From the Angiogenesis Research Center, Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Recent studies suggest that some of the heparan sulfate-carrying proteoglycans may directly participate in signaling via their cytoplasmic tail. The present investigation addresses the potential involvement of syndecan-4, a widely expressed transmembrane proteoglycan, in this process. We found that the cytoplasmic tail of syndecan-4 is phosphorylated on a single serine residue (Ser183) in growth-arrested NIH 3T3 fibroblasts, with a stoichiometry of 0.3 mol Pi/mol syndecan-4. Treatment of the cells with a protein kinase C (PKC)-activating phorbol ester lead to a 2.5-fold increase in Ser183 phosphorylation. This increase was inhibited by a generic PKC inhibitor but not by an inhibitor specific to the calcium-dependent conventional PKCs, suggesting that the cytoplasmic tail of syndecan-4 is phosphorylated by a calcium-independent novel PKC isozyme. Application of 10-30 ng/ml basic fibroblast growth factor (bFGF) produced a 2-3-fold reduction in the phosphorylation of syndecan-4. Because treatment with the phosphatase inhibitor calyculin prevented the bFGF-induced decrease in syndecan-4 phosphorylation, the effect of bFGF appears to be mediated by a protein serine/threonine phosphatase type 1 or 2A. We conclude that the cytoplasmic tail of syndecan-4 is subject to in vivo phosphorylation on Ser183, which is regulated by the activities of a novel PKC isozyme and a bFGF-dependent serine/threonine phosphatase.
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INTRODUCTION |
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Although growth factor signaling generally occurs through specific high affinity receptors, several growth factors interact with additional membrane-anchored co-receptors. In particular, bFGF1 requires binding to a specific sequence of sulfated polysaccharides in the extracellular heparan sulfate glycosaminoglycan (GAG) chain (1) to bind to its high affinity receptor (2) and to exert its effect on target cells (3, 4). The current picture of the role of heparan sulfate in the binding mechanism of bFGF consists of dimerization of the growth factor (2, 5, 6), as well as direct heparan sulfate binding to the high affinity receptor (7, 8). Together, these events lead to receptor multimerization (9) and to tyrosine transphosphorylation of adjacent FGF receptor cytoplasmic tails, followed by phosphorylation of other downstream substrates (10).
Heparan sulfates are primarily associated with two classes of cell surface proteoglycans: syndecans and glypicans. The syndecans are a widely distributed, four-member family of transmembrane proteins capable of carrying both heparan and chondroitin sulfate chains (11-13). Although there are significant differences between the sequences of their ectoplasmic domains, the four syndecans share a highly conserved cytoplasmic tail containing four invariant tyrosines and one invariant serine (14). This degree of conservation may reflect functional similarities between the cytoplasmic tails of all the syndecans. However, unlike the well established involvement of the ectoplasmic domain in growth factor binding through the GAG chains, there is still no consensus regarding the function of the cytoplasmic tail. Several reports (15, 16) point to transient association of the cytoplasmic tail of syndecan-1 to the actin cytoskeleton, which seems to be highly dependent on the presence of one of the four conserved tyrosines (17).
The 28-amino acid-long cytoplasmic tail of syndecan-4 is the least
homologous to the other three syndecans, containing a unique nine-residue sequence (RMKKKDEGSYDLGKKPIYKKAPTNEFYA).
Syndecan-4 is incorporated into focal adhesions of fibroblasts in
a PKC-dependent manner (18), and its cytoplasmic tail
appears to bind and activate PKC (19). These capacities are probably
special to the cytoplasmic tail of syndecan-4 and not shared by the
other syndecans, because they are mediated through oligomerization of
its unique nine-residue sequence (20).
The presence of the five conserved phosphorylatable residues in the cytoplasmic tails of all the syndecans suggests that the cytoplasmic tail could be a kinase substrate in vivo. However, although in vitro phosphorylation by calcium-dependent PKC of serine residues in partial or complete synthetic cytoplasmic tails was reported for syndecan-2 and syndecan-3, it could not be produced for syndecan-1 or syndecan-4 (21, 22). Serine phosphorylation in situ was detected in syndecan-2 of carcinoma cells cultured in the presence of serum (23). This phosphorylation was attributed to a serine residue in the cytoplasmic tail of syndecan-2, contained within a sequence that conforms to a phosphorylation motif of cAMP and cGMP-dependent kinases. In situ phosphorylation of the cytoplasmic tail of syndecan-1 was produced in mammary gland cells by treatment with orthovanadate or pervanadate, both of which inhibit tyrosine phosphatase (24). Accordingly, this treatment resulted predominantly in tyrosine phosphorylation, although a lesser degree of serine phosphorylation was also detected. One of the four tyrosines in the cytoplasmic tail of syndecan-1 is contained within a tyrosine kinase phosphorylation motif (25) conserved in all the syndecans and may at least partially account for the orthovanadate and pervanadate-produced phosphorylation.
Given recent reports of the involvement of syndecan-4 in PKC binding and activation (19, 20), we set out to investigate whether the syndecan-4 molecule itself is subject to phosphorylation and whether this phosphorylation is affected by bFGF binding to the cell surface.
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EXPERIMENTAL PROCEDURES |
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Materials-- Calyculin, chelerythrine, PMA, and bFGF were purchased from Sigma. Gö 6976 was purchased from Calbiochem (La Jolla, CA). Chelerythrine, PMA, and Gö 6976 were dissolved in Me2SO.
Isolation of Syndecan-4 Core Proteins--
NIH 3T3 cells
(American Type Culture Collection, Bethesda, MD) were grown to
confluence in 100-mm plates in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum (Life Technologies Inc.) at
37 °C in a 5% CO2 humidified atmosphere. The cells were
harvested by scraping in 1 ml of lysis buffer (150 mM NaCl,
20 mM NaF, 20 mM
Na4P2O7, 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 50 mM HEPES, pH 7.4). The lysate was cleared by centrifugation
at 9000 × g for 30 min and then subjected to
DEAE-chromatography as described (26). The eluates were dialyzed twice
against 10 mM NH4HCO3, 1 mM -mercaptoethanol and concentrated by evaporation under vacuum. The concentrated samples were resuspended in 50 µl of
digestion buffer (50 mM NaCl, 4 mM
CaCl2, 20 mM Tris, pH 7.4), and GAG chains were
cleaved off the proteoglycan core proteins by 4 h of incubation in
a mixture of 0.06 unit of chondroitinase ABC and 1 unit each of
heparinases I, II, and III (Sigma) at 37 °C.
Radiolabeling of Cultured Cells-- Confluent NIH 3T3 cells were washed twice in phosphate-free DMEM and incubated for 24 h at 37 °C in a 5% CO2 humidified atmosphere in phosphate-free DMEM supplemented with 0.5% fetal bovine serum. The cells were washed twice with methionine, phosphate, and serum-free DMEM and incubated for 6 h in the same medium, supplemented with 400 µCi/ml [35S]methionine (New England Nuclear, Boston, MA). At the onset of the last 2 h of incubation, 500 µCi/ml [32P]orthophosphoric acid (New England Nuclear) was added to the medium.
Immunoprecipitation of Cytoplasmic and Ectoplasmic Syndecan-4 Domains-- Cells were washed with PBS (137 mM NaCl, 10 mM Na2HPO4, 3.6 mM KCl, 1.8 mM KH2PO4, pH 7.4), dissociated by 0.05% trypsin, 0.5 mM EDTA (Life Technologies Inc.) in PBS for 10 min at 22 °C, and sedimented by 2000 × g centrifugation at 4 °C for 5 min. The syndecan-4 ectoplasmic domain was immunoprecipitated from 0.5 ml of medium collected before cell trypsinization or from 0.5 ml of supernatant of the latter centrifugation. The cytoplasmic tail was immunoprecipitated from the pellet after a 30-min extraction at 4 °C in 0.5 ml of lysis buffer supplemented with 100 µM leupeptin, 2 µM pepstatin, and 10 nM okadaic acid (Sigma). Total protein concentrations in each fraction were measured by spectrophotometry at 595 nm (DU 640, Beckman, Fullerton, CA) of an aliquot developed for 10 min in Protein Assay Dye Reagent (Bio-Rad). Bovine serum albumin (Life Technologies Inc.) was used as standard. The medium, trypsinization supernatant, and extracted pellet fractions were precleared by adding 30 µl of 1:1 (v/v) slurry of protein G plus/protein A-agarose beads (Calbiochem), and 10 µl of nonimmune rabbit serum (Life Technologies Inc.). After a 2-h incubation at 4 °C in rotating tubes, the beads were sedimented by 5 min, 5000 × g centrifugation at 4 °C. The cleared samples were supplemented with 40 µl of 1:1 (v/v) slurry of the above beads and 10 µl of rabbit polyclonal antiserum (syndecan-4 ectoplasmic domain-specific antiserum was added to the medium and trypsinization supernatant samples, and cytoplasmic tail-specific antiserum was added to the extracted pellet fraction; both antisera were generous gifts of Dr. N. W. Shworak, MIT (26)) and incubated in rotating tubes for 18 h at 4 °C. The agarose beads were collected by centrifugation as above, washed three times in heparinase digestion buffer, and resuspended in 40 µl of digestion buffer, and the GAG chains of the bead-attached ectoplasmic domains from the medium and from the trypsinization-supernatant were cleaved as above. The ectoplasmic and cytoplasmic tails were dissociated from the beads by a 10-min incubation in SDS buffer at 95 °C, and the beads were sedimented by a 5 min, 13,000 × g centrifugation at 4 °C.
Electrophoresis, Transfer, Autoradiography, and
Immunoblotting--
Immunoprecipitated full-length syndecan-4 core
proteins were resuspended in Laemmli sample buffer (2% SDS, 10%
glycerol, 0.5% -mercaptoethanol, 0.004% bromphenol blue, 50 mM Tris-HCl, pH 6.8) resolved by SDS-PAGE on a 10% slab
gel, and transferred to a polyvinylidene fluoride (PVDF) membrane
(Immobilon-P, Millipore, Bedford, MA) for 12 h at 25 mA in 150 mM glycine, 20 mM Tris-HCl, and 20% methanol.
The ectoplasmic and cytoplasmic syndecan-4 domains were resolved on a
15% slab gel and transferred for 90 min at 20 mA in 150 mM
glycine, 20 mM Tris-HCl, and 30% methanol to a low
porosity PVDF membrane (Immobilon-PSQ, Millipore).
Radiolabeled bands detected by exposure to film (XAR, Kodak, Rochester,
NY) were excised, and their radioactivity was measured in both the
32P and 35S spectra by scintillation counting
(LS 6000IC, Beckman, Fullerton, CA). In some cases, the same membranes
were used for immunoblotting prior to band excision. After blocking in
PBS containing 5% nonfat milk powder for 1 h at 22 °C, the
membrane was incubated in the same solution supplemented with 1:3000
(v/v) dilution of either ectoplasmic or cytoplasmic tail-specific
antiserum for 2 h, washed with PBS, and incubated for 1 h in
5% milk powder-PBS containing 1:2000 diluted goat anti-rabbit IgG
conjugated to peroxidase (Vector Laboratories, Burlingame, CA). The
secondary antibody was detected, after an additional PBS wash, by
chemiluminescence (Western Blot Chemiluminescence Reagent Plus, New
England Nuclear). Molecular weights were estimated by comparison with
the electrophoretic mobility of standards (Kaleidoscope Prestained
Standards, Bio-Rad). Densitometry of digitized images of immunoprobed
membranes (ScanJet 4c, Hewlett Packard) was performed using ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
Thin Layer Chromatography-- Bands excised from PVDF membranes were hydrolyzed for 75 min in 6 N HCl at 110 °C. Solvent was evaporated under vacuum, and the sediment was washed thrice with H2O. The sediment was resuspended in 5 µl of H2O after the third evaporation, applied to a thin layer cellulose acetate plate (Sigma-Aldrich), and underwent electrophoresis at 1000 V in 5% acetic acid, 0.5% pyridine, pH 3.0. The radiolabeled phosphoamino acids were detected by phospholuminescence (PhosphorImager, Molecular Dynamics). Phosphorylated, unlabeled Ser, Thr, and Tyr (Sigma) were used as standards and were detected by spraying with ninhydrin.
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RESULTS |
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Phosphorylation of Syndecan-4 Cytoplasmic Tail in Vivo-- To determine the presence and extent of phosphorylation of the syndecan-4 cytoplasmic tail, full-length heparan and chondroitin sulfate-carrying core proteins were isolated from serum-starved, 32P-labeled NIH 3T3 cells. Autoradiography of NIH 3T3 GAG-lysed core proteins is shown in Fig. 1A (lane 1). To identify the syndecan-4 band, the autoradiogrpahed membrane was probed with an antiserum specific to the cytoplasmic tail of the syndecan-4 core protein (26). The immunoblotting highlighted a single band that ran at an approximate molecular mass of 36 kDa (Fig. 1A, lane 2). A similar syndecan-4 electrophoretic mobility lower than its predicted molecular mass of 20 kDa (14) was observed before with the same antiserum (26). As illustrated in Fig. 1A, the antiserum-detected band superimposed precisely on the second band from the bottom in the autoradiograph.
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Regulation of Syndecan-4 Phosphorylation by bFGF and PKC-- The participation of the syndecan ectoplasmic domain in bFGF binding (2, 3) raises the question whether this binding is accompanied by intracellular modifications of syndecan-4, such as phosphorylation of its cytoplasmic tail. Treatment with 10 ng/ml of bFGF during the last 5 h of the serum-free starvation decreased the phosphorylation stoichiometry of syndecan-4 to 0.16 ± 0.02 (n = 5), approximately half its basal level (Fig. 3A). Larger bFGF dosages of 20 and 30 ng/ml further decreased the phosphorylation stoichiometry of syndecan-4 to 0.12 ± 0.06 (n = 3), but this decrease was not statistically different from the effect of 10 ng/ml bFGF (Fig. 3B). To test for the possible involvement of a phosphatase in the bFGF-induced decrease of syndecan-4 phosphorylation, we applied the phosphatase 1/2A inhibitor calyculin (5 nM) to bFGF (10 ng/ml)-treated cells. Calyculin countered the effect of bFGF, maintaining the syndecan-4 phosphorylation at its basal level (Fig. 3B). Moreover, when the same calyculin dose was applied to cells in the absence of bFGF, syndecan-4 phosphorylation was increased more than 2.5-fold relative to the basal level. If, contrary to our assumption, the incorporation efficiency of 35S is higher than that of 32P, the bFGF-induced decrease in syndecan-4 phosphorylation could solely result from bFGF up-regulation of syndecan-4 synthesis. To address this possibility, the syndecan-4 expression levels in control and in bFGF-treated cells processed identically to those in the phosphorylation assays were compared by immunoblotting cell lysates containing equal amounts of total protein. The syndecan-4 bands, which similar to immunoprecipitated samples (Fig. 2A) ran at an approximate molecular mass of 5 kDa, were detected with the antiserum specific to the ectoplasmic domain, and the amount of protein in each band was quantified by densitometry. In cells treated by 10 and by 30 ng/ml bFGF, the level of syndecan-4 expression was 85% (Fig. 3B, inset) and 93% of the control cells, respectively.
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DISCUSSION |
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We have shown that the cytoplasmic tail of syndecan-4 is
phosphorylated in cultured fibroblasts and that the extent of its phosphorylation is determined by activities of a nPKC enzyme and a
bFGF-activated phosphatase. The phosphorylation site was localized to
Ser183, immediately upstream of a nine-amino acid segment
involved in binding to and activation of PKC (19). Phosphorylation
of a cytoplasmic serine residue was previously detected in the
cytoplasmic tail of syndecan-2 (23) and to a lesser extent in
syndecan-1 (24). In our studies we observed a relatively high degree of syndecan-4 phosphorylation in growth-arrested cells, which could be
further increased by treatment with PMA or decreased by bFGF. Because
Ser183 is part of an invariant seven-residue sequence
(KKDEGSY), these findings may be relevant to all four
members of the syndecan family.
The PMA-induced increase in the phosphorylation of syndecan-4 and its
decrease by chelerythrine strongly suggest the involvement of PKC in
this phosphorylation. Syndecan-4 could not be phosphorylated in
vitro, however, by cPKC isozymes (21, 22). In agreement with this
observation, we were unable to suppress the PMA-induced phosphorylation
of syndecan-4 by a cPKC-specific inhibitor, pointing to the
participation of a nPKC isozyme in the phosphorylation. Although the
amino acid sequence around Ser183 in the cytoplasmic tail
of syndecan-4 does not ideally fit a particular PKC isozyme-specific
substrate sequence motif (32), it does contain the glycine residue that
immediately precedes the phosphorylatable serine/threonine in the
motifs of all the PKC isozymes. The nPKC isozymes ,
, and
were observed to be membrane-associated in NIH 3T3 fibroblasts (33),
the same cell type used in the present study. The substrate sequence
motif of PKC
(AKRKRKGSFFYGG, (32)) has the highest similarity among all PKC isozymes to the amino acid sequence around Ser183
in syndecan-4.
A phosphatase inhibitor reversed the bFGF-induced reduction in
syndecan-4 phosphorylation observed in our study. This suggests that
bFGF binding up-regulates a phosphatase and/or down-regulates a kinase
involved in controlling the level of Ser183
phosphorylation. This effect could be mediated either through the bFGF
high affinity tyrosine kinase receptor or through the syndecan-4
molecule. Tyrosine phosphorylation was reported to inhibit PKC (34),
although an opposite stimulatory effect of this phosphorylation has
also been observed (35). Alternatively, dimerization of bFGF molecules
bound to heparan sulfate chains of adjacent syndecan-4 core proteins
could cross-link these molecules. This would facilitate
trans-dephosphorylation of their cytoplasmic tails by a putative
tail-associated phosphatase, similar to the association of tyrosine
phosphatase to the cytoplasmic tails of growth factor receptors (10).
Although the identity of the phosphatase cannot be determined from
our data, its susceptibility to calyculin indicates that it is
likely to be a serine/threonine phosphatase type 1 or 2A.
Although these findings demonstrate multi-factorial regulation of
syndecan-4 cytoplasmic tail phosphorylation, the functional impact of
this event is not known. Recent findings have suggested that syndecan-4
may play an important role in regulating the distribution and activity
of PKC. These functions are mediated via the oligomerization of a
unique nine-amino acid domain (Leu186-Lys194)
starting three residues downstream of the phosphorylated serine (19).
The state of Ser183 phosphorylation may conceivably affect
syndecan-4 oligomerization. Indeed, phosphorylation of a cysteine
residue appended to the amino terminus of a
Leu186-Lys194 synthetic peptide reduced the
tendency of this peptide to oligomerize in vitro (20). The
location of the phosphorylated cysteine is only two residues downstream
of Ser183. Thus, the bFGF-induced dephosphorylation of the
syndecan-4 cytoplasmic tail on Ser183, which we have
detected in vivo, may be required for oligomerization of the
core protein, as well as for other possible processes. These may
include co-assembly of syndecan and high affinity bFGF receptors into
signaling complexes (9, 12), association with the actin cytoskeleton
(17), and recruitment into focal adhesions (18). These and other
potential consequences of syndecan-4 phosphorylation are currently
being investigated.
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ACKNOWLEDGEMENT |
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We thank Dr. Colleen Sweeney Crovello for expert help with TLC.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-53793 (to M. S.), National Institutes of Health Training Grant HL-07374 (to A. H.) and American Heart Association Scientist Development Grant 9730282N (to A. H.).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: Cardiovascular Div.,
RW453, Beth Israel Deaconess Medical Center, 330 Brookline Ave.,
Boston, MA 02215. Tel.: 617-667-5364; Fax: 617-975-5201; E-mail:
msimons{at}bidmc.harvard.edu.
1 The abbreviations used are: bFGF, basic fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; GAG, glycosaminoglycan; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; PVDF, polyvinylidene fluoride.
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REFERENCES |
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