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Originally published In Press as doi:10.1074/jbc.M305695200 on August 11, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42679-42685, October 24, 2003
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Functional Regulation of Tissue Plasminogen Activator on the Surface of Vascular Smooth Muscle Cells by the Type-II Transmembrane Protein p63 (CKAP4)*

Tahir M. Razzaq{ddagger}, Rosemary Bass, David J. Vines§, Finn Werner, Simon A. Whawell||, and Vincent Ellis**

From the School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom

Received for publication, May 30, 2003 , and in revised form, July 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have demonstrated that tissue plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing plasminogen activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555-21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with 125I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed plasminogen activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the plasminogen activation system in the vessel wall.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular smooth muscle cells (VSMC)1 play a key role in the response of blood vessels to various types of injury or insult (1), partly as a consequence of their ability to migrate and to remodel their surrounding extracellular matrix (2-4). Proteolytic activity is fundamental to these processes (5, 6), with the serine protease plasmin particularly implicated in the vessel wall. Plasmin mediates multiple effects in experimental models of vascular injury, including direct degradation of matrix components (7, 8), propagation of proteolysis by activation of pro-matrix metalloproteases (9), and the activation of latent or matrix-associated growth factors (10, 11). In most tissues undergoing pathological remodeling, uPA is responsible for the generation of plasmin (12). However, in the vessel wall, tPA also plays a prominent role. tPA has a very limited expression pattern in vivo but is expressed by VSMC and up-regulated in response to arterial injury, both in animal models (13, 14) and human atherosclerotic disease (15, 16). The two plasminogen activators are differentially regulated in VSMC in response to vascular injury (13), and observations in gene-ablated mice suggest that they have different roles in this response (14).

Pericellular proteases involved in matrix degradation are often associated with cellular binding sites or receptors (6), which localize them and potentially regulate their activity (17). Perhaps the best described example is the regulation of plasmin generation by the interaction of uPA with its specific glycosylphosphatidylinositol-anchored cellular receptor uPAR (12, 17-19). Although uPAR is expressed by VSMC (20, 21), we have found that these cells also specifically bind tPA and that this binding regulates the functional activity of the bound protease by multiple mechanisms, potentially leading to greater levels of plasmin generation than those achieved by the uPA/uPAR system (21, 22).

The interaction of tPA with VSMC displays unique characteristics. In addition to increasing the activation of cell-associated plasminogen by >100-fold (21), bound tPA also becomes refractory to inhibition by its cognate inhibitor PAI-1 (22). This effect is not observed with other proteins known to stimulate tPA activity, including fibrin, and is due to the involvement of the catalytic domain in the binding. As a consequence, conformational changes in this domain caused by the binding of certain inhibitors render tPA unable to bind to VSMC (22). Furthermore, the lysine binding kringle-2 domain of tPA, analogous to those of plasminogen and allowing tPA to interact promiscuously with proteins exposing C-terminal lysine residues, is not involved in the binding (21). Consequently tPA binding to VSMC is not competed by plasminogen, a phenomenon that limits the functionality of other proposed cellular binding sites for tPA.

We have now isolated the protein that binds tPA on VSMC by using an affinity purification scheme based on these unique binding characteristics. This protein was identified by mass spectrometry (MS) as the type-II transmembrane protein p63 (Human Genome Organization (HUGO) gene name CKAP4, cytoskeleton-associated protein 4). Evidence that this protein is responsible for the specific binding of tPA includes the detection of p63 on the surface of VSMC by immunofluorescence microscopy, inhibition of tPA binding by a monoclonal antibody to p63, and a specific increase in tPA-catalyzed plasminogen activation when overexpressed in COS cells. This represents the first demonstration of the direct involvement of a transmembrane protein in plasminogen activation and suggests that p63 may have an important role in the functional regulation of pericellular proteolytic activity in the vessel wall.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human VSMC were prepared from aortic explants as described previously (21) and used between passage 2-6. Rat primary VSMC were isolated from the thoracic aorta of Wistar rats by enzymatic digestion (23) and used between passage 4-7. The immortalized rat VSMC line, SV40LT-SMC (CRL 2018), was obtained from the American Type Culture Collection (Manassas, VA) and cultured according to their recommendations. All cell culture media and sera were obtained from Invitrogen.

Determination of Plasminogen Activation by VSMC-associated tPA—Plasmin generation by tPA bound to VSMC was determined as described previously (21, 22). Briefly, cells in 24-well plates were incubated with 10 nM tPA (Boehringer Ingelheim GmbH, Ingelheim, Germany) for 20 min, washed extensively, and incubated with 0.2 µM Lys-plasminogen (Enzyme Research Laboratories, Swansea, UK) and 0.2 mM H-D-Val-Leu-Lys-7-amido-4-methylcoumarin (Bachem, Bubendorf, Switzerland). Plasmin generation was determined by continuous monitoring of fluorescence in a SPECTRAmax Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA), and expressed as {Delta}F/min. The binding of tPA was determined by varying the initial concentration of tPA (0-250 nM) as described previously (21). Specific binding was calculated as the difference in plasmin generation in the presence and absence of 1 mM {epsilon}-aminocaproic acid ({epsilon}-ACA). Data were analyzed by non-linear regression to obtain an apparent Kd for tPA binding.

Preparation of Affinity Matrices—tPA was first inactivated with either DFP (Sigma) or PPACK (Bachem) as described previously (22). Inactivated tPA was coupled to CNBr-activated-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions, with the exception that tPA was dissolved in 0.5 M sodium thiocyanate prior to coupling to aid solubility.

Surface Iodination of VSMC—Ten T-175 flasks of VSMC approaching confluence (~2 x 107 cells) were washed in serum-free medium and gently scraped from the culture flasks. The cells were resuspended in 2 ml of PBS at room temperature, labeled with 1 mCi of Na125I (Amersham Biosciences) for 1 h using Iodo-Beads (Pierce), pelleted, and resuspended in 1 ml of ice-cold PBS containing 200 mM n-octyl {beta}-D-glucopyranoside (Sigma) and protease inhibitor mixture (Roche Diagnostics). The cells were passed five times through a 25-gauge needle and solubilized for 30 min on ice before centrifugation for 60 min at 100,000 x g.

Affinity Isolation of 125I-labeled VSMC tPA Receptor—Cleared cell lysates were incubated with PPACK-inactivated tPA-Sepharose for 1 h at 4 °C with mixing. The non-binding supernatant was decanted and applied to a column of DFP-inactivated tPA-Sepharose. The column was washed sequentially with the buffers PBS, PBS containing 1 M NaCl, and PBS containing 100 mM L-lysine, all of which contained 0.1% Triton X-100, and was finally eluted with 0.05 M glycine, pH 3.0, and 1 M NaCl. Column fractions were precipitated with 20% trichloroacetic acid, washed, and resolubilized in SDS/PAGE sample buffer. Samples were resolved on 4-12% SDS/PAGE gels, and the gels were dried and subjected to autoradiography using BioMax MS film (Kodak).

Purification of 63-kDa VSMC tPA Receptor—Large scale purification of tPA-binding proteins from rat VSMC was performed using 5 x 108 SV40LT-SMC cells. These were prepared in the same way as described above, with the exception that they were not radiolabeled and that cleared cell lysates were stored at -80 °C prior to further processing to allow accumulation of sufficient starting material.

Mass Spectrometric Analyses—SDS-PAGE gels were stained with Coomassie Blue, and protein bands were excised. These gel plugs were washed, reduced, alkylated, and digested in situ with trypsin. The generated peptides were analyzed by matrix-assisted laser desorption ionization mass spectrometry on a Bruker REFLEX reflector time-of-flight mass spectrometer (Bruker Daltonics, Billerica, MA). Partial sequencing of individual peptides was performed by nano-electrospray tandem mass spectrometry using a QSTAR quadrupole time-of-flight instrument (PE Sciex, Toronto, Canada). Mass spectroscopic analyses were performed by Protana A/S, Odense, Denmark. Peptide maps and sequence data were searched against non-redundant protein and expressed sequence tag data bases and also against rat genomic sequence data (BAC 2002-11-22 assembly, Human Genome Sequencing Center, Baylor College of Medicine).

Immunoblotting—Fractions eluted from the DFP-tPA affinity purification step were subjected to SDS/PAGE, and proteins were transferred to polyvinylidene difluoride membranes. After blocking membranes were probed with a rabbit polyclonal antibody to human p63 (a gift of Drs. Jack Rohrer and Anja Schweizer), peroxidase-labeled goat anti-rabbit IgG (DAKO, Ely, UK) and detected with 3,3-diaminobenzidine tetrahydrochloride (Sigma). When eluted fractions were derived from surface-iodinated cell preparations, the blots were subsequently subjected to autoradiography.

Immunofluorescence Microscopy—Subconfluent layers of human VSMC grown on glass coverslips were fixed with 4% formaldehyde, blocked, and incubated at 4 °C with 1:100 G1/293, a monoclonal antibody to p63 (24). The secondary antibody was fluorescein isothiocyanate-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Controls included incubation with isotype-matched mouse IgG (DAKO) or secondary antibody alone. Mounted slides were visualized by epifluorescence, and images were captured digitally.

Expression of Recombinant p63—COS-1 cells (CRL-1650, ATCC) at ~80% confluency in 24-well plates were transfected using 3 µl of FuGENE-6 (Roche Diagnostics) and 1 µg of DNA per well with the expression plasmids pECE-p63 or pECE-{Delta}2-101AA (25) coding for wild-type human p63 and an N-terminal deletion mutant of p63, respectively. Cells were analyzed 48 h after transfection for tPA binding, cell surface plasminogen activation, and p63 immunofluorescence in the same way as described for VSMC.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
tPA Binding to VSMC—We have demonstrated previously that human VSMC bind tPA specifically and that this binding greatly stimulates the activation of cell-associated plasminogen (21, 22). The limited potential of these cells as a source for the isolation of the binding protein led us to investigate more suitable sources of starting material. Primary rat VSMC and the immortalized cell line SV40LT-SMC were found to have characteristics similar to those demonstrated previously for human VSMC. Bound tPA activated cell-associated plasminogen >90-fold more efficiently than plasminogen in solution (Fig. 1), primarily due to a reduction in the Km for plasminogen from >10 µM to 80 nM (data not shown), and the specific binding associated with this high-efficiency plasminogen activation had an apparent Kd of 15 nM (Fig. 1, inset).



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  		FIG. 1.
tPA binding and stimulation of plasminogen activation on rat VSMC. Rat primary VSMC were incubated with tPA, washed extensively, and incubated with Lys-plasminogen and a plasmin-specific fluorogenic substrate. Plasmin generation is shown in the absence (•) and presence ({circ}) of {epsilon}-aminocaproic acid as a competitor of the cellular binding of plasminogen. By varying the initial tPA concentration, similar data were used to determine the apparent affinity of tPA binding (inset). The difference between the data obtained in the absence and presence of {epsilon}-aminocaproic acid ({blacksquare}) represents plasmin generation by specifically bound tPA (21). Similar data were also obtained using SV40LT-SMC cells.

 

Isolation of VSMC tPA-binding Protein—To identify the functional tPA binding protein, rat VSMC were surface-labeled with 125I prior to affinity chromatography of cell lysates on tPA-Sepharose. Preliminary experiments suggested that multiple proteins interact with immobilized tPA under these conditions, suggesting a relatively high level of nonspecific interaction. To overcome this, we sought to exploit two unique characteristics of the specific binding of tPA to VSMC that we had previously identified in functional studies. The first is its independence from the kringle-2 lysine-binding site (21). The second is the differential binding of two catalytically inactivated forms of tPA, i.e. DFP-inactivated tPA, which binds to VSMC, and tPA inactivated by the tripeptide chloromethane inhibitor PPACK, which does not (22).

To remove nonspecifically bound proteins, surface-labeled cell lysates were pre-adsorbed with immobilized PPACK-tPA, and the non-binding fraction was applied to immobilized DFP-inactivated tPA and washed extensively in lysine-containing buffer. The proteins remaining specifically bound were subsequently eluted at low pH (Fig. 2). The eluted proteins were analyzed by SDS-PAGE and autoradiography, which revealed a single major radiolabeled band with a molecular mass of 63 kDa (Fig. 2, inset). To establish that this protein was distinct from the abundant proteins migrating with similar mobilities in the starting material and non-bound fractions, the latter were pooled and re-chromatographed. A greatly reduced level of radioactivity was eluted under these conditions (Fig. 2, open circles), which was not observable by autoradiography (data not shown), demonstrating that the specifically bound radiolabeled protein had been quantitatively bound by the affinity matrix. Therefore, in these experiments a radiolabeled 63-kDa protein, which did not bind to PPACK-inactivated tPA, bound specifically and quantitatively to DFP-inactivated tPA in a lysine-independent manner.



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  		FIG. 2.
Affinity purification and detection of 125I-labeled tPA-binding proteins. SV40LT-SMC cells were surface-labeled with 125I, lysates were pre-adsorbed with immobilized PPACK-tPA, and the non-binding supernatant was subjected to chromatography on immobilized DFP-tPA. The elution profile of radioactivity from this column is shown (•) washed in PBS (fractions 1-9), high salt buffer (fractions 10-14), and lysine-containing buffer (15-18) and eluted at low pH (fractions 19 onwards). Pooled elution fractions were analyzed by SDS-PAGE. The inset show an autoradiogram of the starting material applied to the DFP-tPA column (lane 1), fractions specifically eluted at low pH (lane 2), and the run-through fractions (lane 3). The samples in lanes 1 and 3 are diluted 50-fold compared with that in lane 2. Similar observations were made using rat primary VSMC. Also shown is the elution profile of pooled run-through fractions re-chromatographed on the DFP-tPA column ({circ}).

 

Identity of the 63-kDa tPA-binding Protein—To identify the 63-kDa tPA-binding protein, large scale non-radiolabeled lysates of rat VSMC were prepared and subjected to the same chromatographic procedure. Specifically eluted proteins were separated on SDS-PAGE and stained with Coomassie Blue (Fig. 3, lane 1). Two bands were observed at 44 and 63 kDa, the latter major band corresponding to the single radiolabeled band observed by autoradiography. Both bands were excised from the gel, digested in situ with trypsin, and subjected to matrix-assisted laser desorption ionization MS.



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  		FIG. 3.
Isolation of 63-kDa tPA-binding protein. Large scale purification of non-radiolabeled rat SV40LT-SMC lysates were made using the same procedure as outlined in the Fig. 2 legend. The specifically eluted fractions were pooled, concentrated, and subjected to SDS-PAGE. The gel was stained with Coomassie Blue, and the 44- and 63-kDa protein bands were excised and processed for MS analysis. Lane 1 represents the first purification attempt in which more than one protein was subsequently detected in the 63-kDa band. Lane 2 represents an independent purification in which the DFP-tPA column was washed more extensively in lysine-containing buffer and in which only one protein was detected in the 63-kDa band.

 

The peptide mass map obtained from the 63-kDa protein band contained 97 individual peptides masses, which, when searched against protein and expressed sequence tag data bases, identified the presence of two proteins. Seven of the peptide masses matched the sequence of a 66-kDa human type-II transmembrane protein known as p63 or CKAP4. Sequencing of six of the peptides by nano-electrospray tandem MS either confirmed their assignment or gave positive hits to mouse or rat ESTs homologous to p63. Subsequent searching of rat genome data revealed a p63 coding sequence which, when compared with the original peptide mass map, gave a greatly improved match with 32 of 97 peptides covering 52.2% of the sequence of a 63.6-kDa protein (Table I), unequivocally identifying this protein as the rat ortholog of p63 (sequence comparison is shown in the supplemental data available in the on-line version of the article). Fifteen of the remaining peptide masses matched the peptide map of a 69.8-kDa mouse RNA-binding protein, GRY-RBP (covering 29.7% of the sequence). This protein appeared to be a contaminant, as we failed to detect a GRY-RBP peptide mass signature in a second protein isolation, which included a more extensive lysine wash (Fig. 3, lane 2).


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  		TABLE I
Sequence assignments of tryptic peptides derived from the 63-kDa protein by mass spectrometry

 

Identity of the 440-kDa tPA-binding Protein—The peptide mass map obtained for the 44-kDa protein, which was not labeled in the surface iodination experiments and was therefore most likely intracellular, was unequivocally identified as cytoplasmic {beta}-actin (21 of 90 peptides matched covering 55% of the sequence, data not shown). This is consistent with previous reports of tPA binding to actin, a lysine-dependent interaction (26, 27). Presumably, the abundance of actin has allowed a small proportion of it to evade our attempts to exclude lysine-dependent binding proteins.

Confirmation of the Specific tPA-binding Protein as p63—To confirm the identity of the specific tPA-binding protein as the rat ortholog of p63, 125I-labeled cell lysates were again subjected to DFP-tPA affinity chromatography, and elution fractions were probed by both autoradiography and Western blotting using a polyclonal antibody raised against authentic human p63. The eluted protein was specifically recognized by the antibody (Fig. 4, top panel) and had an exact correspondence with the radiolabeled 63-kDa protein (top panel). Comparison of the loading and intensities of the various lanes suggest that p63 was quantitatively removed from the cell lysate by DFP-tPA affinity chromatography, reinforcing the conclusion that the 63-kDa radiolabeled protein does not represent a small proportion of a nonspecifically bound, highly abundant protein.



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  		FIG. 4.
The 63-kDa tPA-binding protein corresponds to p63 (CKAP4) immunoreactivity. Specific elution fractions from an experiment similar to that shown in Fig. 2 were run on SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. This membrane was then probed both by autoradiography (top panel) and Western blotting for p63 (bottom panel). Lane 1 represents the starting material applied to the DFP-tPA column, and lanes 2-6 represent individual fractions collected across the peak of radioactivity specifically eluted at low pH. The sample in lane 1 is diluted ~5-fold compared with the other lanes. Small scale experiments using human VSMC also showed a similar correspondence between the radiolabeled protein and p63 antigen (data not shown). The minor band of ~57 kDa detected in the Western blot was not detected in the starting cell lysate or in other experiments and most likely represents a degradation product of p63.

 

p63 Is Present at the Surface of VSMC—Although a transmembrane protein, p63 has previously been reported to be retained in the ER (28). To confirm that p63 was present on the surface of VSMC as suggested by the surface iodination experiments, human VSMC were probed with a p63-specific monoclonal antibody (G1/293) by immunofluorescence microscopy. Intact, non-permeabilized cells demonstrated a punctate cell-surface staining for p63, which was also apparent on permeabilized cells (Fig. 5). The latter also demonstrated extensive perinuclear staining consistent with ER localization. The specificity of the cell-surface staining was tested in control experiments using COS cells overexpressing wild-type human p63, which demonstrated only perinuclear staining (data not shown), consistent with previous reports (25).



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  		FIG. 5.
Detection of p63 (CKAP4) on the surface of human VSMC by immunofluorescence microscopy. Human VSMC were probed with the monoclonal antibody G1/293 and a fluorescein isothiocyanate-conjugated secondary antibody. Panel A shows surface staining for p63 on non-permeabilized cells. Panel C shows extensive intracellular p63 staining with saponin-permeabilized cells. Isotype-matched, non-immune mouse IgG controls for non-permeabilized and permeabilized cells are shown in panels B and D, respectively.

 

p63 Binds tPA on the Surface of VSMC—To determine whether p63 on the surface of VSMC was responsible for the binding that we identified originally, anti-p63 antibodies were tested for their ability to inhibit tPA binding and subsequent cell-surface plasminogen activation. Fig. 6 shows that increasing concentrations of G1/293 led to a decrease in cell surface plasminogen activation. At the highest concentrations of antibody used, this inhibition approached that observed using DFP-tPA as a specific competitor of tPA binding.



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  		FIG. 6.
Inhibition of tPA binding to human VSMC by an anti-p63 monoclonal antibody. Cells were incubated with varying concentrations of the anti-p63 monoclonal antibody G1/293 prior to incubation with tPA and determination of cell surface plasminogen activation in experiments similar to those shown in Fig. 1. The dashed line represents the level of plasmin generation observed in the presence of a 100-fold molar excess of DFP-inactivated tPA as a competitor of tPA binding. The solid line represents the best fit of the experimental data (•) to a single-site competition model by non-linear regression and is compared with an equivalent concentration of isotype-matched, non-immune mouse IgG ({circ}). In control experiments, the antibody G1/293 was shown to have no direct inhibitory effect on tPA activity.

 

To further confirm the role of cell-surface p63 in tPA binding, COS cells were transfected with either wild-type p63 or the N-terminal deletion mutant p63 {Delta}2-101AA. The latter lacks the sequence necessary for retention in the ER, lacking residues 2-101 and with the adjacent di-Arg motif mutated to di-Ala (R102A,R103A), and has been shown previously to traffic preferentially to the plasma membrane (25). p63-transfected cells did not display a significant increase in tPA-catalyzed plasminogen activation (Fig. 7), and p63 could not be detected at the cell surface by immunofluorescence microscopy (data not shown). By contrast, transfection with p63 {Delta}2-101AA, which was readily detectable on the cell surface, led to a >7-fold increase in plasminogen activation, which could be competed to similar extents by either DFP-inactivated tPA or G1/293.



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  		FIG. 7.
Expression of plasma membrane-targeted p63 increases tPA binding to COS cells. COS-1 cells were transfected with various p63 constructs and assayed at 48 h post-transfection. All cells were incubated with tPA, and plasminogen activation was determined as described in the legend to Fig. 1. Data are shown for untransfected COS cells (Control), cells transfected with empty vector, cells transfected with wild-type (WT) p63, cells transfected with the plasma membrane targeted {Delta}2-101AA deletion mutant, and the latter cells in the presence of the competitors DFP-tPA and anti-p63 monoclonal antibody (mAb), respectively. Wild-type p63 and {Delta}2-101AA-transfected cells both expressed similar levels of p63 as determined by immunofluorescence staining of permeabilized cells (data not shown).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The plasminogen activation system has many potential roles in the pericellular environment, and multiple mechanisms exist to regulate its activity in this situation (6, 17). Binding of tPA to specific cell surface binding sites appears to be one of these mechanisms, as we have demonstrated both the specific binding of tPA to VSMC and the functional regulation of tPA activity as a consequence of this binding (21, 22). In the present study we have identified the protein responsible for this binding as the type-II transmembrane protein p63 (CKAP4). The evidence that p63 is the specific binding site regulating tPA activity on VSMC includes the following observations: (i) antibodies to p63 quantitatively blocked the binding of tPA; (ii) tPA bound specifically to COS cells expressing a plasma membrane-targeted p63 mutant; and (iii) p63 was detected on the surface of VSMC by immunofluorescence. However, the key observation that links p63 with the specific cellular binding site is the close correspondence between the tPA binding characteristics of p63 and those unique characteristics identified previously for the cellular site (21, 22).

The first of these characteristics, which were exploited for the isolation of p63, is the differential binding of inactivated forms of tPA. Previously, we have shown that tPA modified at the active site Ser478 residue by DFP competes effectively for the binding of active tPA (as did a Ser478 -> Ala active site mutant), but tPA inactivated with the tripeptide chloromethane inhibitor PPACK does not, an effect due to conformational rearrangements in the catalytic domain of the protease (22). Consistent with these observations, p63 bound quantitatively to a DFP-tPA affinity matrix but not to a PPACK-tPA matrix. The second characteristic is that, in contrast to the binding of tPA to other identified binding proteins, the functional interaction of tPA with VSMC is independent of the lysine-binding kringle-2 module of tPA (21). Again, p63 was quantitatively retained on the affinity matrix in the presence of lysine. The relative nonspecificity and promiscuity of lysine-dependent, kringle-2 mediated interactions is highlighted by the presence of two contaminating proteins in the eluate from the DFP-tPA column. The contaminant protein GRY-RBP has a C-terminal lysine residue, the preferred ligand for lysine-binding kringles, and {beta}-actin has previously been reported to bind tPA in a lysine-dependent manner (26, 27). Small amounts of these proteins (which were not detected in the surface-labeling experiments) presumably evaded our attempts to exclude them because of their abundance. Notably, other proteins reported to bind tPA at the surface of various cell types, including annexin II (29, 30), cytokeratin 8 (31), and LRP (32), were not detected by Western blotting in the elution fractions from the DFP-tPA affinity matrix, although they were readily detectable in the cell lysates (data not shown).

The lysine-independence of tPA binding to p63 has functional consequences in that p63 would not be expected to bind plasminogen. Plasminogen has been shown to bind to other tPA-binding proteins and, although this is believed to be part of a mechanism for stimulating plasmin generation (29, 31), competition between tPA and plasminogen for binding sites is also apparent (33). Consistent with the lysine-independence of the p63/tPA interaction, we found no evidence for plasminogen binding to p63. 125I-labeled plasminogen did not detect isolated p63 in ligand blotting experiments, and p63 did not bind to a plasminogen affinity matrix as detected either by immunoblotting or ligand blotting, although the plasminogen binding protein {alpha}-enolase (34) was readily detected by both methods (data not shown). These observations suggest that at least part of the mechanism for p63 enhancement of plasminogen activation is similar to that employed by the uPA/uPAR system in which the binding of plasminogen to distinct cell surface binding sites is required (12).

The affinity of tPA binding to VSMC determined here and elsewhere (21) is relatively modest compared with the affinity of the uPA/uPAR interaction (Kd of 15 nM cf. 0.3-1 nM), suggesting that it may only have a minor impact on plasmin generation. However, tPA levels in atherosclerotic lesions have been shown to be up to 50-fold higher than those of uPA (16, 35). Therefore, despite this differential affinity, similar levels of receptor occupancy and subsequent plasmin generation may be achieved by both activators in this setting.

The role of p63 as a specific cellular receptor for tPA is dependent on its plasma membrane localization as demonstrated here on VSMC, both by immunofluorescence staining and the effect of an anti-p63 monoclonal antibody on tPA-catalyzed plasminogen activation. However, p63 has been characterized previously in fibroblasts as an intracellular protein associated with the ER (28), with this retention being determined by its 108-residue N-terminal cytoplasmic domain (25), which is deleted in the {Delta}2-101AA mutant. The mechanism whereby p63 reaches the plasma membrane in VSMC is unclear, but there are precedents for the presence of normally ER resident proteins at the plasma membrane, including the integral membrane proteins kinectin (36) and calnexin (37). Previous studies of trafficking and localization have been performed in cells overexpressing p63, which leads to rearrangements of both the ER and microtubules mediated by the cytoplasmic domain of p63 (38). At endogenous expression levels p63 may behave differently and, possibly, in a cell type-specific manner (39). Without the close association with microtubules as a consequence of overexpression, a proportion of p63 may escape its sub-domain-specific localization and be free to reach the plasma membrane. Consistent with this, the protein we isolated from VSMC was readily soluble in Triton X-100, a characteristic reported for mutants of p63 that traffic to the cell surface but not for ER-associated p63, which is largely insoluble in this detergent (25). Alternatively, the cytoplasmic domain of p63 may remain associated with microtubules and direct a sub-population of the protein to the cell surface. As microtubule dynamics are known to be involved in cytoskeletal rearrangement during cell movement (40), this could represent a mechanism for the targeting of p63 and the proteolytic activity of tPA during cell migration. A final and intriguing possibility is that p63 trafficking to the plasma membrane is dependent on its binding to tPA in the secretory apparatus in a "chaperone-like" function and, therefore, that it reaches the surface only of those cells, such as VSMC, that express a significant amount of tPA. It seems unlikely that p63 reaches the plasma membrane as a consequence of a lack of the N-terminal retention signal, for example by alternative splicing or proteolysis. The cytoplasmic and transmembrane domains are encoded by a single exon (covering residues 1-161; see supplemental data in the on-line version of this paper) and, although a slightly lower Mr band was detected on Western blots (e.g. Fig. 4, bottom panel), this was not detected by surface labeling (Fig. 4, top panel), suggesting that it was not present at the cell surface.

From our data it is not possible to predict how tPA interacts with p63. The three-dimensional structure of p63 is not known, but the C-terminal extra-cytoplasmic part of the protein (residues 132-602) has been suggested to have a largely {alpha}-helical structure based on its CD spectrum (39). This is consistent with sequence analysis (41), which suggests that up to 30% of this domain will form {alpha}-helical coiled-coils due to repeating heptad motifs. Other proteins known to interact with tPA either in vivo or in vitro also contain coiled-coil structures, most notably fibrin, but also the cytoplasmic proteins myosin (42) and cytokeratin 8 (31). Fibrin contains multiple binding sites for tPA, at least one of which (AA 148-160) is contained within a coiled-coil region of the protein (43), and the binding sites for tPA in the other two proteins are retained in fragments containing their coiled-coil regions. The isolated extra-cytoplasmic domain of p63 expressed in Escherichia coli has been shown to form insoluble oligomers and rod-like multimeric structures (39). The p63 that we detected on the surface of VSMC appeared to be highly clustered, which may similarly be due to oligomerization. This may be involved in the interaction with tPA, as oligomer formation has been shown to enhance the binding of tPA to both amyloid {beta} and endostatin (44).

In addition to its identification as a component of the ER and its role in binding tPA, p63 has also been independently identified as a transcriptionally regulated protein involved in cellular stress responses. In fibroblasts p63 expression is cell cycle-regulated, but this regulation is lost in senescent cells that have constitutively elevated expression levels (45). p63 has also been shown to be identical to a gene transcriptionally induced in the liver in response to circulatory shock (46). Similar responses in VSMC would lead to increased levels of bound tPA and increased proteolytic activity. Because both cellular stress responses and senescence of VSMC are important factors in the development and progression of atherosclerosis (47), this may suggest a mechanism by which cell surface levels of tPA can be regulated by VSMC. In conclusion, our data demonstrate that p63 binds and regulates the function of tPA on the surface of VSMC and suggest that it may have an important role in the generation of pericellular proteolytic activity in the vessel wall.


   FOOTNOTES
 
* This work was funded by British Heart Foundation Grants PG/96041 and PG/98172. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains a sequence comparison of rat and human p63 (CKAP4). Back

{ddagger} Present address: Dept. of Experimental Haematology, The Royal London Hospital Medical School, London E1 2AD, United Kingdom. Back

§ Present address: Ludwig Inst. for Cancer Research, University College London, London W1W 7BS, United Kingdom. Back

Present address: Dept. of Biological Sciences, Imperial College London, London SW7 2AZ, United Kingdom. Back

|| Present address: Eastman Dental Inst., University College London, London WC1X 8LD, United Kingdom. Back

** Senior Research Fellow of the British Heart Foundation and to whom correspondence should be addressed. Tel.: 44-1603-592570; Fax: 44-1603-592250; E-mail: v.ellis{at}uea.ac.uk.

1 The abbreviations used are: VSMC, vascular smooth muscle cells; CKAP4, cytoskeleton-associated protein 4; DFP, diisopropyl fluorophosphate; ER, endoplasmic reticulum; MS, mass spectrometry; PBS, phosphate-buffered saline; PPACK, H-D-Phe-Pro-Arg-chloromethane; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator; uPAR, uPA receptor. Back


   ACKNOWLEDGMENTS
 
We would like to thank Drs. Jack Rohrer and Anja Schweizer, Friedrich Miescher Institute, Basel, Switzerland for antibodies to p63.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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