Tyrosine Phosphorylation of Syndecan-1 and -4 Cytoplasmic Domains in Adherent B82 Fibroblasts*

The syndecans, a family of cell surface proteoglycans, have highly conserved cytoplasmic domains that bind proteins containing PDZ domains and co-localize with the actin cytoskeleton. The syndecan cytoplasmic domains contain four conserved tyrosine residues, two of which are located within favorable sequences for phosphorylation. Endogenous tyrosine phosphorylation of syndecans-1 and -4 is detected in adherent B82 fibroblasts. Approximately 1.5% of total syndecan is endogenously phosphorylated, while most, if not all, cell surface syndecan is phosphorylated following treatment with the tyrosine phosphatase inhibitor pervanadate. Syndecan phosphorylation is also detected in Raji-S1 and NMuMG cells, but only following treatment with vanadate or pervanadate, suggesting that endogenous phosphorylation is maintained in an “off” state in these cells. Endogenous syndecan phosphorylation in B82 cells is rapidly blocked by genistein (IC 50 < 10 m M ) con- firming the presence of a constitutively active kinase and a corresponding tyrosine phosphatase. Phosphorylation is also inhibited by herbimycin A (IC 50 < 1.0 m M ) and staurosporine (IC 50 < 1.0 n M ), suggesting a role for Src family kinases in regulating syndecan phosphorylation. Together, these data suggest an important role for tyrosine phosphorylation of the syndecan cytoplasmic domains in regulating downstream signaling events in response

Interactions between extracellular matrix molecules and cell surface adhesion receptors play an important role in the regulation of cell behavior (1,2). The syndecans are a family of cell surface receptors that are characterized by highly conserved transmembrane and cytoplasmic domains, and extracellular domains bearing heparan sulfate glycosaminoglycan (GAG) 1 chains (3,4). The syndecan family contains four vertebrate syndecans, namely, syndecans-1 through -4. The syndecans bind to a variety of extracellular matrix molecules and growth factors in a heparan sulfate-dependent manner (5)(6)(7)(8)(9), and associate with the actin cytoskeleton through a mechanism dependent on their cytoplasmic domains (10,11). These interac-tions suggest a role for the syndecans in transducing signals from the extracellular environment to the cell interior.
Protein phosphorylation plays a critical role in a variety of cellular processes, including the generation of intracellular signals in response to external stimuli (12). The syndecans do not display intrinsic enzymatic activity; however, they are postulated to assemble protein complexes at the plasma membrane that contain structural and/or signaling proteins capable of initiating downstream signals (13,14). The best characterized example of adhesion receptors that function in this manner are the integrins, which initiate the assembly of focal adhesions in response to cell adhesion to extracellular matrix (14). These specialized sites of adhesion contain structural proteins such as talin, vinculin, and paxillin, and a variety of kinases such as FAK, Src, and protein kinase C. Integrin engagement results in enhanced kinase activity, phosphorylation of numerous focal adhesion components, the recruitment of proteins containing SH2 domains, and the activation of downstream signaling pathways resulting in changes in gene expression, cell cycle progression, and cell morphology (15,16).
Cell surface proteoglycans have been implicated in initiating downstream signaling events in response to cell adhesion to extracellular matrix. Engagement of the chondroitin sulfate proteoglycan NG2 is required along with ␣4␤1 integrin to initiate cell spreading and focal adhesion formation in melanoma cells (17). Similarly, fibroblast spreading and focal adhesion formation in response to fibronectin requires the engagement of both integrins and heparan sulfate proteoglycans (18,19). In this system, engagement of proteoglycans can be replaced by activation of protein kinase C (20). Syndecan-4 has been shown to localize to focal adhesions containing ␤1 or ␤3 integrins (21,22), and binds directly to protein kinase C␣ (23). The cytoplasmic domain of syndecan-3 binds a protein complex containing c-Src and Fyn, along with the cytoskeleton associated proteins cortactin and tubulin, and ligation of syndecan-3 results in the phosphorylation of c-Src and cortactin (24).
In contrast to their extracellular domains, the cytoplasmic domains of the syndecans are highly conserved (4). This domain contains two regions that are 100% conserved among all vertebrate syndecans (Fig. 1). One is a conserved juxtamembrane region consisting of 11 amino acids (YRMR/KKK-DEGSY), and the second (EFYA) is located at the extreme carboxyl terminus of the cytoplasmic domain, and has recently been shown to bind to the PDZ domains of syntenin (25) and CASK (26,27). The syndecan cytoplasmic domains also contain four conserved tyrosine residues, two of which are in favorable sequences for phosphorylation (28,29). Tyrosine phosphorylation of syndecan-3 has been demonstrated in vitro (30).
In this report, we demonstrate the endogenous tyrosine phosphorylation of syndecans-1 and -4, but not syndecan-2, in B82 fibroblasts. Phosphorylation is tightly regulated and is not readily detected in other cell types tested except following treatment with tyrosine phosphatase inhibitors. This is consistent with our previous report of syndecan-1 phosphorylation in mammary epithelial (NMuMG) cells in response to pervanadate (31), and demonstrates that syndecan phosphorylation is regulated differently depending on cell type. Using a variety of kinase inhibitors, Src family kinases are implicated in regulating the tyrosine phosphorylation of both syndecan-1 and -4; although data also suggest that the two proteins may be phosphorylated on different tyrosine residues. This work suggests an important function for tyrosine phosphorylation of the syndecans, which may vary in different cell types and among the different syndecan family members.
Inhibitor Treatments-Sodium orthovanadate, hydrogen peroxide, staurosporine, and genistein were purchased from Sigma. Herbimycin A was purchased from Calbiochem. Stock solutions of genistein (10 mg/ml), herbimycin A (1 mg/ml), and staurosporine (1 mM) were prepared in Me 2 SO or methanol and diluted to the indicated concentrations in cell culture medium. Inhibitors were incubated with cells at 37°C for the indicated lengths of time. Control samples were treated with matching concentrations of vehicle, and in all cases showed no significant effect on syndecan phosphorylation.
Biotinylation of Cell Surface Proteins-Cells were washed with biotinylation buffer (10 mM Hepes (pH 8.0), 150 mM NaCl, 0.2 mM MgCl 2 , 0.2 mM CaCl 2 ) and 0.2 mg/ml EZ-Link TM Sulfo-NHS-LC-biotin (Pierce) in biotinylation buffer was added to the cells for 30 min at 4°C. The reaction was terminated by the addition of 1 M glycine to a final concentration of 40 mM.
Potato Acid Phosphatase Treatment-Cells were lysed in extraction buffer and syndecans immunoprecipitated as described above. The precipitates were washed and resuspended in 25 mM MES (pH 6.0) containing 30 units/ml potato acid phosphatase (Calbiochem). Samples were incubated for 3 h at 37°C, with fresh enzyme addition after each hour. Precipitates were washed again with TBS/EDTA and resuspended in sample buffer.

Tyrosine Phosphorylation of Syndecan Core Proteins in B82
Fibroblasts-Most adherent cells express multiple syndecans at the cell surface. To determine which syndecan family members are expressed in B82 fibroblasts, cell surface proteins were biotinylated and syndecans were immunoprecipitated with a panel of syndecan antibodies specific for the cytoplasmic domains of different syndecan family members ( Fig. 2A). Using a pan-syndecan antibody (Pan) that recognizes the conserved C2 region in the cytoplasmic domains of all the syndecan family members (4), three distinct syndecan core proteins migrating at about 75, 48, and 35 kDa are detected following digestion of extracellular GAG chains with heparitinase and chondroitin ABC lyase. In some experiments, the three core proteins appear to migrate as doublets. The reason for this is not clear, but may be the result of incomplete digestion of the heparan sulfate GAG chains. Without enzymatic digestion of the GAG chains, the syndecans migrate in a diffuse, high molecular weight distribution on SDS-PAGE and are difficult to identify by immunostaining. The sizes of the core proteins suggest that they represent syndecans-1, -2, and -4. This is confirmed by detection with specific antibodies. The 75-kDa core protein is identified as syndecan-1 by precipitation with mAb 281.2, which is specific for the mouse syndecan-1 extracellular domain (data not shown). Syndecan-1 is also the predominant core protein isolated by immunoprecipitation with mAb 2E9, although this antibody also precipitates a very minor amount of the 35-kDa protein identified as syndecan-4. The 35-kDa syndecan-4 protein is precipitated with antiserum raised against a syndecan-4 cytoplasmic domain peptide (S4CD). In addition, S4CD precipitates a minor amount of syndecan-1. The final antibody used in this study, mAb 6G12, is the only antibody, other than the pan-syndecan antibody, that precipitates the 48-kDa protein which is predicted to be syndecan-2 based on its molecular mass. However, this antibody also precipitates syndecan-4, and, to a lesser extent, syndecan-1.
Tyrosine phosphorylation of intact syndecan core proteins is not easily detected. The core proteins remain somewhat diffuse even after digestion of the GAG chains, which makes it extremely difficult to detect the low level of endogenous syndecan phosphorylation that occurs in these cells (see Fig. 6). In the best of cases, endogenous phosphorylation can be detected by immunostaining with an anti-phosphotyrosine antibody (PY20), as shown for syndecan-1 in Fig. 2B (left panel), although the detection is very weak. The phosphotyrosine band is confirmed to be syndecan-1 by subsequent immunostaining with the pansyndecan antibody (data not shown). In this particular assay, no phosphotyrosine was detected on the syndecan-2 and -4 core proteins.
To facilitate the detection of syndecan phosphorylation, cells were treated with mild trypsin, which specifically cleaves the syndecan extracellular domains, leaving cell-associated fragments consisting of the syndecan transmembrane and cytoplasmic domains (COOH-terminal fragments) (31). Because of the extreme sensitivity of the syndecans to trypsin, this cleavage can be conducted rapidly on ice (37). Unlike the full-length syndecan core proteins, the COOH-terminal fragments migrate as discrete reproducible bands on SDS-PAGE, and tyrosine phosphorylation of multiple COOH-terminal fragments is readily detected (Fig. 3). Four trypsin-generated COOH-terminal fragments migrating between 12 and 18 kDa are precipitated using the pan-syndecan antibody, and are referred to as bands a through d (Fig. 3A). Without trypsin treatment, the pan-syndecan antibody precipitates smaller COOH-terminal fragments (see Fig. 3A, no trypsin) that migrate between 8 and 12 kDa. These fragments are generated by endogenous shedding of the syndecan extracellular domains, which is characteristic of the syndecans and has been described previously (38). Immunostaining with PY20 demonstrates endogenous tyrosine phosphorylation of multiple COOH-terminal fragments, with the majority of phosphotyrosine detected on bands a and d, and a minor amount on band c (Fig. 3B).
To determine which syndecans are phosphorylated, syndecan COOH-terminal fragments were isolated using the specific syndecan antibodies characterized in Fig. 2, followed by immunostaining with PY20. Syndecan-1 COOH-terminal fragments are precipitated with mAb 2E9 and account for three of the four trypsin-generated fragments (bands a-c). Immunostaining with PY20 demonstrates that two of the syndecan-1 COOH-terminal fragments (bands a and c) are phosphorylated on tyrosine. The majority of phosphotyrosine is detected in band a, with a minor amount in band c. Band b does not appear phosphorylated.
Endogenous tyrosine phosphorylation of syndecan-4 is also detected. A single syndecan-4 COOH-terminal fragment is isolated by immunoprecipitation with S4CD (band d), and subsequent immunostaining with PY20 demonstrates that this fragment is phosphorylated. Together, the two major tyrosine phosphorylated fragments represent syndecan-1 (band a) and syndecan-4 (band d).
To identify the syndecan-2 fragment(s), syndecan COOHterminal fragments were precipitated with mAb 6G12, which recognizes equal amounts syndecans-2 and -4, and a lesser amount of syndecan-1 ( Fig. 2A). Bands c and d are the predominant fragments precipitated with mAb 6G12 and are present in approximately equal amounts. Since band d has been identified as syndecan-4, band c is likely to represent syndecan-2 (Fig. 3A). However, a minor amount of syndecan-1 precipitated with this antibody may also contribute to band c. To confirm that band c represents syndecan-2, cell lysates were depleted of syndecan-1 by multiple rounds of immunoprecipitation with mAb 2E9. Following immunodepletion of syndecan-1, bands c and d are still the predominant fragments precipitated with mAb 6G12, confirming that band c represents syndecan-2 (data not shown). Using mAb 6G12, tyrosine phosphorylation of syndecan-2 (band c) is not detected, whereas tyrosine phosphorylation of syndecan-4 (band d) is readily observed (Fig. 3B).
Characterization of Syndecan Phosphorylation-Tyrosine phosphorylation of syndecans-1 and -4 is also detected in L-M(TK) Ϫ and L929 fibroblasts, demonstrating that endogenous phosphorylation of syndecans is not restricted to the B82 cell line (Fig. 4A). However, B82 cells consistently show the highest  level of endogenous phosphorylation and were therefore chosen for further characterization. Treatment of syndecan COOHterminal fragments with potato acid phosphatase results in the loss of detectable tyrosine phosphorylation, confirming that PY20 immunoreactivity is specific for phosphorylated syndecan (Fig. 4B, compare lanes 7 and 8). Interestingly, band a (syndecan-1) is no longer detected following phosphatase treatment (Fig. 4B, compare lanes 2 and 3), demonstrating that band a represents a syndecan-1 COOH-terminal fragment that migrates at a higher molecular weight when phosphorylated. This shift in migration may be due to hyperphosphorylation of the syndecan-1 cytoplasmic domain, or phosphorylation at a site that alters its migration on SDS-PAGE. Treatment with heatinactivated potato acid phosphatase does not result in the loss of tyrosine phosphorylation, nor does it affect the migration of band a. Interestingly, a comparison of the phosphorylated syndecan-1 (e.g. band a) to the syndecan-4 cytoplasmic domain (e.g. band d) demonstrates that the majority of the syndecan-1 shifts when phosphorylated, while the majority of the syndecan-4 does not (Fig. 3B). This suggests that syndecans-1 and -4 may be phosphorylated on different residues. As expected, when cells are pretreated with pervanadate, syndecan phosphorylation is dramatically enhanced (Fig. 4B). Pervanadate treatment also leads to enhanced shedding of syndecans, and tyrosine phosphorylation of the shed fragments is also detected. These results are consistent with previous studies demonstrating the tyrosine phosphorylation and shedding of syndecan-1 following pervanadate treatment (31).
Syndecan COOH-terminal fragments isolated from B82 cells were further characterized by comparing them to syndecan-1 COOH-terminal fragments isolated from Raji-S1 cells before and after treatment with pervanadate. Raji-S1 cells were generated by transfecting syndecan-1 cDNA into syndecan-negative Raji lymphoblastoid cells as described elsewhere (32). Treatment with trypsin generates two syndecan-1 COOH-terminal fragments that correspond to bands b and c precipitated from B82 cells (Fig. 4C, compare lanes 2 and 4). Without trypsin treatment, a single shed fragment is detected that migrates below bands a-d. Syndecan-4 (band d) is not detected in Raji-S1 cells as expected. In contrast to the endogenous phosphorylation of syndecans seen in B82 cells, syndecan-1 phosphorylation in Raji-S1 cells is only detected following treatment with pervanadate. Furthermore, band a is not endogenously detected in Raji-S1 cells, but appears when syndecan-1 is phosphorylated in response to pervanadate. This is consistent with the results obtained using B82 cells which demonstrates that band a represents a syndecan-1 fragment that migrates at a higher molecular weight only when phosphorylated (Fig. 4B).
Tyrosine Phosphorylation of Syndecans in B82 Versus NMuMG Cells-Treatment of B82 and mammary epithelial (NMuMG) cells with vanadate over a 3-h time course has different effects on syndecan phosphorylation, demonstrating that phosphorylation is regulated differently in the two cell types (Fig. 5). Treatment of B82 cells with vanadate results in enhanced tyrosine phosphorylation of bands a-d. Phosphorylation of syndecan-4 (band d) is enhanced within 15 min but is then diminished. However, at these time points, syndecan-4 protein is no longer detected. This loss of protein is likely the result of vanadate-induced shedding and subsequent internalization or degradation of the protein. In support of this, full-length syndecan-4 core protein is not detected by surface biotinylation in B82 cells following treatment with pervanadate, whereas a significant amount of syndecans-1 and -2 remain at the cell surface (data not shown). Tyrosine phosphorylation of bands a and c is also enhanced within 15 min, and continues to accumulate over the 3-h time course. In contrast, tyrosine phosphorylation of band b is evident only after 60 min of vanadate treatment.  5 and 10). In other conditions, COOH-terminal fragments were treated with potato acid phosphatase (lanes 3 and 8) or heat-inactivated potato acid phosphatase (lanes 4 and 9). C, syndecan COOH-terminal fragments were immunoprecipitated using the pansyndecan antibody from either Raji-S1 (lanes 1-3) or B82 (lane 4) cells. Cells were either not treated (lane 1) or treated with trypsin (lanes 2-4). In one condition, Raji-S1 cells were pretreated with pervanadate for 15 min (lane 3).

FIG. 5. Tyrosine phosphorylation of syndecans isolated from vanadate-treated B82 and NMuMG cells.
Syndecan COOH-terminal fragments were immunoprecipitated using the pan-syndecan antibody from either B82 or NMuMG cells as indicated. Cells were either not treated (Ϫ) or treated with trypsin (ϩ). Cells were pretreated with 1 mM vanadate for the indicated amounts of time. Samples were resolved by gel electrophoresis and membranes probed with either the pan-syndecan antibody (A) or PY20 (B).
NMuMG cells express primarily syndecans-1 and -4, but lower levels of syndecans-2 and -3 are also detected (39). Endogenous phosphorylation of syndecans is not detected in NMuMG cells, even though they have 2-3 times more syndecan at the cell surface compared with B82 cells (data not shown), suggesting that phosphorylation in NMuMG cells is maintained in an "off" state. Three trypsin-generated COOH-terminal fragments are immunoprecipitated from NMuMG cells that correspond to bands b-d from B82 cells (Fig. 5A). Endogenous phosphorylation is not detected on any of these fragments by immunostaining with PY20 (Fig. 5B). Like the B82 cells, tyrosine phosphorylation of band b is detected in NMuMG cells after treatment with vanadate for 60 min. However, in contrast to the B82 cells, phosphorylation of bands c and d is not detected during the 3-h vanadate treatment. Furthermore, band a, which represents phosphorylated syndecan-1, is not detected even after treatment with vanadate. Taken together, these results demonstrate that phosphorylation of specific syndecan family members is regulated differently in NMuMG and B82 cells.
Syndecan Phosphorylation Is Maintained by the Activities of Tyrosine Kinases and Phosphatases-The experiments described above demonstrate that syndecan phosphorylation is enhanced following treatment of cells with vanadate or pervanadate. During these studies, it was observed that pervanadate is more effective at preserving syndecan phosphorylation. Pervanadate has been demonstrated to be a more potent inhibitor of tyrosine phosphatases in vitro than vanadate and other oxidants such as H 2 O 2 (40), and is more effective at enhancing the levels of tyrosine phosphorylation in intact cells (41). To directly compare the effects of these inhibitors on syndecan phosphorylation, B82 cells were treated with vanadate, H 2 O 2 , or pervanadate, and levels of syndecan phosphorylation measured (Fig. 6, A and B). Immunostaining syndecan COOH-terminal fragments with PY20 reveals that pervanadate is most effective at increasing overall levels of syndecan phosphorylation, followed by H 2 O 2 and then vanadate. Syndecan-4 protein (band d) is not detected following treatment with H 2 O 2 or pervanadate, which is likely due to shedding of the syndecan-4 protein in response to treatment with the inhibitors.
The apparent presence of an active kinase and phosphatase(s) that target the syndecan cytoplasmic domains raises the question of how much cell surface syndecan is phosphorylated on tyrosine both endogenously and after treatment with phosphatase inhibitors. To determine the amount of syndecan endogenously phosphorylated on tyrosine, total cell surface syndecan was isolated from B82 cells by multiple rounds of immunoprecipitation using the pan-syndecan antibody, and was set at 100%. Total phosphorylated syndecan was isolated by multiple rounds of immunoprecipitation with PY20. Quantification of the amount of syndecan precipitated with PY20 reveals that approximately 1.5% of cell surface syndecan is endogenously phosphorylated on tyrosine (Fig. 6C). To determine the amount of syndecan phosphorylated in response to vanadate or pervanadate, total syndecan was immunoprecipitated from treated cells using either the pan-syndecan antibody or PY20. Again, total syndecan precipitated with the pan-syndecan antibody was set at 100%. PY20 precipitates 12 and 135% of the amount of syndecan precipitated with the pansyndecan antibody from cells treated with vanadate or pervanadate, respectively. The fact that PY20 precipitates more total syndecan protein than the pan-syndecan antibody in response to pervanadate may be explained by phosphorylation of multiple tyrosine residues. This may create multiple epitopes for PY20 within a single molecule, and therefore make PY20 more effective at precipitating syndecan than the pan-syndecan antibody. The conclusion from these data is that most, if not all, cell surface syndecan in B82 cells is a target for the tyrosine kinase.
To verify the activity of an endogenous tyrosine kinase, the tyrosine kinase inhibitor genistein was used. Genistein is a competitive inhibitor with respect to ATP and shows little inhibitory activity toward serine/threonine kinases (42). Endogenous phosphorylation in B82 cells is effectively inhibited by pretreating cells with genistein for 30 min (IC 50 Ͻ 10 M) (Fig. 7A). Kinetic analysis of the effects of genistein on synde-  PY20 (bottom panel). B, the blot represented in A was quantified using a Storm PhosphorImager and ImageQuant software. Tyrosine phosphorylation is represented by the amount of phosphotyrosine normalized to an arbitrary unit of syndecan protein. C, total syndecan protein was immunoprecipitated with either the pansyndecan antibody or PY20 following pretreatment of cells with the indicated inhibitors and detected with the pan-syndecan antibody. The amount of syndecan precipitated with the pan-syndecan antibody was arbitrarily set to 1.0. FIG. 7. Effects of genistein treatment on tyrosine phosphorylation of syndecans in B82 cells. A, syndecan COOH-terminal fragments were immunoprecipitated with the pan-syndecan antibody following pretreatment of B82 cells with the indicated concentrations of genistein for 30 min. Control lane (C) represents cells pretreated with vehicle only at the highest concentration used. Samples were resolved by gel electrophoresis and membranes probed with either the pansyndecan antibody or PY20. Blots were quantified as described previously. B, syndecan COOH-terminal fragments were immunoprecipitated with the pan-syndecan antibody following pretreatment of B82 cells with 100 M genistein for the indicated amounts of time. Samples were resolved by gel electrophoresis and membranes probed and quantified as described above.
can phosphorylation shows a rapid loss of tyrosine phosphorylation, with approximately 75% loss of phosphorylation within 2 min and ϳ85% loss by 10 min (Fig. 7B). This rapid loss of phosphorylation also verifies the presence of a highly active tyrosine phosphatase.
Endogenous Syndecan Phosphorylation Is Inhibited by Herbimycin A and Staurosporine-To begin to elucidate the kinase and/or signaling pathways involved in syndecan phosphorylation, various kinase inhibitors were used to block endogenous syndecan phosphorylation in B82 cells. Herbimycin A is a potent and irreversible inhibitor of Src related kinases and functions by directly interacting with critical sulfhydryl groups in tyrosine kinases (43). Herbimycin A has also been shown to block cellular tyrosine phosphorylation, cell spreading, and focal adhesion formation in response to cell adhesion to fibronectin (44). Herbimycin A effectively inhibits endogenous syndecan phosphorylation in B82 cells (IC 50 Ͻ 1.0 M) (Fig. 8,  A and B). The effects of herbimycin A are visually represented in Fig. 8A. Immunostaining with PY20 shows complete inhibition at 1.0 M, and as expected, at this concentration band a is no longer detected with the pan-syndecan antibody. It should also be noted that tyrosine phosphorylation of all the syndecan COOH-terminal fragments is similarly blocked by herbimycin A. The phosphorylation of each syndecan fragment is also similarly blocked by the same concentration of genistein (IC 50 Ͻ 10 M) and with the same kinetics (data not shown). Furthermore, tyrosine phosphorylation of each syndecan fragment is blocked by the same concentration of staurosporine (IC 50 Ͻ 1.0 nM) and with the same kinetics (see Fig. 8C and data not shown). These results suggest that the same kinase, or class of kinase, is involved in regulating tyrosine phosphorylation of the different syndecan family members.
Staurosporine is a kinase inhibitor originally described as a potent inhibitor of protein kinase C (45). However, staurosporine displays broad specificity for protein kinases, including both serine/threonine and tyrosine kinases (46). Pretreatment of B82 cells with staurosporine for 15 min blocks tyrosine phosphorylation of syndecans with an IC 50 Ͻ 1.0 nM (Fig. 8C). Inhibition is rapid as phosphorylation is completely lost within 10 min of treatment with 100 nM staurosporine (data not shown). A number of protein kinases, including both serine/ threonine and tyrosine kinases, have been shown to be inhibited by staurosporine in the low nanomolar range (3-30 nM) (46). Of these, Src (IC 50 ϭ 6 nM) and protein kinase C (IC 50 ϭ 2.7 nM) are candidates for having a role in syndecan phosphorylation (45,46). However, protein kinase C inhibitors such as chelerythrine chloride (0.1-3.0 M) and calphostin C (1-300 nM) do not have significant effects on the level of endogenous syndecan phosphorylation in B82 cells (data not shown). DISCUSSION This report demonstrates that cell surface syndecans isolated from B82, L-M(TK) Ϫ and L929 fibroblasts are constitutively phosphorylated on tyrosine. These three cell lines represent different subpopulations of mouse L cells. Although syndecan phosphorylation is not readily detected in other cell types, including NMuMG and Raji-S1 cells, or in other fibroblast cell lines (data not shown), syndecan phosphorylation is detected in these cells in the presence of phosphatase inhibitors. These observations suggest that tyrosine phosphorylation of the syndecan cytoplasmic domains may be a common mechanism for regulating syndecan activity. However, phosphorylation of syndecans appears to be a transient event, tightly controlled by a constitutively active kinase and an associated tyrosine phosphatase. B82 fibroblasts express syndecans-1, -2, and -4 at the cell surface. Using an antibody that recognizes the conserved cytoplasmic domains of all syndecan family members, as well as antibodies specific to individual syndecans, endogenous tyrosine phosphorylation of syndecans-1 and -4 is detected.
The syndecan cytoplasmic domains contain four tyrosine residues that are 100% conserved among all syndecan family members (3,4). Two of the tyrosines are likely targets for phosphorylation based on their surrounding amino acid sequences (28,29). One of the tyrosines is located within the membrane-proximal conserved region (C1), specifically within the sequence DEGSY, and the other is located within a second conserved sequence (EFYA) at the extreme carboxyl terminus (C2) (4). While it is not known which tyrosine(s) is phosphorylated, the presence of two phosphorylated fragments corresponding to syndecan-1, one of which migrates at a higher molecular weight when phosphorylated, suggests that phosphorylation may occur on multiple tyrosine residues. The phosphorylation of multiple tyrosines may be important for mediating downstream signaling. The cytoplasmic domains of several T-cell and B-cell receptor subunits contain two regularly spaced tyrosine residues within sequence motifs known as ITAMs (for immunoreceptor tyrosine-based activation motifs) (47), and phosphorylation of both tyrosines within this motif is required for T-cell receptor binding to the tyrosine kinase ZAP-70, which is a critical event in T-cell activation (48,49). While the syndecans do not contain an ITAM motif per se, they do show some similarity to this motif in the regular spacing of the tyrosine residues and in the amino acid sequences surrounding the tyrosines.
Syndecan phosphorylation is detected endogenously at a low level in B82 cells. This implies that the kinase responsible for syndecan phosphorylation is constitutively active. Treatment with genistein or staurosporine results in a dose-dependent and rapid loss of syndecan phosphorylation, confirming the presence of a constitutively active kinase, as well as an equally active phosphatase. Syndecan phosphorylation is dramatically enhanced by pretreatment of cells with tyrosine phosphatase inhibitors, pervanadate being more effective than either H 2 O 2 or vanadate. The differences in overall potency of these inhibitors can be attributed, at least in part, to the specific mechanisms through which these compounds inhibit tyrosine phosphatases (50). Alternatively, pervanadate has been shown to activate cellular kinases (51,52), and it is possible that pervanadate may also activate a cellular kinase that recognizes the syndecan cytoplasmic domains.
At the present time, the identity of the kinase(s) responsible for syndecan phosphorylation in B82 cells is unknown. Tyrosine phosphorylation is blocked by genistein (IC 50 Ͻ 10 M), herbimycin A (IC 50 Ͻ 1.0 M), and staurosporine (IC 50 Ͻ 1.0 nM). Interestingly, phosphorylation of each COOH-terminal fragment is blocked at the same concentration of inhibitor, and for genistein and staurosporine, with the same kinetics (data not shown). These data suggest that the same kinase, or class of kinase, may be responsible for phosphorylation of the different syndecan family members. Herbimycin A was identified by its ability to reverse cellular transformation induced by Rous sarcoma virus through inactivation of v-Src tyrosine kinase (43), and staurosporine inhibits v-Src and Lyn at low nanomolar concentrations (IC 50 ϭ 6 and 20 nM, respectively) (46), making these and related kinases possible candidates for having a role in syndecan phosphorylation. In support of this, recombinant syndecan-3 cytoplasmic domain binds a complex of proteins containing c-Src and Fyn in vitro, and engagement of syndecan-3 expressed in cells leads to phosphorylation of c-Src (24).
While the different syndecan family members may be phosphorylated by the same kinase, the regulation of syndecan phosphorylation varies in different cell types. Tyrosine phosphorylation of syndecans-1 and -4 is detected endogenously in B82 cells, but not in NMuMG cells. Furthermore, only a single fragment (band b) is phosphorylated in NMuMG cells in response to vanadate, while phosphorylation of multiple syndecan fragments is significantly enhanced in B82 cells under these conditions. However, phosphorylation of multiple syndecan COOH-terminal fragments is detected in NMuMG cells in response to pervanadate (data not shown). Together, these data demonstrate that the syndecans can be phosphorylated in NMuMG cells, but mechanism(s) exist to maintain syndecan phosphorylation at a low level, and these mechanisms appear different in B82 cells.
Tyrosine phosphorylation is an important signaling event in mediating cellular responses to cell-cell and cell-matrix interactions. Tyrosine phosphorylation of cell surface receptors can provide docking sites for proteins containing SH2 domains and regulate interactions with the cytoskeleton. Tyrosine phosphorylation of a ␤1 integrin peptide supports binding to the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase, and tyrosine phosphorylated ␤1 in cells is distinctly localized to podosomes compared with non-phosphorylated ␤1 (53). Similarly, ligation of ␣6␤4 integrin results in the phosphorylation of ␤4 on multiple tyrosines and binding to the Shc/Grb complex, and mutation of specific tyrosine residues blocks ␣6␤4 localization to hemidesmosomes and its association with the cytoskeleton (54).
Through similar mechanisms, tyrosine phosphorylation of the syndecan cytoplasmic domains may provide binding sites for proteins containing SH2 domains and/or regulate syndecan association with the cytoskeleton. Syndecan-1 has been shown to co-localize with actin microfilaments through a mechanism dependent on the cytoplasmic domain, and mutation of a single tyrosine residue within the cytoplasmic domain abrogates this effect (55). The carboxyl-terminal EFYA sequence has recently been shown to bind the PDZ domains of syntenin and CASK (25,26). Cytosolic proteins containing PDZ domains are often localized to adhesion complexes at the plasma membrane, and are proposed to function as adapter molecules, facilitating the formation of structural and/or signaling complexes at the cell surface in response to cell adhesion (56). Tyrosine phosphorylation of the syndecan cytoplasmic domains may have a role in modulating interactions with PDZ domain-containing proteins, possibly by the direct phosphorylation of the tyrosine residue within the carboxyl-terminal EFYA sequence itself, as this tyrosine is in a favorable site for phosphorylation (28,29). There is currently no published report of tyrosine phosphorylation regulating this type of interaction, but phosphorylation of the carboxyl-terminal tail of the potassium channel Kir 2.3 on serine results in receptor dissociation from the PDZ domaincontaining protein PSD-95 (57). While the precise function of syndecan phosphorylation is not known, it likely has a role in binding proteins capable of activating downstream signaling pathways, or in regulating the assembly of a structural complex linking syndecans to the cytoskeleton, in response to cell adhesion and/or growth factor activity.