From the Department of Cell Biology, University of
Alabama, Birmingham, Alabama 35294-0006, § Department of
Bioscience, University of Helsinki, Helsinki FIN-00014, Finland, and
¶ Cell and Molecular Biology Division, Division of Biomedical
Sciences, Imperial College, London SW7 2AZ, United Kingdom
Received for publication, July 16, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Cell adhesion to the extracellular matrix
influences many cellular functions. The integrin family of matrix
receptors plays major roles in the formation of adhesions, but other
proteins modulate integrin signaling. Syndecan-4, a transmembrane
proteoglycan, cooperatively signals with integrins during the formation
of focal adhesions. To date, a direct link between syndecan-4 and the
cytoskeleton has remained elusive. We now demonstrate by Triton X-100
extraction immunoprecipitation and in vitro binding assays
that the focal adhesion component Upon contact with the extracellular matrix
(ECM),1 fibroblasts undergo a
multitude of morphological changes including attachment, spreading, and
the formation of stress fibers (SFs) that span the cell and terminate
at focal adhesions (FAs), which anchor the cell to the extracellular
substrata (1-5). In addition to providing tight adhesion to the ECM,
FAs are supramolecular complexes containing proteins that regulate the
organization of the microfilament cytoskeleton, generate downstream
signals, and act as adaptor molecules for the localization of other
proteins (2-8). Some FA molecules interact directly with integrins,
including Four mammalian syndecans have been identified, and all share some
characteristics (20-26). All members of the family are type 1 transmembrane glycoproteins whose core proteins range in size from 20 to 40 kDa (21-26). The extracellular domain of each syndecan core
protein has 3-5 covalently linked glycosaminoglycan chains, mostly
heparan sulfate (22-26). All syndecans have a short cytoplasmic tail
consisting of a region unique to each syndecan (V region) flanked by
two regions (C1 and C2) of sequence conserved among all family members
(22-27). At the COOH terminus of the membrane distal C2 region is a
FYA tripeptide that enables all syndecans to associate with PDZ
domain-containing proteins (21-27). Despite these similarities,
syndecans-1, -2, and -3 are expressed in a tissue-specific manner (28).
Syndecan-4 is ubiquitously expressed (28) but selectively enriched in
focal adhesions (29).
In addition to binding soluble extracellular ligands such as growth
factors (21, 22), syndecans interact with the ECM, including
fibronectin (20-26). When cells are seeded onto coverslips coated with
the 120-kDa proteolytic fragment of fibronectin (the cell-binding
domain), cells attach and spread but do not form FAs and SFs (1, 14).
Focal adhesion formation requires additional signaling; ligation of
syndecan-4 with the HepII domain of fibronectin (1, 14, 19) or
recombinant peptides derived from HepII (30) or clustering syndecan-4
with antibodies (18) can circumvent the need for intact fibronectin to
form FAs and SFs, implicating syndecan-4 in the process of FA and SF
formation. The direct activation of protein kinase C (PKC) with phorbol
esters in cells spread on the cell binding domain of fibronectin also
bypasses the need for full-length fibronectin in the formation of FAs
and SFs (31). The V region of syndecan-4 binds PKC Although it is clear that syndecan-4 plays an important role in FA and
SF formation, a direct role for the molecule in cytoskeletal organization has remained elusive. Here, we present novel evidence that
syndecan-4 is linked to the microfilament cytoskeleton by association
with the microfilament bundling protein Materials and Antibodies--
All general chemicals were
purchased from Sigma. Monoclonal antibodies used include those against
Cells and Culture Conditions--
Rat embryo fibroblast (REF)
cells and human foreskin fibroblast cells were cultured in Triton X-100 Extraction--
Confluent cells (for
immunoblotting) or 50-75% confluent (for immunocytochemistry) were
extracted for 20 min at 37 °C in extraction buffer containing 0.2%
Triton X-100 (TX100) in phosphate-buffered saline (PBS), pH 7.0, 20 mM EDTA, 10 mM N-ethylmaleimide, 100 mM Immunostaining--
For staining with 150.9 antibody
against syndecan-4, cells were fixed in 100% methanol at Immunoprecipitation--
Co-immunoprecipitation studies were
performed on confluent monolayers of REFs (~107
cells/immunoprecipitation). Cells were scraped into lysis buffer containing 1% TX100 and the protease inhibitors leupeptin,
phenylmethylsulfonyl fluoride, and benzamidine in PBS, incubated on ice
for 30 min, then cleared by centrifugation (1000 × g).
Before immunoprecipitation, lysates were incubated with rabbit
anti-mouse antibodies (1:5000; Dako, Glostrup, Denmark) followed by
protein-A-Sepharose beads (Amersham Biosciences) preblocked in 10%
fetal bovine serum to pre-clear the lysate. Immunoprecipitations were
performed by sequentially incubating lysates with primary antibody
followed by rabbit-anti-mouse antibodies and fresh preblocked
protein-A-Sepharose beads. Beads were washed 4 times in lysis buffer,
once with 1 M NaCl, and 3 times with PBS. Some cells were
lysed in radioimmunoprecipitation assay (RIPA) buffer containing 1%
TX100, 1% deoxycholate, and 0.2% SDS. Co-immunoprecipitated proteins
were eluted by boiling in SDS sample buffer for 5 min and subjected to
SDS-PAGE and immunoblotting.
Immunoblotting--
To identify proteins resistant to TX100
extraction, extracted cells were scraped into SDS sample buffer,
resolved on SDS-PAGE gels, and transferred to nitrocellulose membranes
(Bio-Rad) for 1.5 h at 100 V. Blots were blocked with 5% nonfat
milk and 0.1% Tween 20 in PBS by incubating at room temperature for
1 h. After washing with 1% milk, 0.1% Tween 20 in PBS, the
membranes were incubated with primary antibodies (1:200 for 150.9, 1:1000 for anti- Binding Studies--
Binding studies with synthetic peptides
(Synpep, Dublin, CA) coupled to Sulfolink Coupling Gel (Pierce) were
performed in 1 ml of REF cells lysed in 1% TX100 immunoprecipitation
lysis buffer at 4 °C for 30 min. Binding assays using 0.5 µg of
chicken gizzard Syndecan-2 and Syndecan-4 Have Distinct Cell Surface Distributions
and Susceptibilities to TX100 Extraction--
Syndecan-2 and
syndecan-4 are involved in cell-ECM communication; syndecan-2 modulates
the assembly of the ECM (47), and syndecan-4 regulates FA and SF
formation (19, 34, 35). When REF cells are examined by indirect
immunofluorescence microscopy, syndecan-2 labeling revealed a punctate
pattern on the cell body (Fig.
1A). In contrast, syndecan-4
staining is restricted to FAs and the membrane overlying SFs (Fig.
1B). The amount of labeling over stress fibers is variable,
dependent on cell type, and also dependent on whether the syndecan-4
antibody is directed against the cytoplasmic domain (29) or the
extracellular domain (19, 34, 56). Syndecan-2 and syndecan-4 also show
differential susceptibility to TX100 extraction. After TX100 extraction
of live cells, syndecan-2 labeling is lost (Fig. 1C), but
most syndecan-4 remains (Fig. 1D). These results confirm
that syndecan-4 resides in the TX100-resistant cytoskeleton and matrix
residue, whereas syndecan-2 does not.
FA Components Exhibit Different Susceptibilities to TX100
Extraction--
Because syndecan-4 is resistant to TX100 extraction,
we attempted to determine whether the FA proteins Syndecan-4 and Syndecan-4 Association with Syndecan-4 Synthetic Peptides Are Able to Capture
Syndecan-4 Association with PKC The role of syndecans in the regulation of cell morphology and
cytoskeletal organization has been addressed in previous studies (18,
34, 35, 56). Although these studies implicated a link to the
microfilament cytoskeleton, the nature of an association to the
cytoskeleton had not been determined. Here, we report the association
of the transmembrane heparan sulfate proteoglycan syndecan-4 with
The interaction with Synthetic peptides encompassing the V region of syndecan-4 can compete
for binding of Previous studies have indicated a possible interaction between
-actinin interacts with syndecan-4
in a
-integrin-independent manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin (9), focal adhesion kinase (10), talin (11), and
paxillin (10, 12, 13). Integrins transduce signals from the ECM through a variety of mechanisms, and they are the primary catalyst in FA and SF
formation (5-8). However, data from our laboratory and others show
that signals from integrins alone are sufficient for attachment and
spreading but not FA formation (1, 14). Integrin signals are thought to
be modulated through lateral associations within the plasma membrane
with other transmembrane proteins (15) such as the integrin-associated
protein (16) and the TM4SF proteins (17). Indeed, signaling through a
transmembrane proteoglycan, syndecan-4, has been shown to be required
for FA formation in primary fibroblasts (18, 19).
and potentiates
its enzymatic activity (32, 33). Furthermore, overexpression of
syndecan-4 increases FA and SF formation (34, 35), whereas expression of syndecan-4 truncated within its V region prevents their formation (34).
-actinin. Originally
characterized in muscle cells,
-actinin cross-links actin stress
fibers in both muscle and non-muscle cells (36-38).
-Actinin is 100 kDa in size, and it contains a globular actin binding domain attached
to a rod domain consisting of four spectrin-like repeats (36, 37).
-Actinin associates with
integrins (9), vinculin (39), zyxin
(40), the cysteine-rich protein (41), and palladin (42) within FAs, and
potential signaling roles for the molecule have emerged with the
reports of interactions with phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2) (43), MEKK1 (44), actinin-associated LIM
proteins (45), and focal adhesion kinase (46).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin, vinculin, and talin, (Sigma),
1 integrin
(Transduction Labs, Lexington, KY, and Invitrogen),
3
integrin (ATCC; Manassas, VA), paxillin (Zymed Laboratories
Inc., San Francisco, CA), PKC
(Upstate Biotechnology, Lake
Placid, NY), PI(4,5)P2 (Assay Design, Ann Arbor, MI), and mouse monoclonal 150.9 recognizing the NH2-terminal portion
of syndecan-4 (32). Polyclonal antibodies include anti-
-actinin (Sigma), a rabbit polyclonal antibody raised against recombinant syndecan-2 extracellular domain (47), and anti-actin antisera (48).
PI(4,5)P2 (Avanti Polar Lipids, Alabaster, AL) was dried under nitrogen, resuspended in ice-cold H2O, and sonicated
to form micelles. To bind PI(4,5)P2 to
-actinin, 1 µg
of
-actinin was incubated with 10 µg of PI(4,5)P2 in
PBS for 30 min on ice.
-minimal
essential medium (Mediatech; Fisher) containing 5% fetal bovine serum
(Atlanta Biologicals; Norcross, GA, or Summit Biotechnology, Ft.
Collins, CO) in 10% CO2. Cells were used before passage 20 and were shown to be free of mycoplasma by labeling with Hoechst 33258. All cell culture equipment was from Fisher. For immunostaining, cells
were seeded onto 12-mm-diameter coverslips; for extractions and
immunoprecipitations, cells were cultured in 25-, 75-, or
150-cm2 culture flasks. For some experiments, cells were
allowed to adhere to substrates coated with fibronectin (Collaborative
Biomedical Products, Bedford, MA) at either 100 µg/ml for 1 h at
room temperature or 50 µg/ml for 2-3 h in serum-free medium. No
differences were seen in the results between the two coating concentrations.
-amino caproic acid, 0.2 mM
phenylmethylsulfonyl fluoride, 5 mM benzamidine, and 1 µg/ml leupeptin (49). After extraction, detergent-resistant fractions
were washed three times with PBS. Some cells were treated with 200 nM PMA for 15 min at 37 °C as indicated in the figures.
PMA was diluted into medium from 4 mM stocks in
Me2SO. Control cells were treated with Me2SO alone.
20 °C
for 20 min and blocked with 2% whole goat serum (ICN/Cappel; Aurora,
OH) for 45 min. Cells stained with all other antibodies were fixed for
10 min at 37 °C with 3.5% paraformaldehyde. Nonextracted cells were
permeabilized with 0.1% Tween 20 (Bio-Rad) after fixation. Primary
antibodies were incubated with fixed cells (1:50 in PBS) for 45 min at
37 °C or overnight at 4 °C. After washing with PBS, fluorescein
isothiocyanate- or Texas Red-conjugated goat-anti-mouse or anti-rabbit
antibodies (1:50, Organon Tekenika Corp., Durham, NC) were added for 45 min at 37 °C. Preparations were examined on a Nikon Optiphot
microscope equipped for epifluorescence, and images were recorded on
Ilford HP-5 film. Co-localization studies were performed using a
combination of 150.9 and rabbit anti-
-actinin. Co-localization was
analyzed on a Leitz Orthoplan microscope equipped for epifluorescence, and digital images were captured with a Vario-Orthomat II (High Resolution Imaging Facility, University of Alabama at Birmingham).
-actinin, anti-paxillin, anti-PKC
, anti-talin,
and anti-vinculin, and 1:500 for anti-PI(4,5)P2) in the
same buffer overnight at 4 °C. After washing for 3 × 5 min,
blots were incubated for 1 h at room temperature with horseradish
peroxide-conjugated goat-anti-mouse or goat-anti-rabbit antibodies
(Bio-Rad) at 1:3000 dilution. Detection was performed with enhanced
chemiluminescence (Amersham Biosciences).
-actinin (Sigma) were performed at room temperature
in 0.01% TX100 in PBS. Bound material was eluted with SDS sample buffer and analyzed by immunoblotting. In competition experiments, V
region peptides (50) were added to a final concentration of 50 µg/ml
to cells immediately after lysis and were present throughout.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Syndecan-2 and -4 exhibit different
localizations within the cell and different susceptibilities to
TX100 extraction. A, syndecan-2 labeling in
nonextracted cells. B, syndecan-4 labeling in nonextracted
cells. C, syndecan-2 staining is removed after TX100
treatment. D, syndecan-4 labeling is undisturbed after TX100
treatment. The bar represents 10 µM.
-actinin,
paxillin, talin, and vinculin were also resistant.
-Actinin staining
decorates FAs and SFs (Fig.
2A), whereas paxillin (Fig.
2C), talin (Fig. 2E), and vinculin (Fig.
2G) staining is localized exclusively to FAs. After TX100
treatment, most
-actinin labeling remains (Fig. 2B), but
paxillin (Fig. 2D), talin (Fig. 2F), and vinculin (Fig. 2H) does not. These results were confirmed by
immunoblotting the TX100-resistant fractions (data not shown).
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Fig. 2.
TX100 extraction of cytoskeletal
proteins. Shown is labeling for -actinin (A),
paxillin (C), talin (E), and vinculin
(G) in nonextracted cells. After TX100 extraction
-actinin staining is present (B) whereas paxillin
(D), talin (F), and vinculin (H) are
removed. The bar represents 10 µM.
-Actinin Co-localize and
Co-immunoprecipitate--
These data suggest that syndecan-4 and
-actinin share a similar subcellular localization within the cell.
To verify this, we employed double labeling and immunofluorescence
microscopy. Antibodies directed against
-actinin (Fig.
3A) and syndecan-4 (Fig.
3B) label FAs and SFs in nonextracted cells, and the merged image (Fig. 3C) shows co-localization of the two molecules.
Triton X-100 extraction decreases FA staining of
-actinin (Fig.
3D) and syndecan-4 (Fig. 3E), although syndecan-4
and
-actinin labeling remains colinear with SFs. The merged image
(Fig. 3F) confirms preserved co-localization after detergent
treatment. To test whether
-actinin and syndecan-4 interact, we
immunoprecipitated syndecan-4 and
-actinin from cells and examined
the immunoprecipitates by immunoblotting. We first analyzed syndecan-4
immunoprecipitates with antibodies against paxillin (Fig.
4A), since an indirect association between syndecan-4 and paxillin through syndesmos has been
demonstrated (52). In the presence of TX100, paxillin co-immunoprecipitates with syndecan-4. However, when RIPA buffer is
used to lyse the cells, paxillin is no longer detected in syndecan-4 immunocomplexes. Syndecan-4 immunoprecipitates were also analyzed for
-actinin and vinculin (Fig. 4B). In both Triton and RIPA buffers,
-actinin co-immunoprecipitates with syndecan-4, whereas vinculin does not. To corroborate the association with syndecan-4,
-actinin immunoprecipitates were examined by Western blotting with
syndecan-4 antibodies (Fig. 4C). Syndecan-4
co-immunoprecipitated with
-actinin in either TX100 or RIPA
buffers.
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Fig. 3.
Double labeling reveals that syndecan-4
and -actinin share a similar location within
the cell. A, syndecan-4 labeling decorates FAs and SFs.
B,
-actinin also localizes to FAs and SFs. C,
yellow in the merged image confirms co-localization.
D, syndecan-4 is distributed along SFs after TX100
extraction. E,
-actinin also localizes along SFs after
TX100 extraction. F, co-localization of
-actinin and
syndecan-4 is evident in the merged image.
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Fig. 4.
Syndecan-4 and
-actinin co-immunoprecipitate. The detergent
present in the lysis buffer as well as whether each lane is a sample of
beads used to pre-clear (PC) or immunoprecipitate
(IP) is listed above each blot (WB).
A, paxillin co-immunoprecipitates with syndecan-4 in TX100
but not RIPA buffer. B,
-actinin (
-A)
co-immunoprecipitates with syndecan-4 (S4) in both TX100 and
RIPA buffer, but vinculin is not detected. C, syndecan-4
co-immunoprecipitates with
-actinin in TX100 and in RIPA buffer.
Samples in A and B were boiled in reducing SDS
sample buffer, whereas those in C were boiled in nonreducing
buffer.
-Actinin Is Integrin
1-independent--
Previous studies indicated that
integrins
1 and
3 associate with
-actinin (9), and syndecan-4 co-localizes with both integrins in
nonextracted cells (29), leading to the possibility that the
association between syndecan-4 and
-actinin requires the presence of
integrins. Cells grown on fibronectin substrates in the absence of
serum contain
1 integrins in their FAs (53). Human
foreskin fibroblast cells were grown on fibronectin-coated coverslips
and extracted to determine whether
1 integrin was resistant to TX100 treatment. Labeling for
1 integrin is
seen in FAs in unextracted cells (Fig.
5A), but this is lost after TX100 treatment (Fig. 5B). This was confirmed by
immunoblotting with anti-
1 integrin antibodies (Fig.
5C); Western blotting of REF cell lysate reveals two bands,
probably mature glycanated and immature nonglycanated forms of
1 integrin (54), both of which are lost after TX100
treatment. Similar results were seen for
3 integrin,
which is the integrin found most prominently in focal adhesions when
cells were grown in serum (53) (data not shown). A
integrin-independent association between syndecan-4 and
-actinin was
confirmed by preserved co-immunoprecipitation of
-actinin with
syndecan-4 in TX100-resistant fractions that lack
integrins (Fig.
5D).
-Actinin co-immunoprecipitates with
1
integrin in REF cells lysed in CHAPS buffer (51). However, we are
unable to detect
1 integrin in
-actinin
immunoprecipitates under more stringent conditions using TX100 buffer
(data not shown), confirming that an association between syndecan-4 and
-actinin does not require the presence of integrin
subunits.
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Fig. 5.
Syndecan-4 associates with
-actinin independently from
1 integrin. A,
1 integrin labeling in nonextracted human foreskin
fibroblast cells grown on fibronectin. B,
1
integrin staining is lost after TX100 extraction. C, REF
cells were grown on fibronectin and extracted, and detergent-insoluble
fractions were Western-blotted with antibodies against
1
integrin. D, after TX100 extraction, syndecan-4 antibodies
were used to co-immunoprecipitate (IP)
-actinin from REF
cell remnants. Immunoprecipitation conditions are listed
above each blot. PC, pre-clear.
-Actinin--
To investigate the interaction between syndecan-4 and
-actinin, we utilized in vitro binding assays similar to
those used to characterize associations between
-actinin and
integrins (9), zyxin (40), vinculin (39), and the cysteine-rich protein
(41). Synthetic peptides mimicking the cytoplasmic tails of syndecan-4 and syndecan-2 (Fig. 6A) were
coupled to beads. Immunoblotting of material bound to beads coated with
syndecan-2 or syndecan-4 cytoplasmic domain peptides reveals that
-actinin is captured specifically by syndecan-4 from cell lysates
(Fig. 6B).
-Actinin from a chicken gizzard preparation is
also captured specifically by syndecan-4 cytoplasmic domain (Fig.
6C). Because both syndecan-4 (33) and
-actinin (43)
associate with PI(4,5)P2, we attempted to determine whether
this chicken gizzard preparation of
-actinin was already
PI(4,5)P2-bound, possibly contributing to the
interaction. Anti-PI(4,5)P2 antibodies were used to
detect the presence of PI(4,5)P2 in Western blots of REF
lysate and chicken gizzard
-actinin (Fig. 6D).
Immunodetection reveals that the
-actinin preparation is not bound
to PI(4,5)P2; however, immunoblotting a REF cell lysate or
-actinin that had been preincubated with PI(4,5)P2 reveals a 100-kDa band that corresponds to
-actinin.
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Fig. 6.
In vitro binding assays with
synthetic syndecan-4-derived peptides. A, amino acid
sequences of syndecan-2 and -4 cytoplasmic domains. The V regions are
underlined. For each immunoblot, added material is labeled
in each lane, and the antibody used to Western blot (WB) is
listed below. In B and D, cell lysates
were used as positive controls. B, Cys4L, but not Cys2L or
uncoated beads, specifically captures -actinin from REF lysate.
C, Cys4L, but not Cys2L, captures
-actinin purified from
chicken gizzard. D, gizzard
-actinin preparations were
analyzed for PI(4,5)P2 presence. A 100-kDa immunopositive
band is detected in REF lysate and in gizzard
-actinin after
pretreatment with PI(4,5)P2.
-Actinin Can Occur through the V
Region--
To map the site of interaction between syndecan-4 and
-actinin, binding assays were performed with peptides encompassing the cytoplasmic domains of syndecan-2 (Cys2L) or -4 (Cys4L), the V
region of syndecan-4 (Cys4V), or a truncated syndecan-4 that lacks the
PDZ domain binding FYA (21-27) tripeptide (Cys4E). Cell lysates were
incubated with syndecan cytoplasmic domain-coated beads, and bound
material was immunoblotted with
-actinin antibodies (Fig.
7B). Syndecan-2 cytoplasmic
domain, which contains homologous C1 and C2 domains, fails to capture
-actinin, although all syndecan-4 synthetic peptides bind
-actinin. This suggests the syndecan-4 V region plays a significant
role in the interaction of
-actinin, but the FYA region is not
required. To verify the importance of the V region with respect to
-actinin binding, competition assays with mutated V region peptides
were used (Fig. 7C). REF cells were lysed in the presence of
competing peptide, Cys4L beads were added to the lysates, and bound
material was examined for the presence of
-actinin. Native V region
peptides (Cys4V) and V region peptides harboring a proline to alanine
mutation (4VPA) or a tyrosine to phenylalanine mutation (4VYF) competed
for
-actinin binding. However, a V region peptide with a scrambled
sequence (4Vscr) and a peptide containing two lysine to arginine
mutations (4VKR) failed to compete.
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Fig. 7.
Binding assays with syndecan-4 variants.
A, amino acid sequences of peptides used in assays. Cys2L
and Cys4L are listed in Fig. 6. Cys4E lacks the COOH-terminal FYA
tripeptide. B, REF lysates were incubated with
peptide-coupled beads as indicated above each lane, and bound fractions
were immunoblotted with anti- -actinin antibodies. C,
peptides derived from syndecan-4 V region (noted above each
lane) were used to compete for
-actinin binding from REF
lysates to Cys4L beads as monitored by immunoblotting with
anti-
-actinin antibodies. REF lysates were immunoblotted as
controls.
and
-Actinin Translocation after PMA
Treatment--
PKC
binds to the V region of syndecan-4 (33) and
co-precipitates with syndecan-4 from cells pretreated with PMA to
activate PKC (32). Our data here demonstrate that
-actinin also
associates with the V region of syndecan-4, suggesting that competition
for binding to syndecan-4 may occur. Localization of PKC
and
-actinin was monitored after PKC activation by PMA. Triton
X-100-insoluble fractions ± PMA were analyzed by Western blotting
with antibodies against PKC
,
-actinin, syndecan-4, and actin.
When compared with Me2SO-treated control cells, pretreating
cells with PMA decreases the amount of
-actinin in TX100-resistant
fractions (Fig. 8A) and causes
the translocation of PKC
to TX100-resistant preparations (55) (Fig.
8B). However, the amount of syndecan-4 present does not
appear to be affected by PMA treatment (Fig. 8C). These
differences are not due to unequal protein loading, as revealed by
immunoblotting by actin antibodies (Fig. 8D).
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Fig. 8.
PMA treatment affects the distribution
of -actinin and PKC
.
Detergent-insoluble fractions from REFs treated with Me2SO
(D) or PMA (P) were Western-blotted for
-actinin, PKC
, syndecan-4, and actin. A, PMA treatment
reduces the amount of
-actinin detected in TX100-resistant
preparations. B, PMA treatment induces the translocalization
of PKC
to the detergent-insoluble fraction. C, syndecan-4
localization is unaffected by PMA treatment. D, actin
labeling reveals equal protein loading.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin. These components co-localize in cells, show similar
resistance to TX100 extraction, co-immunoprecipitate, and interact in
in vitro binding assays. It should be noted that both
-actinin and syndecan-4 are retained after TX100 extraction at
37 °C, which appears to be more stringent than at 4 °C where lipid rafts (57), paxillin, talin, and vinculin (data not shown) are
retained in TX100-resistant fractions. The specific association between
syndecan-4 and
-actinin was confirmed by co-immunoprecipitation since other FA components co-precipitate with syndecan-4 in milder conditions (in TX100), but only
-actinin co-immunoprecipitates in
RIPA buffer. This was not dependent on the presence of integrin
1 or
3, because the interaction between
syndecan-4 and
-actinin is preserved after the removal of
integrins, and unlike
-actinin/
1 integrin interaction
(9), is retained after washes with 1 M NaCl.
-actinin was specific for syndecan-4.
Syndecan-2 and syndecan-4 have distinct subcellular distributions and
differential susceptibilities to TX100 extraction, indicating different
intracellular binding partners. Syndecan-2, through its C1 region,
interacts with ezrin (58), a member of the ERM family of proteins that
mediate cytoskeleton-cell membrane associations (59), but this does not
result in resistance to extraction. The cytoplasmic tails of syndecan-2
and -4 share a COOH-terminal FYA motif that interacts with several PDZ
domain proteins (60-62) whose overexpression can alter cellular
morphology. However, our studies indicate this motif does not mediate
SF and FA formation. When full-length syndecan-4 is overexpressed in
Chinese hamster ovary cells (34) or REF
cells,2 spreading and FA and
SF formation are promoted. A similar effect is observed in cells
overexpressing the FYA deletion.2 Deletion of this sequence
in 4E peptides does not prevent
-actinin association in in
vitro binding assays. The V region of syndecan-4 appears to
control FA and SF formation, because a syndecan-4 construct that is
truncated within this region acts as a dominant negative for spreading
and FA formation (34). The V region of syndecan-4 binds and
superactivates PKC
(32, 33), and PKC activity is required for FA and
SF formation (31, 63, 64). However, our in vitro binding
assays indicate that
-actinin also binds the V region, and this may
also contribute to the lack of FAs and SFs.
-actinin to beads coated with the entire cytoplasmic
domain. Peptides where the sequence of 4V was scrambled (4Vscr) or
where two lysines were replaced with arginines (4VKR) did not compete
for binding, whereas 4VPA and 4VYF did. Previous studies demonstrated
that 4VPA and 4VYF also lack the ability to activate PKC
, possibly
because of a reduced ability to form oligomers (50). This indicates
there are similarities between syndecan-4 binding to
-actinin and
PKC
. However, neither 4VPA nor 4VYF activated PKC
, although they
do compete for
-actinin binding. These data imply that the sites of
interaction within syndecan-4 cytoplasmic domain V region for
-actinin and PKC
have some similarities but also some
differences. This suggests that there may be some competition for
binding, and further studies are under way. Interestingly. PMA-mediated
PKC activation causes the translocation of PKC
to
detergent-resistant preparations with a concomitant decrease in the
amount of
-actinin.
-actinin and syndecan-4. Both FA components can also be present in
the myosin sheath found in some cells (65), both components are
up-regulated in proliferative renal disease (56), and both components
move coordinately into FA when cells spread on the cell binding domain
of fibronectin are treated with soluble HepII domain that binds
syndecan-4 (19). Embryonic fibroblasts from syndecan-4-deficient mice
lack the ability to form FAs in response to HepII treatment (66), and a
preliminary analysis of the distribution of FA and SF components
indicates their cytoskeletal organization is abnormal, particularly
with respect to SF organization and
-actinin distribution. In
addition, several signaling molecules associate with
-actinin,
including PI(4,5)P2 (43), MEKK1 (44), and focal adhesion
kinase (46), and it will be interesting to see how the interaction with
syndecan-4 affects these associations.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grant GM50194 (to A. W.) and by Sankyo, Co., Ltd.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.
To whom correspondence should be addressed: Dept. of Cell
Biology, THT 946, University of Alabama, 1530 3rd Ave. S., Birmingham, AL 35294-0006. Tel.: 205-934-1548; Fax: 205-934-7029; E-mail: anwoods@uab.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M207123200
2 R. L. Longley, J. R. Couchman, and A. Woods, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: ECM, extracellular matrix; FA, focal adhesion; SF, stress fiber; PDZ, post-synaptic density-95, disks large, zonula occludens-1; PKC, protein kinase C; REF, rat embryo fibroblast; TX100, Triton X-100; PBS, phosphate-buffered saline; RIPA, radioimmunoprecipitation assay; PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PMA, phorbol 12-myristate 13-acetate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Woods, A., Couchman, J. R., Johansson, S., and Hook, M. (1986) EMBO J. 5, 665-670[Abstract] |
2. | Sastry, S. K., and Burridge, K. (2000) Exp. Cell Res. 261, 25-36[CrossRef][Medline] [Order article via Infotrieve] |
3. | Burridge, K., and Chrzanowska-Wodnicka, M. (1996) Annu. Rev. Cell Dev. Biol. 12, 463-518[CrossRef][Medline] [Order article via Infotrieve] |
4. | Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve] |
5. | Yamada, K. M., and Miyamoto, S. (1995) Curr. Opin. Cell Biol. 7, 681-689[CrossRef][Medline] [Order article via Infotrieve] |
6. | Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve] |
7. | Kolanus, W., and Seed, B. (1997) Curr. Opin. Cell Biol. 9, 725-731[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Giancotti, F. G.,
and Ruoslahti, E.
(1999)
Science
285,
1028-1032 |
9. | Otey, C. A., Pavalko, F. M., and Burridge, K. (1990) J. Cell Biol. 111, 721-729[Abstract] |
10. | Schaller, M. D., Otey, C. A., Hildebrand, J. D., and Parsons, J. T. (1995) J. Cell Biol. 130, 1181-1187[Abstract] |
11. | Horwitz, A., Duggan, K., Buck, C., Beckerle, M. C., and Burridge, K. (1986) Nature 320, 531-533[Medline] [Order article via Infotrieve] |
12. | Liu, S., Thomas, S. M., Woodside, D. G., Rose, D. M., Kiosses, W. B., Pfaff, M., and Ginsberg, M. H. (1999) Nature 402, 676-681[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Liu, S.,
Slepak, M.,
and Ginsberg, M. H.
(2001)
J. Biol. Chem.
276,
37086-37092 |
14. |
Bloom, L.,
Ingham, K. C.,
and Hynes, R. O.
(1999)
Mol. Biol. Cell
10,
1521-1536 |
15. |
Woods, A.,
and Couchman, J. R.
(2000)
J. Biol. Chem.
275,
24233-24236 |
16. | Lindberg, F. P., Gresham, H. D., Schwarz, E., and Brown, E. J. (1993) J. Cell Biol. 123, 485-496[Abstract] |
17. |
Berditchevski, F.,
Bazzoni, G.,
and Hemler, M. E.
(1995)
J. Biol. Chem.
270,
17784-17790 |
18. |
Saoncella, S.,
Echtermeyer, F.,
Denhez, F.,
Nowlen, J. K.,
Mosher, D. F.,
Robinson, S. D.,
Hynes, R. O.,
and Goetinck, P. F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2805-2810 |
19. | Woods, A., Longley, R. L., Tumova, S., and Couchman, J. R. (2000) Arch. Biochem. Biophys. 374, 66-72[CrossRef][Medline] [Order article via Infotrieve] |
20. | Carey, D. J. (1997) Biochem. J. 327, 1-16[Medline] [Order article via Infotrieve] |
21. | Simons, M., and Horowitz, A. (2001) Cell Signal. 13, 855-862[CrossRef][Medline] [Order article via Infotrieve] |
22. | Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo, R. L., and Lose, E. J. (1992) Annu. Rev. Cell Biol. 8, 365-393[CrossRef] |
23. | Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729-777[CrossRef][Medline] [Order article via Infotrieve] |
24. | Woods, A., and Couchman, J. R. (1998) Trends Cell Biol. 8, 189-192[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Zimmermann, P.,
and David, G.
(1999)
FASEB J.
13 (suppl.),
91-100 |
26. | Couchman, J. R., Chen, L., and Woods, A. (2001) Int. Rev. Cytol. 207, 113-150[Medline] [Order article via Infotrieve] |
27. | Rapraeger, A. C., and Ott, V. L. (1998) Curr. Opin. Cell Biol. 10, 620-628[CrossRef][Medline] [Order article via Infotrieve] |
28. | Kim, C. W., Goldberger, O. A., Gallo, R. L., and Bernfield, M. (1994) Mol. Biol. Cell 5, 797-805[Abstract] |
29. | Woods, A., and Couchman, J. R. (1994) Mol. Biol. Cell 5, 183-192[Abstract] |
30. | Woods, A., McCarthy, J. B., Furcht, L. T., and Couchman, J. R. (1993) Mol. Biol. Cell 4, 605-613[Abstract] |
31. | Woods, A., and Couchman, J. R. (1992) J. Cell Sci. 101, 277-290[Abstract] |
32. |
Oh, E. S.,
Woods, A.,
and Couchman, J. R.
(1997)
J. Biol. Chem.
272,
8133-8136 |
33. |
Oh, E. S.,
Woods, A.,
Lim, S. T.,
Theibert, A. W.,
and Couchman, J. R.
(1998)
J. Biol. Chem.
273,
10624-10629 |
34. |
Longley, R. L.,
Woods, A.,
Fleetwood, A.,
Cowling, G. J.,
Gallagher, J. T.,
and Couchman, J. R.
(1999)
J. Cell Sci.
112,
3421-3431 |
35. |
Echtermeyer, F.,
Baciu, P. C.,
Saoncella, S., Ge, Y.,
and Goetinck, P. F.
(1999)
J. Cell Sci.
112,
3433-3441 |
36. | Lazarides, E., and Burridge, K. (1975) Cell 6, 289-298[Medline] [Order article via Infotrieve] |
37. | Blanchard, A., Ohanian, V., and Critchley, D. (1989) J. Muscle Res. Cell Motil. 10, 280-289[Medline] [Order article via Infotrieve] |
38. | Burridge, K., Nuckolls, G., Otey, C., Pavalko, F., Simon, K., and Turner, C. (1990) Cell Differ. Dev. 32, 337-342[CrossRef][Medline] [Order article via Infotrieve] |
39. | Belkin, A. M., and Koteliansky, V. E. (1987) FEBS Lett. 220, 291-294[CrossRef][Medline] [Order article via Infotrieve] |
40. | Crawford, A. W., Michelsen, J. W., and Beckerle, M. C. (1992) J. Cell Biol. 116, 1381-1393[Abstract] |
41. |
Pomies, P.,
Louis, H. A.,
and Beckerle, M. C.
(1997)
J. Cell Biol.
139,
157-168 |
42. |
Parast, M. M.,
and Otey, C. A.
(2000)
J. Cell Biol.
150,
643-656 |
43. | Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150-152[CrossRef][Medline] [Order article via Infotrieve] |
44. | Christerson, L. B., Vanderbilt, C. A., and Cobb, M. H. (1999) Cell Motil. Cytoskeleton 43, 186-198[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Pomies, P.,
Macalma, T.,
and Beckerle, M. C.
(1999)
J. Biol. Chem.
274,
29242-29250 |
46. |
Izaguirre, G.,
Aguirre, L., Hu, Y. P.,
Lee, H. Y.,
Schlaepfer, D. D.,
Aneskievich, B. J.,
and Haimovich, B.
(2001)
J. Biol. Chem.
276,
28676-28685 |
47. |
Klass, C. M.,
Couchman, J. R.,
and Woods, A.
(2000)
J. Cell Sci.
113,
493-506 |
48. | Badley, R. A., Lloyd, C. W., Woods, A., Carruthers, L., Allcock, C., and Rees, D. A. (1978) Exp. Cell Res. 117, 231-244[Medline] [Order article via Infotrieve] |
49. |
Woods, A.,
Couchman, J. R.,
and Hook, M.
(1985)
J. Biol. Chem.
260,
10872-10879 |
50. |
Oh, E. S.,
Woods, A.,
and Couchman, J. R.
(1997)
J. Biol. Chem.
272,
11805-11811 |
51. |
Sampath, R.,
Gallagher, P. J.,
and Pavalko, F. M.
(1998)
J. Biol. Chem.
273,
33588-33594 |
52. |
Denhez, F.,
Wilcox-Adelman, S. A.,
Baciu, P. C.,
Saoncella, S.,
Lee, S.,
French, B.,
Neveu, W.,
and Goetinck, P. F.
(2002)
J. Biol. Chem.
277,
12270-12274 |
53. | Fath, K. R., Edgell, C. J., and Burridge, K. (1989) J. Cell Sci. 92, 67-75[Abstract] |
54. |
Akiyama, S. K.,
Yamada, S. S.,
and Yamada, K. M.
(1989)
J. Biol. Chem.
264,
18011-18018 |
55. | Kiley, S. C., and Jaken, S. (1990) Mol. Endocrinol. 4, 59-68[Abstract] |
56. |
Yung, S.,
Woods, A.,
Chan, T. M.,
Davies, M.,
Williams, J. D.,
and Couchman, J. R.
(2001)
FASEB J.
15,
1631-1633 |
57. | Hooper, N. M. (1999) Mol. Membr. Biol. 16, 145-156[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Granes, F.,
Urena, J. M.,
Rocamora, N.,
and Vilaro, S.
(2000)
J. Cell Sci.
113,
1267-1276 |
59. | Louvet-Vallee, S. (2000) Biol. Cell 92, 305-316[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Grootjans, J. J.,
Zimmermann, P.,
Reekmans, G.,
Smets, A.,
Degeest, G.,
Durr, J.,
and David, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13683-13688 |
61. |
Cohen, A. R.,
Woods, D. F.,
Marfatia, S. M.,
Walther, Z.,
Chishti, A. H.,
Anderson, J. M.,
and Wood, D. F.
(1998)
J. Cell Biol.
142,
129-138 |
62. | Gao, Y., Li, M., Chen, W., and Simons, M. (2000) J. Cell. Physiol. 184, 373-379[CrossRef][Medline] [Order article via Infotrieve] |
63. |
De Nichilo, M. O.,
and Yamada, K. M.
(1996)
J. Biol. Chem.
271,
11016-11022 |
64. | Lewis, J. M., Cheresh, D. A., and Schwartz, M. A. (1996) J. Cell Biol. 134, 1323-1332[Abstract] |
65. | Zigmond, S. H., Otto, J. J., and Bryan, J. (1979) Exp. Cell Res. 119, 205-219[Medline] [Order article via Infotrieve] |
66. |
Ishiguro, K.,
Kadomatsu, K.,
Kojima, T.,
Muramatsu, H.,
Tsuzuki, S.,
Nakamura, E.,
Kusugami, K.,
Saito, H.,
and Muramatsu, T.
(2000)
J. Biol. Chem.
275,
5249-5252 |