Requirement of the p130CAS-Crk Coupling for Metastasis Suppressor KAI1/CD82-mediated Inhibition of Cell Migration*

Xin A. Zhang {ddagger}, Bo He, Bin Zhou and Li Liu

From the Vascular Biology Center and Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, March 25, 2003 , and in revised form, April 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
KAI1/CD82 protein is a member of the tetraspanin superfamily and has been rediscovered as a cancer metastasis suppressor. The mechanism of KAI1/CD82-mediated suppression of cancer metastasis remains to be established. In this study, we found that migration of the metastatic prostate cancer cell line Du145 was substantially inhibited when KAI1/CD82 was expressed. The expression of focal adhesion kinase (FAK) and Lyn, a Src family tyrosine kinase and substrate of FAK, was up-regulated at both RNA and protein levels upon KAI1/CD82 expression. The activation of FAK and Lyn, however, remained unchanged in Du145-KAI1/CD82 cells. As a downstream target of FAK-Lyn signaling, the p130CAS (Crk-associated substrate) protein was decreased upon the expression of KAI1/CD82. Consequently, less p130CAS-CrkII complex, which functions as a "molecular switch" in cell motility, was formed in Du145-KAI1/CD82 cells. To confirm that the p130CAS-CrkII complex is indeed important for the motility inhibition by KAI1/CD82, overexpression of p130CAS in Du145-KAI1/CD82 cells increased the formation of p130CAS-CrkII complex and largely reversed the KAI1/CD82-mediated inhibition of cell motility. Taken together, our studies indicate the following: 1) signaling of FAK-Lyn-p130CAS-CrkII pathway is altered in KAI1/CD82-expressing cells, and 2) p130CAS-CrkII coupling is required for KAI1/CD82-mediated suppression of cell motility.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Understanding the molecular determinants that govern cancer invasiveness and metastasis is fundamentally important for successfully diagnosing and treating metastatic cancers. KAI1/CD82 was rediscovered as a prostate cancer metastasis suppressor through genetic screening (1). Reduced KAI1/CD82 expression is associated with malignant progression of human prostate cancer (2). Advanced prostate tumors show decreased KAI1/CD82 expression compared with normal prostate and benign prostatic hyperplasia, and the down-regulation usually does not involve mutation or allelic loss of KAI1/CD82 (3). Both Gleason grades and clinical stages of prostate cancers have an inverse correlation with the percentage of KAI1/CD82-positive cells (4). Then, it was found that the expression of KAI1/CD82 was actually down-regulated in many other poorly differentiated or metastatic cancers (513). Expression of KAI1/CD82 in cell lines derived from advanced or metastatic cancers substantially inhibits cell motility and/or invasiveness of these cancer cells in vitro and dramatically suppresses metastasis in animal models (1, 10, 14, 16). These observations indicate that the KAI1/CD82 expression level may determine the invasive and metastatic potential of various malignant tumors. However, the mechanism by which KAI1/CD82 suppresses cancer invasiveness or metastasis remains unclear.

KAI1/CD82 protein belongs to the tetraspanin or transmembrane 4 superfamily (TM4SF)1 and contains four presumed transmembrane domains, a short N- and a C-terminal cytoplasmic domain (11 and 12 amino acids), a small intracellular loop (4 amino acids), and two extracellular loops (23 and 114 amino acids) (1922). TM4SF proteins participate in many biological events ranging from cellular fusion, cell adhesion, and cell migration to cell proliferation, synapse formation, and neurite outgrowth (1924). Like other TM4SF proteins, KAI1/CD82 has been implicated in regulating cell motility (1922). Also, the KAI1/CD82 protein in T cells behaves as a costimulatory molecule during T cell activation (23, 24), indicating that KAI1/CD82 plays a role in signal transduction. Recent studies have revealed that KAI1/CD82 attenuates epidermal growth factor (EGF) signaling by accelerating EGF receptor endocytosis through an interaction with the EGF receptor (25), thereby confirming that KAI1/CD82 is functionally connected to the intracellular signaling machinery. Clustering KAI1/CD82 with its monoclonal antibody (mAb) induces cytoskeletal rearrangement and elongated cellular extension (26, 27). This signaling event is dependent on the activities of protein kinase A, protein kinase C (PKC), and Rho small GTPases but independent of Src kinase activity (26, 27). These studies suggest that KAI1/CD82 may directly initiate signaling events. Thus, how the KAI1/CD82-initiated or -mediated signaling intercepts the signaling required for cancer cell motility and invasiveness becomes an essential question.

Recently, progress has been made in understanding the signaling pathways that control cell migration. For example, the signaling pathway that involves Shc, MEK, and MAP kinase ERKs determines random cell migration through regulating actin-myosin assembly and cell contraction (28, 29). Another pathway that involves FAK, Src, p130CAS, and CrkII determines directional persistence of cell migration by regulating actin reorganization, focal contacts, and membrane ruffling (2831). The small GTPases of the Rho family regulate multiple aspects of cell motility such as generation of lamellipodia, assembly of focal adhesion, retraction of tail, and formation of stress fiber by either directly acting on cytoskeleton reorganization or by cross-talking with the above signaling pathways (32, 33). For example, Rac GTPase activity is required to propagate the signals generated from the FAK-Src-p130CAS-CrkII pathway to the actin cytoskeleton (28). The phosphatidylinositol 3-kinase (PI3-K)-Akt/PKB pathway also plays an important role in the events of cell movement, such as cellular polarization during chemotaxis, by modulating FAK and Rho signaling pathways (3436).

In the FAK-Src-p130CAS-CrkII pathway, the association of p130CAS with CrkII induces cell migration and enhances invasiveness of carcinoma cells (30). The p130CAS-CrkII coupling depends on the interaction of the CrkII SH2 domain with phosphotyrosine residues present in the substrate domain of p130CAS (37). Recent evidence indicates that p130CAS-CrkII coupling occurs through a tightly regulated balance of tyrosine phosphorylation of p130CAS by Src and/or FAK and dephosphorylation by phosphatases such as PTP-PEST (37). In a proposed model, p130CAS-CrkII coupling induces the CrkII SH2 domain to recruit DOCK180 to the cell membrane and to activate Rac1, thus causing the induction of membrane ruffling (28, 30). Therefore, the formation of p130CAS-CrkII complex provides a molecular switch leading to cell migration and invasion (30).

This study sought to identify the signaling pathway responsible for the KAI1/CD82-mediated suppression of cancer cell movement. Using Du145 metastatic prostate cancer cells as the experimental model, we demonstrated that the FAK-Src-p130CAS-CrkII pathway is crucial for the KAI1/CD82-mediated suppression of cell motility of Du145 cells. The signaling derived from KAI1/CD82 alters the levels of FAK and Lyn and attenuates the p130CAS-CrkII coupling. This finding illustrates an important signaling mechanism by which KAI1/CD82 suppresses cell motility.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Extracellular Matrix Proteins—The mAbs used in this study were human integrin {alpha}3 subunit mAb X8 (38), human integrin {alpha}5 subunit mAb PUJ-2 (39), and human CD82 mAb M104 (40) and 4F9 (24). A mouse IgG2 was used as a negative control antibody (Sigma). The mAb against FAK, p130CAS, CrkII, paxillin, Yes, RACK1, or PKC{alpha} was purchased from BD Transduction Laboratory (Lexington, KY), and the mAb against vinculin or tubulin {beta} was from Sigma. The phosphotyrosine mAb 4G10 was from Upstate Biotechnology (Lake Placid, NY). The mAb against Src phosphotyrosine 416 was purchased from Cell Signaling Technology (Beverly, MA); this mAb cross-reacts with the corresponding phosphotyrosine residues of other Src family members such as Lyn and Yes. The mAb to phosphotyrosine 397 of FAK and the mAb to phosphotyrosine 577 of FAK were obtained from BIOSOURCE International (Camarillo, CA). The polyclonal antibody (pAb) against FAK, Lyn, Fyn, and c-Src were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma).

The extracellular matrix (ECM) proteins used in this study were human plasma fibronectin (FN) (Invitrogen), mouse laminin (LN)-1 (Invitrogen), and rat LN-5 (Drs. Chris Stipp and Martin Hemler of the Dana Farber Cancer Institute).

Cell Culture and Transfectants—Prostate cancer cell line Du145 was obtained from ATCC (Manassas, VA) and cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The full-length KAI1/CD82 cDNA was obtained from Dr. Christopher Class (German Cancer Research Center) and subcloned into a eukaryotic expression vector pCDNA3.1 (Invitrogen). Du145 cells were transfected with pCDNA3.1-KAI1/CD82 plasmid DNA using Superfectin (Qiagen, Valencia, CA) and selected with G418 (Invitrogen) at a concentration of 1 mg/ml. Hundreds of G418-resistant clones were pooled, and the KAI1/CD82-positive ones were collected by flow cytometric cell sorting. The pooled Mock or KAI1/CD82-positive clones were the stable transfectants used in the following experiments. In some experiments, plasmid DNA of wild-type p130CAS (from Dr. R. Klemke, Scripps Research Institute) was transiently transfected into Du145-KAI1/CD82 and Du145-Mock cells using LipofectAMINE 2000 (Invitrogen) by following the manufacturer's protocol.

Flow Cytometry—Du145 transfectant cells were incubated with negative control mAb, integrin mAbs, or specific TM4SF mAbs and then stained with FITC-conjugated goat-anti-mouse IgG as previously described (41). The stained cells were analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Fluorescence with negative control mAb was subtracted to give specific mean fluorescence intensity (MFI) units.

Immunoprecipitation and Immunoblotting—Immunoprecipitations were carried out basically as previously described (42). Briefly, an identical number of Du145 transfectant cells were lysed using RIPA lysis buffer containing 1% Nonidet P-40, 0.2% SDS, 150 mM NaCl, 25 mM HEPES, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 2 mM sodium vanadate, and 2 mM sodium fluoride at 4 °C for 1 h. Lysates were precleared with a combination of protein A-Sepharose and protein G-Sepharose beads (Amersham Biosciences) two times at 4 °C after removing the insoluble material by 14,000 x g centrifugation. Then the mAb-preabsorbed protein A-Sepharose and protein G-Sepharose beads were incubated with cell lysate overnight at 4 °C. The precipitates were washed with the lysis buffer three times, dissolved in Laemmli sample buffer, heated at 95 °C for 5 min, separated by SDS-PAGE, and then subjected to immunoblotting.

Immunoblotting was also performed as previously described (42). For Western blotting of total cellular proteins, an equivalent number of cells were lysed using RIPA buffer, the protein concentrations of lysates were normalized, and then the lysates were separated by SDS-PAGE. After being transferred electrically, nitrocellulose membranes (Schleicher & Schuell, Keene, NH) were sequentially blotted with primary antibody and horseradish peroxidase conjugated anti-mouse or -rabbit IgG (Sigma) and then detected with chemiluminesence reagent (PerkinElmer Life Sciences). In some cases, immunoblots were stripped and reblotted with mAbs or pAbs according to the manufacturer's instruction.

Cell Migration Assay—The cell motility assays were performed as described (43) using modified Boyden chambers with Transwell membrane filter inserts in a 24-well tissue culture plate (Corning Costar Corp., Cambridge, MA). The Transwell filters were 6.5-mm in diameter, 8-µM pore size, 10-µM thick polycarbonate membrane. FN (10 µg/ml) or LN-5 (2 µg/ml) was coated on the lower surface of the Transwell inserts at 4 °C overnight and then blocked with 0.1% heat-inactivated bovine serum albumin (BSA) at 37 °C for 45 min before adding cells. Cells were serum-starved overnight and resuspended in serum-free DMEM medium containing 0.1% heat-inactivated BSA and then added to each insert or the top well. For haptotatic migration assays, the migration medium containing serum-free DMEM and 0.1% heat-inactivated BSA was added to the bottom wells of the chambers. For chemohaptotactic migration assays, the migration buffers put in the bottom wells contained either 1% fetal calf serum, EGF (100 ng/ml), or platelet-derived growth factor (PDGF)-BB (40 ng/ml) in addition to plain DMEM and 0.1% heat-inactivated BSA. After overnight incubation at 37 °C, the cells that had not migrated through the filter were removed with cotton swabs from the upper face of the filter, and cells that had migrated to the lower surface of the filters were fixed and stained with Diff-Quick (Baxter Healthcare Corp., McGraw Park, IL). The number of cells per field was counted under a light microscope at magnification x40.

Gene Expression Profile Analysis—Total RNA from Du145-Mock and Du145-CD82 cells was prepared using TRIZOL reagent (Invitrogen) by following the manufacturer's instruction. RNA integrity was assessed by using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). Poly(A)+-RNA was purified from total RNA with Oligotex latex beads (Qiagen). cDNA was synthesized using a T-7 linked oligo(dT) primer, and cRNA was then synthesized with biotinylated UTP and CTP. The labeled cRNA was then fragmented and hybridized onto Affymetrix GeneChip Human Genome U133 probe microarray set (Affymetrix Inc., Santa Clara, CA), which represents over 33,000 known human genes and ESTs, by following Affymetrix protocols (44, 45). Arrays were scanned using a laser confocal scanner (Agilent).

Raw data were analyzed using Affymetrix GeneChip software v.4.0 (for explanation of quantitative analysis, see 44). The probe set intensity (average intensity difference) was proportional to the abundance of the specific mRNA it represented and was calculated by comparing hybridization signal of the perfect match oligonucleotide to that of the mismatch oligonucleotide and averaged over a set of 20 specific oligonucleotide pairs for each gene. Total signal intensity of different probe arrays was scaled to the same value before comparison. Fold change was calculated using the Affymetrix GeneChip software by pairwise comparison of corresponding probe pairs from KAI1/CD82 and Mock probe arrays. To account for high noise at low signal intensities, genes for which the average difference values were below 100 throughout the experiment (in both KAI1/CD82 and Mock) were eliminated from the analysis. To avoid saturation effects at high signal intensities, genes with average differences larger than 10,000 throughout the experiment were also eliminated.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
KAI1/CD82 Inhibits the Migration of Du145 Metastatic Prostate Carcinoma Cells—Consistent with previous observations (1, 2), Du145, a metastatic prostate cancer cell line (46), barely expressed any KAI1/CD82 (Fig. 1). To investigate the effect of KAI1/CD82 expression on cell motility, the stable KAI1/CD82 transfectant of Du145 cell was established by pooling and sorting multiple KAI1/CD82-expressing clones with flow cytometry. As shown in Fig. 1, the cell surface expression level of KAI1/CD82 in the Du145-KAI1/CD82 transfectant was much higher than that in the Du145-Mock transfectant. Integrin {alpha}3{beta}1, a receptor of laminin 5, and integrin {alpha}5{beta}1, a receptor of fibronectin, showed equivalent expression on both transfectants and served as positive controls (Fig. 1). Other TM4SF proteins such as CD81 and CD151 were also equivalently expressed on the cell surfaces of both transfectants, and their levels were not altered by the KAI1/CD82 expression in Du145 cells (data not shown).



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FIG. 1.
Restoration of the KAI1/CD82 expression in Du145 metastatic prostate cancer cells. The KAI1/CD82 stable transfectant was established as described under "Experimental Procedures." KAI1/CD82-expressing Du145 cells and Du145-Mock transfectant cells were detached, incubated with a negative control mAb P3, an integrin {alpha}3 mAb X8, an integrin {alpha}5 mAb PUJ2, or a KAI1/CD82 mAb M104, and then stained with a FITC-conjugated second Ab. The stained cells were analyzed by flow cytometry. The mean fluorescence intensity of KAI1/CD82 staining for Du145-Mock and Du145-KAI1/CD82 was 1.5 and 20.2, respectively.

 

The Du145 transfectants were then tested for their directional motility on FN in a transwell haptotactic migration assay. As shown in Fig. 2A, the ability of Du145-KAI1/CD82 cells to migrate toward ECM was remarkably reduced compared with that of the Du145-Mock cells. The diminished haptotactic cell migration in Du145-KAI1/CD82 cells was observed on both FN- and LN5-coated substrata (Fig. 2A), indicating that Du145-KAI1/CD82 has general suppressive effects on cell migration mediated by both FN- and LM-binding integrins such as {alpha}5{beta}1 and {alpha}3{beta}1, respectively. Also, cell migration on LN1, a ligand for {alpha}6 integrins, was substantially diminished (data not shown).



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FIG. 2.
The KAI1/CD82 expression attenuates the migration of Du145 cells. A, KAI1/CD82 inhibits haptotactic cell migration on fibronectin and laminin. Haptotactic migration of the Du145 transfectants was measured using the transwell inserts coated with FN (20 µg/ml) or LN5 (2 µg/ml). The migration medium in top and bottom wells was DMEM containing 0.1% heat-inactivated BSA and antibiotics. Cells that migrated onto the lower surface were fixed, stained, and photographed. In each individual experiment, cells that migrated through the filters were counted from at least three randomly selected fields. Results were obtained from at least three individual experiments and represented as the cell migration index, which is the number of cells per a high power field. The bar graph represent means ± S.D. p < 0.001 on both FN and on LN5 between the Mock and KAI1/CD82 transfectants. B, KAI1/CD82 inhibits chemohaptotactic cell migration. Chemohaptotactic migration assays were carried out toward chemoattractants on FN (20 µg/ml)-coated filters in the medium of the bottom well. The chemoattractants in the medium of the bottom well were 1% fetal calf serum, EGF (100 ng/ml), or PDGF-BB (40 ng/ml). The results were obtained from three experiments (mean ± S.D.). p < 0.001 toward FCS, EGF, or PDGF between the Mock and KAI1/CD82 transfectants.

 

The defect in cell migration upon KAI1/CD82 expression was also observed in chemohaptotactic migration, i.e. cells migrated toward both the chemoattractant gradient and immobilized gradient. In Fig. 2B, the KAI1/CD82 transfectant still migrated significantly less on FN toward fetal calf serum, but the difference between Mock and CD82 cells was not as large as in the absence of serum, while cell migration on FN toward EGF or PDGF-BB was also substantially decreased upon the KAI1/CD82 expression (Fig. 2B).

Up-regulation of FAK and Lyn Proteins—The goal of this study was to identify the signaling pathways responsible for KAI1/CD82-mediated suppression of cell motility. Regulation of cell motility is a complex process that varies among cell types; differs under individual cellular environments; and involves many signaling molecules such as FAK, Src, p130CAS, Crk, MAP kinases, PI-3 kinase, Rho small GTPases, and phosphatases (2837, 4750). Since 1) TM4SF proteins physically interact with integrins (21), 2) the FAK-Src-p130CAS-Crk pathway plays important roles in integrin-mediated signaling (37, 5155), and 3) the FAK and Src signaling are involved in integrin-mediated motility of prostate cancer cells (48, 56), we investigated effects of KAI1/CD82 expression on the FAK-Src-p130CAS-Crk signaling pathway in Du145 cells.

First, we found that the FAK protein level was substantially enhanced in the KAI1/CD82 transfectant cells compared with the Mock transfectant cells (Fig. 3A). The increase was observed when the cells were either kept in suspension (Fig. 3A) or spread on FN- or LN-coated substratum (data not shown). FAK proteins in Du145-KAI1/CD82 cells are about 2.5-fold more than those in Mock cells (Fig. 3A, bar graph). Pyk2, a FAK homologue, remains equivalent between the Mock and KAI1/CD82 transfectants (Fig. 3A). The {beta}-tubulin was used as a control in these experiments to demonstrate equal protein loading (Fig. 3A). To determine if this difference was due to the differential expression of the FAK gene, we compared FAK gene expression at the RNA level between the Mock and KAI1/CD82 cells by using gene expression profiling technology. According to the statistical analysis of the microarray data, the FAK RNA in Du145-KAI1/CD82 cells was significantly elevated compared with that in Du145-Mock cells (Table I), indicating that FAK gene expression was up-regulated upon KAI1/CD82 expression and the elevated FAK protein in Du145-KAI1/CD82 cells resulted from an increased expression of the FAK gene. The Pyk2 gene expression remained unchanged upon KAI1/CD82 expression as did housekeeping genes such as actin and tubulin (Table I).



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FIG. 3.
KAI1/CD82 regulates the level and activation of FAK. A, FAK was increased at the protein level upon KAI1/CD82 expression. The Du145-Mock and -KAI1/CD82 cells were lysed in RIPA buffer. The expression of FAK, Pyk2, and {beta}-tubulin proteins were analyzed in Western blot using their specific mAbs. Blots show results from a single representative experiment; the graph represents the relative density of the FAK band summarized from five individual experiments (mean ± S.D.), based on the densitometric analyses. p value between Mock and KAI1/CD82 is < 0.01. B, the effect of KAI1/CD82 expression on tyrosine phosphorylation of FAK. Du145-Mock and -KAI1/CD82 cells were spread on FN (10 µg/ml)-coated plates at 37 °C, 5% CO2 for 30 min and then were lysed with RIPA buffer. FAK was immunoprecipitated with its mAb in 24 hr thorough immunoprecipitations at 4 °C. The immunoprecipitates were blotted with anti-phosphotyrosine mAb (4G10) after SDS-PAGE separation and electrotransfer. The membrane was then stripped and reblotted with FAK mAb. Aliquots of cell lysate were loaded on SDS-PAGE in equal amounts and Western blotted with PKC{alpha} mAb to demonstrate equal loading. C, the effect of KAI/CD82 on FAK autophosphorylation. The Mock and KAI1/CD82 transfectant cells were either kept in suspension or allowed to spread on FN (10 µg/ml)-coated plate at 37 °C, 5% CO2 for 30 min and then were lysed with RIPA buffer. The same amount of cell lysate was Western blotted with the antibody against FAK, FAK phosphorylated tyrosine residue 397, and {beta}-tubulin. D, the effect of KAI/CD82 on FAK maximal activity. As described in C, the transfectant cells were spread on FN (10 µg/ml)-coated plate and then lysed. The lysate was analyzed by Western blot using FAK, FAK pTyr 576, or {beta}-tubulin.

 

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TABLE I
Comparison of the gene expression of the FAK-Src-p130CAS-Crk pathway components between the Du145-Mock and Du145-CD82 transfectant cells

 

Upon integrin-ECM interaction, FAK undergoes autophosphorylation on tyrosine residue 397 and then becomes partially activated (5759). Phosphorylation of FAK Tyr-397 creates a high-affinity binding site for SH2 domains of the Src family tyrosine kinases such as c-Src and Fyn (5759). Subsequently, the FAK-bound Src family kinases phosphorylate other tyrosine residues in FAK such as Tyr-576 and Tyr-577 (57, 58, 60). The phosphorylation of Tyr-576/577 is required for maximal FAK activity (60). Thus, we examined the functional status of FAK by measuring tyrosine phosphorylation of FAK. First, total tyrosine phosphorylation of FAK was analyzed. When FAK proteins from the same amount of cell lysate were compared, no difference in total tyrosine phosphorylation level was found between Du145-Mock and Du145-KAI1/CD82 cells (Fig. 3B), indicating that, although the FAK protein level in Du145-KAI1/CD82 cells was enhanced, the global tyrosine phosphorylation of FAK was not elevated in KAI1/CD82 transfectant cells. PKC{alpha}, a ubiquitously expressed protein, was analyzed by Western blot to demonstrate that equal amounts of cell lysate were used for the experiment (Fig. 3B). Second, we compared FAK autophosphorylation in Western blot analyses using a FAK Tyr-397 phosphorylation-specific antibody. When equal amounts of lysate protein were evaluated, the tyrosine 397 of FAK was equally phosphorylated between Du145-Mock and -KAI1/CD82 cells after the cells were spread on FN-coated substratum for 60 min (Fig. 3C). We also obtained similar results at other time points such as 30 min and 120 min (data not shown). Finally, the full activation of FAK was assessed by measuring the phosphorylation of FAK Tyr-576/577 in Western blot experiments. The phosphorylation of FAK tyrosine 576 remained unchanged in Du145-KAI1/CD82 cells after the cells were spread on FN-coated substratum for 60 min, when equal amounts of cell lysate were compared from the Mock and KAI1/CD82 transfectant cells (Fig. 3D). Similar results were observed when cells were spread for 30 and 120 min (data not shown).

To determine the role of Src family tyrosine kinases in KAI1/CD82-mediated inhibition of cell migration, we analyzed the expression and activity of Src kinases. As mentioned earlier, the Src family tyrosine kinases are not only required for the full activation of FAK but they are also directly responsible for FAK activation-induced tyrosine phosphorylation of p130CAS. Src and Fyn, two ubiquitously expressed members in the family, were not expressed in Du145 cell line (Table I). Lyn and Yes are the only Src kinases expressed in Du145 cells (Table I). The absence of Src and Fyn was confirmed by Western blot analyses using specific antibodies (data not shown). In Du145 cells, the expression of Lyn was significantly up-regulated at both RNA and protein levels in Du145-KAI1/CD82 cells (Fig. 4A and Table I). In contrast to Lyn, we found that Yes remained unchanged between the Mock and KAI1/CD82 transfectant cells at both RNA and protein levels (Fig. 4A and Table I). The activation of Lyn was analyzed by measuring the phosphorylation of its tyrosine residue 397 (61). To compare the Lyn activities, all of the Lyn proteins in lysates were collected through 24-hr thorough immunoprecipitations using the same amounts of cell lysate from the Mock and KAI1/CD82 transfectant cells. After the immunoprecipitation, no Lyn protein was detectable in the lysate (data not shown), indicating that all Lyn proteins were isolated by immunoprecipitation. Then, Lyn activity was measured by detecting the phosphorylation level of Lyn Tyr-397. The total Lyn activity in Du145-KAI1/CD82 cells was actually equivalent to that in Du145-Mock cells, although, as expected, more Lyn protein was collected from Du145-KAI1/CD82 cells (Fig. 4B). The functional status of Yes, by measuring the phosphorylation of the tyrosine residue 426, was not altered upon KAI1/CD82 expression (Fig. 4B).



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FIG. 4.
The effects of KAI1/CD82 on Src family tyrosine kinases. A, the Dul45-Mock and -KAI1/CD82 cells were lysed in RIPA buffer. The expression of Lyn, Yes, and {beta}-tubulin proteins were analyzed by Western blot using their specific mAbs. Blots show results from a single representative experiment; the graph represents the relative density of the Lyn band summarized from five individual experiments (mean ± S.D.), based on the densitometric analyses. p value between Mock and KAI1/CD82 is <0.01. B, the Mock and KAI1/CD82 transfectant cells were lysed with 1% Nonidet P-40, and the Lyn proteins were immunoprecipitated from the same amount of cell lysate at 4 °C for 24 h. After SDS-PAGE and electric transferring, the precipitated Lyn proteins were immunoblotted by the Lyn Ab and then by the mAb specific for Lyn pTyr 397 after stripping the membrane. The cell lysates were also detected for PKC{alpha} by Western blot to demonstrate an equal loading between the Mock and KAI1/CD82 transfectant cells. The Yes proteins were analyzed in the same way as described for Lyn.

 

The p130CAS-CrkII Coupling Is Down-regulated by KAI1/CD82—Among the substrates of FAK and Src family kinases, p130CAS directly regulates cell motility. Thus, we analyzed the level and functional status of this major downstream target. The level of p130CAS protein was markedly reduced in the KAI1/CD82-expressing Du145 cells (Fig. 5, A and B). The reduced p130CAS level was observed when Du145-KAI1/CD82 cells were either kept in suspension (Fig. 5B) or spread on FN- and LN-coated plates (Fig. 5B), indicating that the down-regulation of p130CAS by KAI1/CD82 was constitutive but not induced by the integrin-ECM engagement. Paxillin (Fig. 5A) and PKC{alpha} (Fig. 5B) served as internal controls in Western blots to show equal loading of cell lysates. However, the RNA of p130CAS was at equivalent levels, based on the statistical analysis of microarray data, between Du145-Mock cells and Du145-KAI1/CD82 cells (Table I), indicating that the p130CAS proteins were likely reduced post-transcriptionally. The densitometric analyses of the p130CAS protein bands indicated that decrease of the p130CAS protein in Du145-KAI1/CD82 cells was about 2–4-fold and highly statistically significant (bar graph in Fig. 5, A and B). The p130CAS homologues HEF-1/CAS-L and Efs2/Sin were not expressed in Du145 cells (Table I).



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FIG. 5.
KAI1/CD82 down-regulates p130CAS. A and B, a reduced p130CAS level in KAI1/CD82-expressing Du145 cells. The lysate was generated when the transfectant cells were either kept in suspension (A) or spread on the FN (10 µg/ml)- or LN1 (10 µg/ml)-coated plate (B) in serum-free DMEM medium at 37 °C for 60 min. Equal amounts of cell lysate from Du145-Mock and -KAI1/CD82 transfectants were resolved by SDS-PAGE. After electrotransfer, the filter was blotted either with p130CAS mAb and paxillin mAb (A) or with p130CAS mAb and PKC{alpha} mAb (B). Paxillin and PKC{alpha} signals serve as loading controls to demonstrate equal loading. Paxillin is typically seen in Western blots as a smear. Blots show results from a single representative experiment; graphs represent the relative density of bands summarized from four or five individual experiments (mean ± S.D.) based on densitometric analyses. All p values between Mock and KAI1/CD82 are < 0.01. C, the lysates generated from the cells that attached to FN (10 µg/ml)-coated plates were immunoprecipitated with p130CAS mAb. The loading of p130CAS precipitates on SDS-PAGE was justified to achieve equal loading. After electrotransfer, the precipitates were immunoblotted with phosphotyrosine mAb 4G10 and then with p130CAS mAb after stripping the filter membrane. D, after the indicated pretreatments, p130CAS proteins were collected from the same amount of cell lysates in 24-h thorough immunoprecipitations and then detected sequentially with pTyr and p130CAS mAbs as described in C. Aliquots of cell lysates were loaded on SDS-PAGE in equal amounts and Western blotted with {beta}-tubulin mAb to demonstrate equal loading.

 

The substrate domain of the p130CAS protein undergoes FAK/Src-dependent tyrosine phosphorylation upon integrin engagement (37, 57). Therefore, tyrosine phosphorylation of the p130CAS protein directly reflects integrin-induced FAK/Src activities. We found that the tyrosine phosphorylation of p130CAS remained unchanged between the Mock and KAI1/CD82 cells when compared with equal amounts of the p130CAS protein (Fig. 5C). This result suggests that the FAK/Src activity required for p130CAS phosphorylation appears not to be altered between Du145-Mock and Du145-KAI1/CD82 cells. However, when equal amounts of the cell lysate were compared, not surprisingly, p130CAS in total has lower tyrosine phosphorylation in Du145-KAI1/CD82 cells (Fig. 5D), which may be caused by lower total p130CAS protein.

After tyrosine phosphorylation, some p130CAS proteins form complexes with CrkII through the SH2 domain of CrkII (37). FAK/Src-dependent p130CAS-CrkII coupling is required to induce cell migration (22, 30, 31). Decreased p130CAS in total levels, as well as in the tyrosine-phosphorylated form, predicts that fewer p130CAS-CrkII complexes will be formed, which is critical for cell movement (30, 31). To address this issue, we first analyzed the expression of CrkII and found that CrkII remained unchanged at the protein and RNA levels upon KAI1/CD82 expression (Fig. 6A and Table I). In Fig. 6A, RACK1, a ubiquitously expressed protein, served as a loading control. Second, we investigated the formation of p130CAS-CrkII complexes by detecting the presence of p130CAS proteins in CrkII immunoprecipitates. The p130CAS and CrkII proteins were indeed evidently less coupled in Du145-KAI1/CD82 cells (Fig. 6B). The reduction of p130CAS-CrkII complexes is about 2-fold and highly significant (bar graph in Fig. 6B). Since CrkII remained constant in both transfectants, fewer p130CAS-CrkII complexes upon KAI1/CD82 expression likely resulted from the low level of p130CAS in Du145-KAI1/CD82 cells. The reduced p130CAS-CrkII coupling accompanied by the phenotype of decreased cell migration fits very well with the notion that the p130CAS-Crk pathway is the molecular switch of cell movement (30).



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FIG. 6.
KAI1/CD82 down-regulates the formation of p130CAS-CrkII complex. A, CrkII proteins remained unchanged upon KAI1/CD82 expression. The transfectant cells attached to FN (10 µg/ml)- or LN1 (10 µg/ml)-coated plates were lysed in RIPA buffer, the cell lysates were Western blotted with a CrkII mAb or a RACK1 (receptor of activated C kinase 1, a ubiquitously expressed protein) mAb. B, formation of p130CAS-CrkII complex was decreased in KAI1/CD82-expressing Du145 cells. After removing the culture medium, the attached transfectant cells were lysed in RIPA buffer. The lysates were immunoprecipitated with CrkII mAb and then immunoblotted with p130CAS mAb. After being stripped, the filter membrane was reblotted with CrkII mAb. Blots show results from a single representative experiment; the graph represents the relative density of bands summarized from four individual experiments (mean ± S.D.), based on densitometric analyses. p value between Mock and KAI1/CD82 is < 0.01.

 

p130CAS Reverses KAI1/CD82-mediated Suppression of Cell Migration—To confirm that lack of sufficient p130CAS proteins or the p130CAS-CrkII complex is required for KAI1/CD82-mediated suppression of cell motility, we overexpressed the p130CAS protein in both Du145-KAI1/CD82 and Du145-Mock cells. After overexpression, the level of p130CAS in Du145-KAI1/CD82 cells recovered to a level comparable to that in Mock cells (Fig. 7A) and so did the formation of p130CAS-CrkII complexes (Fig. 7B). These p130CAS-elevated Du145-KAI1/CD82 cells regained robust motility on FN (Fig. 7C). Cell migration of the p130CAS-overexpressing KAI1/CD82 cells was not statistically different from that of the p130CAS-overexpressing Mock cells (Fig. 7C). However, the average cell migration index of the p130CAS-overexpressing KAI1/CD82 cells was still about 75% of the p130CAS-overexpressing Mock cells (Fig. 7C). This result indicates that reduced levels of p130CAS and p130CAS-CrkII complex are largely responsible for KAI1/CD82-induced suppression of Du145 cell motility.



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FIG. 7.
An elevated p130CAS level reverses KAI1/CD82-induced reduction of Du145 cell motility. A, overexpression of p130CAS in Du145 transfectants. The p130CAS cDNA was transiently transfected into Du145-Mock and Du145-KAI1/CD82 transfectant cells. The cells were harvested at 48 h after transfection and then lysed. Equal amounts of cell lysates were immunoblotted with p130CAS and CrkII mAbs. B, formation of p130CAS-CrkII complex in p130CAS-overexpressing Du145 cells. The Du145 transfectant cells that were transiently expressing p130CAS were lysed with RIPA buffer. CrkII was immunoprecipitated with its mAb, and the CrkII precipitates were immunoblotted with p130CAS mAb. C, effects of p130CAS overexpression on the cell migration of Du145 transfectants. The haptotactic cell migration on FN (20 µg/ml) was measured with the Du145-Mock and Du145-KAI1/CD82 cells transfected with either p130CAS cDNA or vector alone.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Decreased Cell Motility Is Likely to Be the Cellular Mechanism of CD82-mediated Suppression of Cancer Metastasis— Cellular invasiveness is an enhanced or aberrant cell movement and a major characteristic of poorly differentiated cancers (62). In metastatic cancers, the cancer cells usually acquire the ability to invade surrounding tissues and then metastasize to secondary lesions (63). So, the aberrant motile feature of cancer cells is responsible for the initial phase of cancer metastasis. Cell motility and/or invasiveness, therefore, is one of the multiple steps that a cancer metastasis suppressor inhibits. Studies elsewhere have demonstrated that CD82 inhibits the invasiveness of breast cancer cell MDA-MB-231 and colon cancer cell DLD-1 (11, 16). For the first time, our study showed that CD82 attenuated the directional cell migration of prostate cancer cells in vitro, although CD82 was originally identified as a metastasis suppressor in prostate cancer (1). In terms of CD82-mediated motility inhibition, our result from Du145 cells is consistent with other observations from colon cancer cell DLD-1, mammary epithelial cell HB2, and CHO cell ldlD-14 (11, 14, 26). Thus, decreased cell motility and invasiveness likely play a major role in CD82-mediated suppression of cancer metastasis.

CD82-mediated suppression of cell migration is not limited to a specific integrin-ECM interaction. In the ECM proteins tested in this study, from LN, the constituent of basement membrane, to FN, a component of interstitial tissue, CD82 exerts global inhibitory effects on cell movement, which fits with the complicated surrounding ECM environment that invasive cancers encounter in vivo. Meanwhile, CD82 suppresses cell migration triggered by EGF and PDGF, indicating that CD82 intercepts the signaling that controls cell motility from both integrins and growth factors or acts on steps common to both integrin and growth factor signaling pathways. A previous study found that CD82 inhibited EGF-induced cell migration (26). In Du145 cells, CD82 apparently inhibits not only EGF-induced but also PDGF-induced cell migration.

CD82 Alters the FAK-Src-p130CAS-Crk Signaling—The FAK-Src-p130CAS-Crk pathway plays critical roles in cell migration and invasiveness (37, 55, 57, 58). This pathway is required for the directional persistency of cell migration during haptotaxis and chemotaxis (28, 29). In this study, we demonstrated that CD82 suppresses cell migration through the FAK-Src-p130CAS-Crk pathway. CD82 intercepts the signals transduced through this pathway by down-regulating the p130CAS protein level and subsequent p130CAS-Crk coupling. Although FAK and Lyn protein levels were elevated in Du145-CD82 cells, the FAK and Lyn activation seem to be unchanged upon CD82 expression. Since the p130CAS-Crk complex is situated downstream of the activation of FAK/Src family kinases and p130CAS is the major FAK/Src substrate that is closely involved in cell migration, less p130CAS-Crk coupling is apparently more directly responsible for CD82-mediated suppression of cell motility.

FAK has been implicated in TM4SF signaling (64, 65). The functional status of FAK is altered upon clustering of cell surface TM4SFs (64). FAK signaling pathway is down-regulated in CD9 knockout mice (65). In this study, CD82 signaling increased FAK expression but did not alter FAK activation. The interpretation is that Du145-CD82 cells probably already have all the activated FAK that they need to function properly, and no more FAK proteins are required to translate into the activated from. But many studies have shown that FAK promotes cell migration, and the FAK level and activity parallel with cell motility (57, 58, 66, 67). In Du145-CD82 cells, however, an increased level of FAK proteins was accompanied by the inhibited cell motility. Thus, another interpretation is that FAK is likely upstream of what CD82 targets and that FAK expression is up-regulated to compensate either the compromised FAK activity or the downstream defect. After compensation, FAK activities, including the cell adhesion-induced autoactivation and the Src-dependent full activation, in Du145-CD82 cells reached a level comparable with that in the Mock cells. Since we used stable transfectants in this study, the original defect in FAK activities resulting from CD82 expression may no longer be obvious after a long-term selection process. Both interpretations also apply to our results with Lyn.

The Src family tyrosine kinases transduce the signals derived from FAK activation downstream by phosphorylating tyrosine residues in the substrate domain of p130CAS (37, 5760). In Src family, Src and Fyn have been reported to bind FAK and then use FAK as a docking site to further phosphorylate p130CAS (37, 57, 58). However, Du145 cells do not express Src and Fyn; the Src kinase(s) responsible for this process is likely to be Lyn and/or Yes. Although no study indicates that Lyn transduces the signal from FAK to p130CAS, Lyn reportedly complexes with FAK (68). Since the total activities of Lyn and/or Yes are equivalent between the Mock and CD82 cells, the ability to phosphorylate p130CAS was not changed upon CD82 expression.

The phosphorylation of p130CAS by FAK/Src, which are activated by integrin and growth factor, creates binding sites for adaptor proteins such as CrkII (37, 57, 66). The p130CAS-CrkII complexes are formed through the direct binding of the CrkII SH2 domain with the tyrosine-phosphorylated p130CAS. The FAK- and Src-dependent p130CAS-CrkII coupling is required for inducing cell migration (2831). The co-overexpression of p130CAS and CrkII causes enhanced cellular motility in a process requiring the CrkII SH2 domain (30). The p130CAS(-/-) fibroblast displays reduced cell migration (69). It appears that cells need to maintain a certain level of p130CAS in order to migrate well. Therefore, sufficient tyrosine phosphorylation and an adequate level of p130CAS proteins are important for cell migration. Upon CD82 expression, the formation of fewer p130CAS-Crk complexes could result from either a lower level of the p130CAS protein or less tyrosine phosphorylation of p130CAS. Apparently, an insufficient amount of p130CAS protein rather than less phosphorylation caused the formation of fewer p130CAS-Crk complexes. No alteration in tyrosine phosphorylation of p130CAS upon CD82 expression indicates that sufficient FAK/Lyn activities had been generated in Du145-CD82 cells to give rise to sufficient p130CAS phosphorylation. In Du145-CD82 cells, the reduced level of p130CAS and subsequently less p130CAS-Crk coupling, which are accompanied by the decreased migration phenotype, fit very well with the notion that the p130CAS-Crk complex is a molecular switch for cell movement (30).

How CD82 down-regulates p130CAS proteins is a critical and interesting question. The immediate cause for the decreased p130CAS level is unclear. There is no clear link between the up-regulation of FAK and Lyn proteins and the decreased level of p130CAS. Also, no evidence suggests that FAK and Lyn activities are involved in the down-regulation of p130CAS. The transcription of p130CAS gene was not significantly changed upon CD82 expression, though we cannot exclude the possibility that a significant alteration in translation of p130CAS resulted from this insignificant alteration in transcription. It is more likely that the turnover of the p130CAS protein was affected by CD82; for example, a lower translational rate or a higher degradation rate could result in fewer p130CAS proteins in CD82 transfectant cells. Since CD82 accelerates the turnover of EGF receptor (26), fewer p130CAS proteins in Du145-CD82 cells could be due to an accelerated turnover rate of p130CAS. Whether the degradation of p130CAS proteins is accelerated by CD82 needs to be determined by future studies.

Although the overexpression of p130CAS largely rescued the CD82-inhibited cell migration, it could not fully reverse the inhibition. It appears that the formation of fewer p130CAS-Crk complexes is partially responsible for diminished cell migration in Du145-CD82 cells. Therefore, CD82 may also affect the downstream step(s) of the p130CAS-Crk pathway such as Rac1 or other signaling pathway(s) that controls cell migration. For example, the p130CAS-Crk coupling will recruit Dock180 into the complex and then facilitate the activation of Rac1 (70). Although less p130CAS-Crk coupling will eventually down-regulate the Rac1 activity, Rac1 could be inhibited directly by CD82 signaling. Also, the phosphorylation of FAK Tyr-397 was found to recruit PI-3K into the complex (35). PI-3K-Akt signaling has been implicated in TM4SF signaling (19, 20); meanwhile, it is also important for cell migration (3436). Whether or not this pathway contributes to CD82-mediated inhibition of cell motility remains to be addressed.

The Role of Integrin and Growth Factor in CD82-mediated Suppression of Cell Motility—Since TM4SF proteins associate with integrin and the FAK-Src-p130CAS-CrkII pathway plays an important role in integrin signaling, integrin is very likely involved in the CD82-mediated suppression of cell motility. CD82 has been found in TM4SF-integrin complexes (71, 72), though reportedly CD82 interacts with integrin indirectly through other TM4SFs such as CD81 (73). So, on the one hand, CD82 could act on integrin per se as they are located so proximal to each other. For example, since some CD82 proteins are localized in the intracellular vesicle compartments (74, 75) and CD82 shuttles between plasma membrane and endosomal/lysosomal compartment,2 CD82 may directly regulate integrin trafficking to modulate cell adhesiveness and motility. On the other hand, CD82 may act downstream of integrin signaling as we have observed in this study. The alteration in FAK expression suggests that CD82 regulates integrin signaling. Interestingly, growth factors also solicit the FAK-Src-p130CAS-CrkII pathway. For example, FAK rapidly undergoes tyrosine phosphorylation upon growth factor stimulation (76), and Src kinases are readily activated by growth factors (77, 78). Since CD82 attenuates EGF signaling (25), altered FAK/Lyn levels could also result from the inhibition of growth factor-initiated signaling. Multiple lines of evidence indicate that integrins and growth factors are likely integrated into macrotransmembrane complexes and integrin signaling interweaves with the signaling initiated by growth factors (5254). For example, FAK links between growth factor receptor and integrin signaling pathways to promote cell migration (15). Therefore, CD82 likely intercepts either both signaling pathways or the common steps of both pathways and ultimately inhibits cell migration.


    FOOTNOTES
 
* 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

{ddagger} To whom correspondence should be addressed: Vascular Biology Center of Excellence, UTHSC, Coleman H300, 956 Court Ave., Memphis, TN 38163. Tel.: 901-448-3448; Fax: 901-448-7181; E-mail: xazhang{at}utmem.edu.

1 The abbreviations used are: TM4SF, transmembrane 4 superfamily; BSA, bovine serum albumin; CAS, Crk-associated substrate; ECM, extracellular matrix; EGF, epidermal growth factor; FAK, focal adhesion kinase; FN, fibronectin; LN, laminin; mAb, monoclonal antibody; MFI, mean fluorescence intensity; pAb, polyclonal antibody; PDGF, platelet-derived growth factor; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pTyr, phosphorylated tyrosine; MAP, mitogenactivated protein; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; RIPA, radioimmune precipitation assay buffer; FITC, fluorescein isothiocyanate. Back


    ACKNOWLEDGMENTS
 
We are grateful for discussions with and suggestions by Drs. L. Jennings, A. Hassid, E. Geisert, and D. Armbruster. We also thank Drs. M. Hemler, C. Stipp, C. Claas, and R. Klemke for the reagents used in this study.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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