Syndecan-4 Associates with alpha -Actinin*

Daniel K. GreeneDagger , Sarka Tumova§, John R. Couchman, and Anne WoodsDagger ||

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -actinin interacts with syndecan-4 in a beta -integrin-independent manner.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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).

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 PKCalpha 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).

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 alpha -actinin. Originally characterized in muscle cells, alpha -actinin cross-links actin stress fibers in both muscle and non-muscle cells (36-38). alpha -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). alpha -Actinin associates with beta  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

Materials and Antibodies-- All general chemicals were purchased from Sigma. Monoclonal antibodies used include those against alpha -actinin, vinculin, and talin, (Sigma), beta 1 integrin (Transduction Labs, Lexington, KY, and Invitrogen), beta 3 integrin (ATCC; Manassas, VA), paxillin (Zymed Laboratories Inc., San Francisco, CA), PKCalpha (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-alpha -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 alpha -actinin, 1 µg of alpha -actinin was incubated with 10 µg of PI(4,5)P2 in PBS for 30 min on ice.

Cells and Culture Conditions-- Rat embryo fibroblast (REF) cells and human foreskin fibroblast cells were cultured in alpha -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.

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 epsilon -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.

Immunostaining-- For staining with 150.9 antibody against syndecan-4, cells were fixed in 100% methanol at -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-alpha -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).

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-alpha -actinin, anti-paxillin, anti-PKCalpha , 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).

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 alpha -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

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.


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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.

FA Components Exhibit Different Susceptibilities to TX100 Extraction-- Because syndecan-4 is resistant to TX100 extraction, we attempted to determine whether the FA proteins alpha -actinin, paxillin, talin, and vinculin were also resistant. alpha -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 alpha -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 alpha -actinin (A), paxillin (C), talin (E), and vinculin (G) in nonextracted cells. After TX100 extraction alpha -actinin staining is present (B) whereas paxillin (D), talin (F), and vinculin (H) are removed. The bar represents 10 µM.

Syndecan-4 and alpha -Actinin Co-localize and Co-immunoprecipitate-- These data suggest that syndecan-4 and alpha -actinin share a similar subcellular localization within the cell. To verify this, we employed double labeling and immunofluorescence microscopy. Antibodies directed against alpha -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 alpha -actinin (Fig. 3D) and syndecan-4 (Fig. 3E), although syndecan-4 and alpha -actinin labeling remains colinear with SFs. The merged image (Fig. 3F) confirms preserved co-localization after detergent treatment. To test whether alpha -actinin and syndecan-4 interact, we immunoprecipitated syndecan-4 and alpha -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 alpha -actinin and vinculin (Fig. 4B). In both Triton and RIPA buffers, alpha -actinin co-immunoprecipitates with syndecan-4, whereas vinculin does not. To corroborate the association with syndecan-4, alpha -actinin immunoprecipitates were examined by Western blotting with syndecan-4 antibodies (Fig. 4C). Syndecan-4 co-immunoprecipitated with alpha -actinin in either TX100 or RIPA buffers.


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Fig. 3.   Double labeling reveals that syndecan-4 and alpha -actinin share a similar location within the cell. A, syndecan-4 labeling decorates FAs and SFs. B, alpha -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, alpha -actinin also localizes along SFs after TX100 extraction. F, co-localization of alpha -actinin and syndecan-4 is evident in the merged image.


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Fig. 4.   Syndecan-4 and alpha -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, alpha -actinin (alpha -A) co-immunoprecipitates with syndecan-4 (S4) in both TX100 and RIPA buffer, but vinculin is not detected. C, syndecan-4 co-immunoprecipitates with alpha -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.

Syndecan-4 Association with alpha -Actinin Is Integrin beta 1-independent-- Previous studies indicated that integrins beta 1 and beta 3 associate with alpha -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 alpha -actinin requires the presence of integrins. Cells grown on fibronectin substrates in the absence of serum contain beta 1 integrins in their FAs (53). Human foreskin fibroblast cells were grown on fibronectin-coated coverslips and extracted to determine whether beta 1 integrin was resistant to TX100 treatment. Labeling for beta 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-beta 1 integrin antibodies (Fig. 5C); Western blotting of REF cell lysate reveals two bands, probably mature glycanated and immature nonglycanated forms of beta 1 integrin (54), both of which are lost after TX100 treatment. Similar results were seen for beta 3 integrin, which is the integrin found most prominently in focal adhesions when cells were grown in serum (53) (data not shown). A beta  integrin-independent association between syndecan-4 and alpha -actinin was confirmed by preserved co-immunoprecipitation of alpha -actinin with syndecan-4 in TX100-resistant fractions that lack beta  integrins (Fig. 5D). alpha -Actinin co-immunoprecipitates with beta 1 integrin in REF cells lysed in CHAPS buffer (51). However, we are unable to detect beta 1 integrin in alpha -actinin immunoprecipitates under more stringent conditions using TX100 buffer (data not shown), confirming that an association between syndecan-4 and alpha -actinin does not require the presence of integrin beta  subunits.


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Fig. 5.   Syndecan-4 associates with alpha -actinin independently from beta 1 integrin. A, beta 1 integrin labeling in nonextracted human foreskin fibroblast cells grown on fibronectin. B, beta 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 beta 1 integrin. D, after TX100 extraction, syndecan-4 antibodies were used to co-immunoprecipitate (IP) alpha -actinin from REF cell remnants. Immunoprecipitation conditions are listed above each blot. PC, pre-clear.

Syndecan-4 Synthetic Peptides Are Able to Capture alpha -Actinin-- To investigate the interaction between syndecan-4 and alpha -actinin, we utilized in vitro binding assays similar to those used to characterize associations between alpha -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 alpha -actinin is captured specifically by syndecan-4 from cell lysates (Fig. 6B). alpha -Actinin from a chicken gizzard preparation is also captured specifically by syndecan-4 cytoplasmic domain (Fig. 6C). Because both syndecan-4 (33) and alpha -actinin (43) associate with PI(4,5)P2, we attempted to determine whether this chicken gizzard preparation of alpha -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 alpha -actinin (Fig. 6D). Immunodetection reveals that the alpha -actinin preparation is not bound to PI(4,5)P2; however, immunoblotting a REF cell lysate or alpha -actinin that had been preincubated with PI(4,5)P2 reveals a 100-kDa band that corresponds to alpha -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 alpha -actinin from REF lysate. C, Cys4L, but not Cys2L, captures alpha -actinin purified from chicken gizzard. D, gizzard alpha -actinin preparations were analyzed for PI(4,5)P2 presence. A 100-kDa immunopositive band is detected in REF lysate and in gizzard alpha -actinin after pretreatment with PI(4,5)P2.

Syndecan-4 Association with alpha -Actinin Can Occur through the V Region-- To map the site of interaction between syndecan-4 and alpha -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 alpha -actinin antibodies (Fig. 7B). Syndecan-2 cytoplasmic domain, which contains homologous C1 and C2 domains, fails to capture alpha -actinin, although all syndecan-4 synthetic peptides bind alpha -actinin. This suggests the syndecan-4 V region plays a significant role in the interaction of alpha -actinin, but the FYA region is not required. To verify the importance of the V region with respect to alpha -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 alpha -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 alpha -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-alpha -actinin antibodies. C, peptides derived from syndecan-4 V region (noted above each lane) were used to compete for alpha -actinin binding from REF lysates to Cys4L beads as monitored by immunoblotting with anti-alpha -actinin antibodies. REF lysates were immunoblotted as controls.

PKCalpha and alpha -Actinin Translocation after PMA Treatment-- PKCalpha 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 alpha -actinin also associates with the V region of syndecan-4, suggesting that competition for binding to syndecan-4 may occur. Localization of PKCalpha and alpha -actinin was monitored after PKC activation by PMA. Triton X-100-insoluble fractions ± PMA were analyzed by Western blotting with antibodies against PKCalpha , alpha -actinin, syndecan-4, and actin. When compared with Me2SO-treated control cells, pretreating cells with PMA decreases the amount of alpha -actinin in TX100-resistant fractions (Fig. 8A) and causes the translocation of PKCalpha 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 alpha -actinin and PKCalpha . Detergent-insoluble fractions from REFs treated with Me2SO (D) or PMA (P) were Western-blotted for alpha -actinin, PKCalpha , syndecan-4, and actin. A, PMA treatment reduces the amount of alpha -actinin detected in TX100-resistant preparations. B, PMA treatment induces the translocalization of PKCalpha 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

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 alpha -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 alpha -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 alpha -actinin was confirmed by co-immunoprecipitation since other FA components co-precipitate with syndecan-4 in milder conditions (in TX100), but only alpha -actinin co-immunoprecipitates in RIPA buffer. This was not dependent on the presence of integrin beta 1 or beta 3, because the interaction between syndecan-4 and alpha -actinin is preserved after the removal of beta  integrins, and unlike alpha -actinin/beta 1 integrin interaction (9), is retained after washes with 1 M NaCl.

The interaction with alpha -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 alpha -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 PKCalpha (32, 33), and PKC activity is required for FA and SF formation (31, 63, 64). However, our in vitro binding assays indicate that alpha -actinin also binds the V region, and this may also contribute to the lack of FAs and SFs.

Synthetic peptides encompassing the V region of syndecan-4 can compete for binding of alpha -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 PKCalpha , possibly because of a reduced ability to form oligomers (50). This indicates there are similarities between syndecan-4 binding to alpha -actinin and PKCalpha . However, neither 4VPA nor 4VYF activated PKCalpha , although they do compete for alpha -actinin binding. These data imply that the sites of interaction within syndecan-4 cytoplasmic domain V region for alpha -actinin and PKCalpha 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 PKCalpha to detergent-resistant preparations with a concomitant decrease in the amount of alpha -actinin.

Previous studies have indicated a possible interaction between alpha -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 alpha -actinin distribution. In addition, several signaling molecules associate with alpha -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.

    FOOTNOTES

* 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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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