT) and T558A mutant moesin fused to GST bind the F-actin probe
relatively poorly. A significantly higher amount (5-7-fold increase)
is observed for full-length moesin, when Thr558 is replaced
with aspartate, a negatively charged residue that is widely used
to mimic phosphorylation. Deletion of KYKTL, the pentapeptide that
includes Thr558 (
The weak signal obtained for WT moesin could have been due to the
fusion of GST to the C terminus, and since GST can have potentially
undesirable effects on structure and on the biological activity of
moesin (48), we formally addressed this issue with untagged moesin. WT
and mutant proteins were expressed in E. coli using pET
vectors and examined for F-actin binding by blot overlay assay. Similar
to data with GST fusions, untagged WT moesin binds F-actin poorly, the
T558A mutation binds slightly better, and binding of the T558D mutant
is greatly enhanced (Fig. 1). Deletion of the five residues
KYKT558L (
To distinguish between these possibilities, we have tested an
additional mutant. Earlier observations suggested that in contrast to
WT full-length moesin, the Co-sedimentation of F-actin and Moesin Mutants: Enhancement by
T558D and
PIP2, but not phosphatidyl 4-phosphate, binds to the
N-terminal domain of ezrin (15). It has been proposed to form a complex with and to stabilize the interaction of moesin, ezrin, and radixin with the cytoplasmic segment of CD44 in physiological salt
concentrations (49). To examine whether PIP2 affects
F-actin binding, radiolabeled WT moesin was generated by in
vitro translation and incubated with actin filaments in the
presence or absence of PI or PIP2. As shown in Fig.
3b, enhanced binding to actin filaments is seen when
PIP2, but not phosphatidyl 4-phosphate, is present. The
amount of sedimented WT moesin is similar to that of the mutants shown in Fig. 3a.
Wild Type and T558D Mutant C-terminal Domains of Moesin Have
Comparable F-actin Binding Activity--
To determine if the T558D
mutation affects the interaction with actin filaments directly, we
tested mutant and wild type C domains, prepared by in vitro
translation, in the co-sedimentation assay. As shown in Fig.
4, equal fractions of wild type and
mutant C domain either untagged or fused to GFP, co-sediment with
F-actin. Thus, the high affinity binding site associated with the
C-terminal domain appears to be identical in WT and mutant molecules.
Once uncovered by removal of the N-terminal domain, T558D has no
additional effect. Fig. 4 shows that the untagged form of C-moesin
yields a double band during translation. This additional band of lower molecular mass can be detected also when this domain is expressed in
fibroblasts, E. coli or
yeast.5 The reason for this
and the nature of the second product has not been investigated
further.
T558D Mutation Prevents Interaction between WT N- and Mutated
C-terminal Domains--
The similar F-actin binding activity of WT and
mutant C-terminal domains suggested that the binding site is present
but not exposed in WT moesin and that the activity is uncovered by the T558D mutation. As originally shown by Gary and Bretscher (30), the two
isolated domains of ezrin made by recombinant techniques bind to each
other, but react poorly with full-length ezrin. This was consistent
with a model in which the two domains interact within the structure of
the full-length protein. To further investigate how the T558D mutation
affects the interdomain and intramolecular interactions, we took
advantage of this information and tested for binding using
35S-labeled N and C domains as probes. GST-N-moesin was
immobilized on Sepharose beads and incubated with isotope-labeled WT
N-terminal, WT C-terminal, and T558D mutant C-terminal domains
generated by in vitro translation. After incubation and
extensive washing, SDS was added, and the solubilized proteins were
analyzed by SDS-PAGE and autoradiography. Fig.
5 shows that, as expected, only WT
C-moesin and not N-moesin bind to N-moesin. In contrast, T558D-C-moesin could not be recovered in the bound fraction, suggesting that the
mutation disrupts the N-terminal binding activity of the C domain.
Similar results were obtained with in vitro translated GST-moesin domain fusions (not shown).
Extending these studies to full-length moesin, we asked the question
whether immobilized GST-fusions of C and N domains bind in
vitro translated and radiolabeled WT and T558D mutant moesin. We
observed only weak binding of both domains to full-length WT moesin
(Fig. 6, lanes 3 and 9) but markedly enhanced binding of the C domain to
T558D mutant moesin molecules (Fig. 6, lane 6). A
similar enhancement was also seen in the binding of
DelN11 moesin is able to bind both N and C fragments (not shown here,
but see below), suggesting that the intramolecular N-C interaction is
sufficiently weakened or disrupted to favor binding of extramolecular fragments.
It is of additional interest that PIP2 enhances binding of
the N but not C domain to WT moesin and inhibits the interaction between Thr558 moesin and GST-C-moesin (Fig. 6,
lane 4). This is consistent with PIP2
blocking the site for C domain binding in the N-terminal domain.
To assess whether these interactions occur under perhaps more relevant
in vivo conditions as well, we employed the yeast two-hybrid assay and consistent data could be obtained regardless of whether fragments were expressed as GAL4BD or GAL4AD fusions (Table
I). For instance, WT moesin shows no
interactions with WT C-moesin, delN11-N-moesin, WT N-moesin, WT moesin,
or delN11 moesin (Table I, line C, 1, 5, 6, 7, and 8). The reverse is
true as well (Table I, column 7, A, B, C, and D). The control
experiment shows strong interactions between WT N- and C domains in
both directions (Table I, compare B5, B6, and 1A), while T558D
abolishes this interaction (Table I, 2A).
In full-length moesin, the T558D mutation enhances the interaction with
WT C-moesin (Table I, E1 and B9), while both N and C domains interact
with delN11-moesin molecules. Thus, both mutations affect binding
between fragments and full-length molecules, albeit by different mechanisms.
The present study documents that T558D substitution, which is
expected to mimic phosphorylation of Thr558, activates
F-actin binding of moesin by exposure of the high affinity binding site
in the C-terminal region. Activation is accomplished by a structural
change that affects the arrangement of the N- and C-terminal domains
within the molecule (Fig. 7). This
conformational mechanism could be physiologically relevant in intact
cells, since intra- and extracellular signals determine the state of
phosphorylation of moesin (28, 29). Phosphokinases and phosphatases
thus appear to regulate one important function of this protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical region, and a ~50-residue highly charged C-terminal domain. Based on a large body of evidence, these proteins bind to components of the cell membrane with the N-terminal domain (5-11). Candidate membrane-binding targets include CD44; intercellular adhesion molecules 1, 2, and 3 (12-14);
phosphatidylinositol 4,5-disphosphate (15); cyclic
AMP-dependent protein kinase (16); and intermediary
adapters of the NHERF family of proteins (17, 18). The C-terminal
domain contains a major high affinity actin binding site, presumed to
be located in region H, a sequence that is identical in ezrin and
radixin (19-23). This sequence differs in the tumor suppressor merlin,
and while F-actin binds to the moesin sequence, it does not bind to
merlin under the same conditions (23).
-phosphokinase C (35). Phosphorylation of this
conserved amino acid by Rho-associated kinase prevents interaction of
the phosphorylated C domain with the N-terminal domain (29) but does
not appear to influence binding of the isolated C domain to F-actin.
Other than Thr558, the role of phosphorylation of specific
tyrosine, serine, or other threonine residues in response to
stimulation by growth factors, histamine, or lysophosphatidic acid has
not been established (36-40).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (5'-CCGGATCTGGCGCAGATCTTTGTATTTGTCTCGGCC-3'); C,
MSN T558D Bgl2(+)
(5'-GGCCGAGACAAATACAAAGATCTGCGCCAGATCCGG-3'); D,
GEX3'Aat2(
) (5'-CTCTCAAGGATCTTACCGCTG-3'); E, MSN T558A
HindII(
) (5'-CCGGATCTGGCGCAAAGCTTTGTATTTGTCTCGGC-3'); F, MSN T558A HindII(+)
(5'-GCCGAGACAAATACAAAGCTTTGCGCCAGATCCGG-3'); G,
MSN3'SalI(
)
(5'-AATTTTAGGTCAGTCGACATCCCTGGAG-3'); H, MSN DK5
PvuI(
)
(5'-GCGCAGGGTCTTGTATCGATCGCGGCCCAGTCGCATGT-3'); I, MSN DK5
PvuI(+)
(5'-CGAGACAAATACAAGACGATCGCCAGATCCGGCAG-3').
K5 (GST-MSN
K5).
C19
(GST-MSN
C19).
(L198-T558) (GST-MSN
m).
K5 was cloned into pETNde to generate
MSN/pET, MSNT558D/pET, and MSN
K5/pET, respectively. The longer
NdeI-HindIII fragment, obtained from
pKG-MSNT558A by partial digest with HindIII, was cloned into
pETNde to generate MSNT558A/pET. The shorter
NdeI-HindIII fragment was cloned into pETNde to
make MSN
C19. The NcoI-NcoI fragment from UIII
was cloned into pETNco to generate MSN
N11/pET.
n11 (G4BD-HA-moesin12-577) and
pACT2-MSN
n11 (G4AD-HA-moesin12-577), respectively.
n11 and pACT2-MSN
n11 were digested with SmaI
to delete the C-terminal domain and recircularized to generate
pAS1-MSNn
11 (G4BD-HA-moesinN12-310) and pACT2-MSNn
(G4AD-HA-moesinN12-310), respectively.
K5 (G4AD-HA-moesin C/KYKTL deleted) and pACT2-MSNc
c19
(G4AD-HA-moesin C-(1-558)/19 residues truncated) were constructed by
subcloning the EcoRI-XhoI fragment from
GFP-MSNc
K5/pCR3 and GFP-MSNc
c19 into pACT2.2
-32P]ATP- or [
-35S]ATP-labeled
F-actin (27). A. Spudich (Department of Biochemistry, Stanford
University) generously supplied rabbit skeletal muscle actin. The probe
was prepared as follows. 66 µM G-actin was gel-filtered into G buffer (2 mM Tris-HCl, pH 8.0, 50 µM
CaCl2, 0.5 mM dithiothreitol, 50 µM NaN3) to reduce the concentration of free
ATP and then incubated for 120 min at room temperature with 125 µCi
([
-32P]ATP at 800 Ci/mM, or
[
-35S]ATP at >1000 Ci/mM). Actin
polymerization was initiated by the addition of 0.1 volume 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 F-actin was sedimented at
100,000 × g for 30 min. An estimated 70% of the
isotope was incorporated into the F-actin pellet. The F-actin was
resuspended at 20 µg/ml in F buffer containing 5 mM phalloidin and 1 mM dithiothreitol. For blot overlay, the
protein was resuspended at 20 µg/ml in Western blocking buffer (TTBS; 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.2%
Tween 20, 5% fat-free milk powder) containing 5 µM
phalloidin and 1 mM dithiothreitol and incubated with
preblocked Western blots for 2 h at room temperature. Blots were
washed in TTBS four times for 5 min at room temperature and then
exposed to film at
80 °C.
-galactosidase activity (46, 47) and onto selective
plates lacking His and containing 25 mM
3-amino-1,2,4-triazol. Incubation was done at 30 °C for 7 days to
assay for growth (46). While results of both assays were in good
agreement, those of the
-galactosidase assay are presented.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
KYK(T558)L), reduces
F-actin binding as compared with WT moesin, while truncation of the
C-terminal 19 residues (
C19) downstream of Thr558 or of
361 residues (
(L198-T558)) upstream of Thr558 eliminates
binding completely.
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Fig. 1.
T558D mutation and N-terminal deletion of 11 amino acid residues result in enhancement of F-actin binding of moesin
on blot overlay. a, top, Coomassie
Blue-stained SDS gel of lysates from bacteria expressing GST fusion
proteins of moesin (WT, T558A, T558D, KYKTL (deletion of
Lys555-Leu559),
C19 (deletion of C-terminal
19 residues), and
L198-T558). Bottom,
32P-labeled F-actin blot overlay of lysates shown in the
top gel. Note the difference in signal intensity of T558D
mutant moesin in comparison with WT, T558A, and
KYKTL. No binding is
seen to C-terminal truncated forms of moesin (lanes
5 and 6). b, top, Coomassie
Blue-stained SDS gel of lysates of bacteria expressing untagged WT and
mutant forms of moesin (lanes 1-5, same as in
a; lane 6,
N11 (deletion of 11 N-terminal residues)). Bottom, 32P-labeled
F-actin blot overlay of lysates shown in the top gel.
Numbers on the left denote molecular masses of
standards. The difference in signal intensity between WT, T558A,
KYKTL, and T558D is similar to that in a. No binding is
seen to
C19.
N11 binds as strongly as T558D mutant moesin.
KYKTL) largely diminishes, and deletion of the
C-terminal 19 residues in
C19 totally abolishes, F-actin binding,
although four times the amount of protein was analyzed. This result
clearly shows that the strong enhancement of F-actin binding is caused by the replacement of Thr558 with Asp and is similar in its
effect to phosphorylation of this threonine residue in platelet moesin
(28, 63). Furthermore, the addition of GST to moesin in the fusion
protein does not affect the actin binding property of moesin. However,
this result does not permit conclusive interpretations regarding
mechanism. The mutation could have increased the affinity for F-actin
of the previously determined binding site in the C-terminal domain (20) simply by the addition of a negative charge, it could have caused exposure of a previously masked binding site, or it could have even
created binding sites de novo.
N11 mutant, made by truncation of 11 N-terminal residues, binds N- and C-terminal fragments (see below). As
shown in Fig. 1B (lane 6), this mutant
binds approximately 3-4-fold higher amounts of F-actin in comparison
with WT. This mutation is located opposite to where the high affinity
F-actin binding site is presumed to reside in the molecule. Thus, it is highly unlikely that, at least for this mutant, the enhancement of
F-actin binding is due to an increase in the affinity of the C-terminal
binding site for F-actin. Additional binding site(s) for actin have
been detected in the N-terminal domain of ezrin with a solid phase
binding assay (48). In contrast to these data, we have not detected
these sites by blot overlay assaying GST fusions or untagged N- and
C-terminal domains of moesin, ezrin, radixin, and merlin. As shown in
Fig. 2 and as expected from previous results (20, 23), the C-terminal domains of moesin, ezrin, and radixin,
but not merlin, bind F-actin, while none of the N-terminal domain
fusions exhibits binding.
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Fig. 2.
The C domains of moesin, ezrin, and radixin
bind 35S-labeled F-actin equally well on blot overlay.
Top left, Coomassie Blue-stained SDS gel of
lysates from bacteria expressing GST fusions of the C-terminal domain
of merlin (NF2c), merlin (NF2), ezrin
(EZR), radixin (RDX), and moesin
(MSN). Top right, Coomassie
Blue-stained SDS gel of lysates from bacteria expressing GST fusions of
N- and C-terminal domains of ezrin, radixin, and moesin.
Bottom, autoradiograms of 35S-labeled F-actin
blot overlays of the gels shown on top. Relatively weak
binding of the F-actin probe is seen with full-length protein, with
radixin showing a stronger signal in comparison with ezrin and moesin
(lanes 3-5), but none of the N domains binds the
probe (lanes 6, 8, and 10).
In contrast, the C domain of all of three proteins interacts
strongly.
N11 Mutation and by PIP2--
The effects of
mutations on F-actin binding were examined independently with a
co-sedimentation assay (43). To perform this experiment, WT moesin and
two of the critical mutants were subcloned into a T7-driven expression
vector and translated in vitro in the presence of
[35S]methionine. Radiolabeled proteins were then
incubated with actin filaments and after centrifugation analyzed by
SDS-PAGE and autoradiography. As shown in Fig.
3a, only a very small amount
(<2%) of WT moesin co-sediments with actin. In contrast, considerably
higher amounts of moesin are recovered in the pellet with actin, when
Thr558 is mutated to aspartate, and somewhat lesser amounts
of the N-terminal deletion mutant (
N11).
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Fig. 3.
Co-sedimentation of F-actin and in
vitro translated T558D and N11
moesin mutants and of wild type moesin in the presence of
PIP2. a, in vitro translated WT,
T558D, or
N11 moesin was incubated with actin filaments and
centrifuged, and supernatants (S) and pellets (P)
were analyzed by autoradiography of the SDS gel. A larger fraction of
T558D moesin co-sediments in comparison with WT and
N11.
b, in vitro translated 35S-labeled WT
moesin was preincubated with PIP2 or PI and F-actin. After
centrifugation, supernatants and pellets were analyzed by SDS gel
electrophoresis and autoradiography. PIP2 but not PI
enhances binding of WT moesin to F-actin.
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Fig. 4.
T558D mutation abolishes the
interaction between N-and C-terminal domains of moesin.
GST-N-moesin was immobilized on a glutathione matrix and in
vitro translated, and labeled N-moesin, C-moesin, or
T558D-C-moesin was added. After washing the beads, the bound material
was solubilized with SDS sample buffer and analyzed by gel
electrophoresis and autoradiography. Lanes 1,
3, and 5 ( lanes) show the
translation products prior to incubation. The arrowheads
indicate molecular masses of N-moesin (left) and C- or
C-T558D-moesin (right). Lanes 2,
4, and 6 (+ lanes) show the bound
material. N-moesin does not interact with itself (lane
2) but binds C-moesin (lane 4) unless
Thr558 is mutated to Asp (lane
6).
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Fig. 5.
In vitro translated wild type and
T558D mutant C-terminal domains of moesin co-sediment equally with
F-actin. In vitro translated 35S-labeled
GFP fusions of WT or T558D mutant C domain (left
panel) or untagged WT and mutant C domains (residues
404-577) (right panel) were incubated with actin
filaments and centrifuged, and supernatants and pellets were analyzed
by SDS-gel electrophoresis and autoradiography. The polyacrylamide gel
concentration was 8% (lanes 1-4) and 15%
(lanes 5-8). Both WT and mutant C domains
co-sediment with actin filament, and this does not appear to be
influenced by GFP. Note the double band in lanes
5-8. The more slowly migrating peptide has the expected
molecular mass. The second peptide may represent a degradation product,
since its amount varied in different experiments.
N11-moesin to
GST-N domain. These results suggest that WT moesin is in a "closed"
configuration, in which the intramolecular interaction sites are
blocked. The T558D mutation shifted molecules to an "open" form,
which can bind an extramolecular C, but not N domain, presumably
because the mutation disrupts the interdomain interaction present in WT
moesin.
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Fig. 6.
PIP2 enhances
intermolecular interaction of full-length moesin with both N and C
domains, while T558D-moesin binds only N-moesin. GST-C- or
GST-N-moesin was immobilized on a glutathione matrix, and
35S-labeled in vitro translated WT moesin,
T558D-moesin, or N11-moesin was added. After washing, the
SDS-soluble material was analyzed by gel electrophoresis and
autoradiography. Lanes 2, 5,
8, and 10 show the in vitro translated
labeled full-length protein. A comparison of lanes
1 and 3 shows that PIP2 does not
change binding between WT moesin and GST-C-moesin. Binding of
GST-C-moesin is much stronger to the Thr558 mutant moesin
(lane 6), and PIP2 partially inhibits
this interaction (cf. lanes 4 and
6). GST-N-moesin binds poorly to WT moesin in the absence of
PIP2 (lane 9), and this binding is
enhanced in the presence of PIP2 (lane
7) and after N-terminal deletion of 11 residues
(lane 11).
Interactions of full-length and domains of moesin in yeast two-hybrid
system: T558D and N11 mutations disrupt intramolecular head (N)
to tail (C) association
-galactosidase assays were performed on yeast Y190 cells
co-expressing pairs of GAL4-moesin fusions as described under
"Experimental Procedures" to assess interdomain and intermolecular
interactions of moesin.
-Galactosidase activity was scored visually,
with +++++ denoting dark blue and
denoting white colonies. The
hybrid constructs used were, pAS1-MSNn
(A), pAS1-MSNc (B), pAS1-MSN
(C), pAS1-MSN
N11 (D), pAS1-MSNT558D (E), pGBT9 (F), pACT2-MSNc (1),
pACT2-MSNcT558D (2), pACT2-MSNc
K5 (3), pACT2-MSNc
C19 (4),
pACT2-MSNn
(5), pACT2-MSNn (6), pACT2-MSN (7), pACT2-MSN
N11 (8),
pACT2-MSNT558D (9), and pACT2 (10).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Model illustrating conformational change
induced by mutations and PIP2. On the left,
the N domain (amino acid residues 1-310) interacts with a segment of
the C domain (residues 404-577). On the right, mutations
(indicated by an asterisk) and PIP2
(open rectangle) disrupt this interaction to
expose a high affinity binding site for F-actin and the sites of
contact of the two domains (solid bars). The site
on the N domain becomes accessible for an additional C domain, but the
C domain site is blocked for N domain binding by T558D. The interaction
of F-actin with the C domain site is not affected by this
mutation.
Extending earlier observations on ezrin and moesin (19,20), we confirm with in vitro translated C-terminal fragments that a high affinity binding site for F-actin is localized in the C-terminal domain and that the T558D mutation does not significantly alter its binding activity (Fig. 4). Two F-actin binding tests indicate that full-length moesin molecules either made in bacteria or in a cell-free system interact with actin filaments, but do so only weakly. Substituting threonine with the negatively charged amino acid residue aspartate, but not alanine markedly enhances binding. The weak binding of the full-length protein is evidently due to the inaccessibility of the binding site because truncation of the entire N domain or of only 11 N-terminal amino acid residues leads to strong F-actin binding (Figs. 1 and 2). This result agrees with previous data demonstrating that radixin C domains made in insect cells prior to and after phosphorylation with Rho-associated kinase in vitro exhibit similar F-actin binding activity (29). Taken together, the current results provide compelling evidence in support of our conclusion that the negative charge introduced at the Thr558 position controls F-actin binding not by changing affinity but rather by exposing the C-terminal binding site.
T558D Mutation Exposes F-actin Binding Site by Disrupting N and C Domain Interaction-- Our data demonstrate a striking link between the unmasking of F-actin binding activity and of interdomain binding sites. Gary and Bretscher (30) have shown that isolated N and C domain fragments of ezrin bind each other and that sites for interdomain binding are not accessible in the intact molecule when probing bacterially made ezrin and ezrin isolated from tissues with N and C domain fragments or with specific peptide antibodies. Partial unfolding was required to recognize the binding sites for domain fragments. Likewise, we observe strong binding between N and C domains of moesin and only weak or absent interaction between these fragments and full-length moesin. The C domain fragment carrying the T558D mutation, on the other hand, does not bind the N domain fragment any longer (Figs. 5 and 6), while the full-length T558D mutant protein binds an additional C domain fragment. Both observations are in agreement with the proposal that N- and C-terminal domains interact within the native protein structure. The mutation disrupts the intramolecular domain interaction, and a conformational shift results that presumably is large enough to allow binding of the extramolecular C-terminal fragment (Fig. 7).
It is of interest in this context that full-length moesin is a poor substrate for Rho-associated kinase in vitro, while this enzyme phosphorylates two threonine residues, Thr558 and Thr567, when the C domain fragment of moesin is used for the reaction (29). In its modified form, the C domain fragment is no longer capable of binding to the N domain. It is clear from our data that the modification of only one site, Thr558, is sufficient to inhibit binding.
We have identified two additional mechanisms that apparently accomplish activation as well: deletion of 11 amino acid residues at the N-terminal end and binding of phosphatidylinositol 4,5-diphosphate (Fig. 7). PIP2 binds to a GST fusion protein of ezrin consisting of the N-terminal 309 amino acid residues but not to the C-terminal fragment (positions 310-586) (19). If this is true for moesin as well, PIP2 may activate F-actin binding of moesin by interfering with interdomain binding as seen by the inhibition of the interaction between the C domain fragment with T558D moesin in its presence (Fig. 6). It is less clear at the moment, how N-terminal truncation activates F-actin binding. Very recent structural data derived for a co-crystal of N and C domains of moesin show that two of the three subregions of the N domain contact the C domain. This suggests that interdomain interaction in the molecule is complex and may depend on the structural integrity of regions of the protein that are separate from identified binding sites (49).
The binding sites for F-actin and the N domain appear to be rather
close to each other, both located near the C terminus. We have shown
that the C19 mutation abolishes both activities,
KYKTLR largely
diminishes the former and eliminates the latter, and T558D retains the
former and abolishes the latter. This suggests that the respective
binding sites do not completely overlap, but we do not know whether
there is competition for binding.
Model for Activation of Moesin in Vivo--
Binding between the C
domain of moesin and actin filaments can be observed in cells most
clearly by co-distribution with stress fibers (5, 6). This
co-distribution is not seen with full-length moesin, suggesting that
activation of the actin-binding site may not occur in the cytoplasm. On
the contrary, recent evidence with GFP fusions of moesin in live NIH3T3
cells have shown that the protein is distributed along the plasma
membrane, where it may be bound to a variety of receptor proteins (5).
It is presumably at these sites and in the vicinity of the plasma
membrane that activation of moesin occurs. In platelets, protein
phosphokinase and phosphatase inhibitors can rapidly change the
phosphorylation state of moesin, suggesting its dependence on a balance
of their activities during cell stimulation (28). Similar findings have been made for other cells. Thus, lysophosphatidic acid stimulates a
rapid but transient increase in the phosphorylation of moesin, ezrin,
and radixin in Swiss 3T3 cells, and it has been suggested that Rho
kinase is responsible for this increase (29). However, a change with
similar kinetics can presumably occur by inhibition of a phosphatase.
The myosin-binding subunit of myosin phosphatase binds moesin, and its
activity is inhibited when phosphorylated by Rho kinase (50).
Phosphokinase C- also phosphorylates moesin at the
Thr558 site, but this kinase is expressed primarily in
blood cells (35).
We demonstrate here that F-actin and interdomain binding characteristics of moesin change in the presence of PIP2. This is of interest, since it was previously shown that PIP2 stabilizes the interaction of moesin with the membrane protein CD44 (51). PIP2, however, has other effects as well. It induces actin polymerization in permeabilized platelets (55), presumably by uncapping barbed ends (56). In platelets, turnover of PIP2 is increased upon stimulation, while overall levels of PIP2 decrease (52). This raises the possibility that local changes in PIP2 concentration in the vicinity of the membrane influence moesin function. The role of PIP2 could thus be to sensitize moesin to become a target for phosphokinases. Moesin is necessary for and localized in protruding filopodia, and this is where most likely activation and linkage to actin filaments occurs (8, 50, 53, 54). Phosphorylated moesin is detectable in protrusions (34, 50). Moesin phosphorylation transiently increases in response to stimuli (28, 29), and the increase in number of phosphorylated molecules presumably reflects the average change and is the result of a simultaneous action of kinase and phosphatase on different molecules. According to this model, changes in the phosphorylation state may also reflect whether moesin is bound to the actin cytoskeleton.
Moesin, ezrin, and radixin appear to be necessary for functions in the cortex of cells. Perturbation of microvillus formation and adhesion was observed in moesin/ezrin/radixin antisense oligonucleotide-treated fibroblasts (53), radixin/moesin-suppressed neurons exhibit abnormal growth cones and short neurites and fail to develop an axon (54), and micro-CALI ablation of ezrin causes collapse of ruffles (57). Recent studies also suggest that expression of the N domain of ezrin in epithelial cells causes loss of microvilli, a change in localization of endogenous ezrin, and perturbation of human growth factor-induced differentiation (58). Expression of the N domains of moesin, ezrin, or radixin in NIH3T3 cells causes subtle changes in behavior that are characterized by more numerous and longer filopodia, which are poorly attached and are retracted abnormally, but even more prominent is a defect in retraction of lamellipodial membrane (5). This suggests that it is not merely the existence of these proteins at these sites that is critical, but the fact that they have to be present in a functionally active form. As the cells respond to stimuli by protruding and retracting different areas of the surface and as new actin filaments are formed and others disassemble, some moesin molecules are activated and are enabled to interact with filaments, while others return to an inactive state but remain bound to the membrane. Parts of this hypothesis have been tested recently by expression of constitutively active and inactive moesin molecules (50).
The concept proposed here of moesin changing its overall configuration
is not completely novel. Such a mechanism has been proposed for other
cytoskeletal proteins, such as vinculin (59), myosin II (60), and Src
kinase (61), which phosphorylates tyrosine residues of proteins at
cell-substratum contacts. It still remains to be seen whether the
folded, jackknife configuration stabilized by head-tail interaction
postulated for vinculin provides an adequate model for moesin, ezrin,
or radixin.
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ACKNOWLEDGEMENTS |
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We thank U. Francke from the Howard Hughes Medical Institute at Stanford University for valuable assistance with sequencing; A. Spudich from the Department of Biochemistry at Stanford University for generously providing rabbit skeletal muscle actin; M. R. Amieva, F. Nakamura, and P. Litman for helpful discussions; and C. S. Hwang for assistance in subcloning and yeast two-hybrid experiments.
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FOOTNOTES |
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* This work was supported in part by California Tobacco-related Disease Research Program Grant 4RT-0316.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a President's Undergraduate Scholarship Award from
Stanford University.
§ To whom correspondence should be addressed: Dept. of Pathology, Stanford University Medical School, Stanford, CA 94305-5324. Tel.: 650-725-8223; Fax: 650-725-4905; E-mail: hfurthmayr{at}pathology.stanford.edu.
1 F. Nakamura and H. Furthmayr, unpublished observations.
2 L. Huang and H. Furthmayr, unpublished observations.
3 E. Ichimaru and H. Furthmayr, unpublished observations.
5 L. Huang, T. Y. W. Wong, R. C. C. Lin, and H. Furthmayr, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-diphosphate; kb, kilobase pair(s).
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REFERENCES |
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