©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell Spreading in Colo 201 by Staurosporin Is 31 Integrin-mediated with Tyrosine Phosphorylation of Src and Tensin (*)

(Received for publication, February 28, 1994; and in revised form, October 17, 1994)

Masafumi Yoshimura (1) (2) Atsushi Nishikawa (1) Tetsuo Nishiura (2) Yoshito Ihara (1) Yoshio Kanayama (2) Yuji Matsuzawa (2) Naoyuki Taniguchi (1)(§)

From the  (1)Department of Biochemistry and the (2)Second Department of Internal Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Staurosporin, a broad-spectrum kinase inhibitor, induced cell spreading in a human colon cancer cell line, Colo 201. On collagen and laminin, cell spreading was induced in more than 90% of the cells and was dependent on very late activation antigen-3, as shown by an antibody inhibition assay. Cell spreading required divalent cations and showed the order of preference Mn > Mg > Ca. On fibronectin, only about 30% of the cells were observed to spread, and spreading occurred via a non-integrin, RGD-independent pathway. Staurosporin-induced spreading was inhibited by treatment with tyrosine kinase inhibitors herbimycin A and methyl 2,5-dihydroxycinnamate. Despite the presence of staurosporin, seven proteins (220, 175, 150, 98, 62, 58, and 45 kDa) showed increased levels of tyrosine phosphorylation in association with cell adhesion. Two of these (58 and 220 kDa) were identified by immunoprecipitation as Src product and tensin, respectively. Flow cytometric analysis showed that the Colo 201 cells expressed the alpha2, alpha3, alpha6, and beta1 chains of integrin, but expression of these chains was not influenced by staurosporin. Immunofluorescence microscopy revealed that the alpha3 chain, diffusely expressed on the cell surface in the absence of staurosporin, was concentrated at focal adhesion plaques after staurosporin treatment. Neither alpha2 nor alpha6 was focalized by the treatment.


INTRODUCTION

Adhesion to extracellular matrix (ECM) (^1)regulates cell growth, differentiation, development, and tumorigenesis(1, 2, 3, 4) . Contact with the ECM is in part mediated through a family of heterodimeric surface molecules called integrins. A heterodimer composed of one of several alpha subunits and a beta1 subunit acquires the ability to interact with collagen, fibronectin, and laminin in clusters on the cell surface called focal adhesion sites(5, 6) . The short cytoplasmic tails of integrins are associated with talin, vinculin, and alpha actinin, and still other proteins probably link integrins with actin-containing cytoskeletons(7, 8) . Therefore, integrins, located between the extracellular matrix and the cytoskeleton, transmit biological signals into the cell, thereby influencing the modulation of intracellular pH (9) , activation of T-lymphocytes (10) and neutrophils(11) , and tumorigenicity(12) . Many studies have shown that tyrosine kinases and protein kinase C play critical roles in signal transduction through integrins. Tyrosine phosphorylation of very late activation antigen (VLA)-5 decreased during retinoic acid-induced differentiation of teratocarcinoma cells(13) , and tyrosine phosphorylation of specific proteins increased in association with alphaIIbbeta3-mediated platelet aggregation induced by thrombin(14) . Phorbol ester-induced cell adhesion in a human leukemia cell line was triggered by protein kinase C and mediated by VLA-5(15) . In addition, cross-linking of integrins on the cell surface with antibodies (16) has been shown to cause tyrosine phosphorylation of proteins, including focal adhesion kinase (17) , paxillin(17) , and tensin(18) , that are in contact with the short cytoplasmic tail of integrin and the cytoskeleton at the focal adhesion sites.

Colo 201, isolated from an ascites of an individual with carcinoma of the colon(19) , is a unique cell line in that it has lost its epithelial appearance and exhibits a round, floating, non-adherent morphology. Cell-to-cell and cell-to-matrix interactions have probably been lost in Colo 201 due to disruption of the adhesion system and the connection between focal adhesion sites and the cytoskeleton during tumor progression and metastasis.

We have been investigating the effects of kinase modulators on cellular adhesion and spreading and have found that staurosporin, a broad spectrum kinase inhibitor, induced VLA-3-mediated cell spreading in Colo 201. The cell spreading was accompanied by elevation of tyrosine phosphorylation in a set of intracellular proteins despite the presence of staurosporin.


EXPERIMENTAL PROCEDURES

Cell Lines and Reagents

A human colon cancer cell line, Colo 201, was obtained from the Japanese Cancer Research Resources Bank. HT1080 (human fibrosarcoma) was obtained from the American Type Culture Collection. These cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Nalgene, Victoris, Australia) and antibiotics. Bovine collagen type I and polylysine were purchased from Collaborative Research, and human plasma fibronectin and mouse laminin produced by Engelbreth-Holm-Swarm sarcoma were from Life Technologies, Inc. A synthetic peptide, GRGDSP, was kindly provided by Takara Shuzou Co., Ltd. (Kyoto, Japan). Staurosporin, KT-5720, KT-5823, herbimycin, calphostin, and 2,5-MeC were purchased from Kyowa Medex Co. (Tokyo, Japan). H-7, H-89, and W-7 were from Seikagaku Kogyo (Tokyo, Japan). Cycloheximide, actinomycin D, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, iodoacetamide, and sodium orthovanadate were from Sigma.

Antibodies

Monoclonal antibodies used in this study were as follows. Gi9 (anti-alpha2) and GoH3 (anti-alpha6) were obtained from Immunotech (Marseille, France). P1B5 (anti-alpha3), anti-Src, and anti-alpha actinin were purchased from Oncogene Science. SG/17 (anti-alpha4), KH/72 (anti-alpha5), and SG/19 (anti-beta1) were kindly provided by Dr. K. Miyake(20) . Anti-phosphotyrosine was generously supplied by Brian Drucker (Dana-Farber Cancer Institute, MA)(21) . 2A7 (anti-focal adhesion kinase) was purchased from Upstate Biotechnology. Anti-tensin and anti-paxillin were from Chemicon International, Inc., and G1CL (anti-human IgG) was from Becton Dickinson.

Cell Spreading and Monoclonal Antibody Inhibition Assays

Well plates were coated in triplicate with type I collagen, fibronectin, laminin, or polylysine. Substrates were diluted in phosphate-buffered saline (PBS) and added to the wells at final concentrations of 1.25, 2.5, 5.0, and 10.0 µg/ml. Following overnight incubation of the well plates at 4 °C, the unbound substrates were removed by washing three times with PBS. The wells were then blocked with 1% bovine serum albumin (BSA) in PBS for 2 h at 37 °C. Cells (3 times 10^4 cells) that had been harvested from culture in exponential growth were resuspended in RPMI 1640 medium supplemented with 1% BSA and seeded in the matrix-coated wells. Staurosporin was prepared as a 35 mM stock solution in dimethyl sulfoxide and added at 1:1000 dilution for all the experiments. After a 4-h treatment with staurosporin (35 nM), the cells were photographed at random using phase contrast microscopy (Nikon, Japan). Spread cells displayed an epithelia-like appearance and were clearly distinguishable from round, floating, non-spread cells. The percentage of spread cells was estimated from the counts of 500 cells photographed at several randomly selected fields. For inhibition experiments, cells that had been harvested and suspended in RPMI 1640 medium supplemented with 1% BSA were incubated with each monoclonal antibody at a concentration of 10 µg/ml or synthetic GRGDSP peptide at concentrations of 0.2 mM, 1.0 mM, and 5.0 mM for 30 min at 37 °C. Thereafter, the cells were transferred to collagen-coated (10 µg/ml), laminin-coated (5 µg/ml), and fibronectin-coated (10 µg/ml) wells. Staurosporin was then added, and the spreading assay was carried out as described above. Divalent cation sensitivity was evaluated as previously described(22) . In brief, cells were washed with PBS containing with 1 mM EDTA to deplete pre-existing divalent cations in the medium. Next, cells were resuspended in 10 mM Tris-HCl, pH 7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM glucose, and 1% BSA and supplemented separately with CaCl(2), MgCl(2), and MnCl(2). The cells were seeded in matrix-coated wells, and cell spreading was assayed as described above. All assays were run in triplicate and presented as mean values. Variation between assays was less than 10%.

Immunoprecipitation and Immunoblotting

Cells were harvested from culture, washed with PBS, and lysed in cold lysis buffer consisting of Tris-buffered saline (140 mM NaCl, 20 mM Tris-HCl, pH 7.8) containing 0.1% sodium azide, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, 10 mM EDTA, 10 µg/ml leupeptin, 100 mM sodium fluoride, and 2 mM sodium orthovanadate at 4 °C for 20 min. Insoluble material was removed by centrifugation at 15,000 rpm for 15 min at 4 °C. The protein content of the lysates was determined with a BCA kit using bovine serum albumin as the standard. Cell lysates (20 µg) were absorbed with normal rabbit serum and protein G-Sepharose beads (Pharmacia, Uppsala, Sweden) for 2 h at 4 °C. The lysates were then incubated with anti-Src and protein G-Sepharose beads to collect antigen-antibody complex. The immunoprecipitates were washed five times with lysis buffer containing inhibitors of proteases and phosphatases and boiled at 100 °C for 5 min in a sample buffer containing 125 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol. The released proteins were subjected to 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a nitrocellulose membrane. The following procedures were carried out at room temperature. Nonspecific binding sites on the filter were blocked by incubating the membrane in PBS containing 0.05% Tween 20 (PBS-T) supplemented with 3% BSA. The filters were then probed overnight with anti-tyrosine phosphate monoclonal antibody in PBS-T at a dilution of 1:5000, washed with PBS-T several times, and incubated for 1 h with biotinylated horse anti-mouse IgG (Vector Laboratories Inc.). The blots were washed as above and incubated with avidin-horseradish peroxidase complex (Vector) for 30 min. After washing, the membranes were developed using enhanced chemiluminescence (Amersham, United Kingdom) according to the manufacturer's protocol. To completely remove the anti-tyrosine phosphate antibody and avidin-biotin complex, the blots were stripped for 1-2 h at 55 °C in 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol. The blots were then re-equilibrated in PBS-T, blocked, and reprobed separately with anti-Src antibody at a dilution of 1:2000 or with 2A7 (anti-focal adhesion kinase), anti-paxillin, or anti-tensin at a dilution of 1:1000 in the manner described above.

Flow Cytometry

Colo 201 cells were incubated in the presence or absence of staurosporin for 4 h at 37 °C. The cells were then harvested, washed three times with PBS, resuspended, and stained for 30 min on ice with primary antibodies to the integrin subunits (5 µg/ml). After washing two times with PBS supplemented with 1% BSA and 0.1% sodium azide, cells were incubated with either fluorescein isothiocyanate (FITC)-conjugated goat ant-mouse immunoglobulin (Becton Dickinson) or FITC-conjugated MAR18.5 (monoclonal antibody against rat IgG) (Organon Teknika) as second antibody for mouse monoclonal antibody and rat monoclonal antibody, respectively. G1CL (anti-human IgG1) was used as the negative control.

Immunofluorescence Microscopy

Plastic 3.5-cm dishes with inserted glass coverslips at the bottom were coated with either collagen type I or laminin (5 µg/ml) in PBS for 1 h at 37 °C and blocked with 1% BSA in PBS for 1 h at 37 °C. Colo 201 cells were harvested, washed three times with serum-free RPMI 1640 medium, and plated on the matrix-coated dishes. After treatment with staurosporin (35 nM) and confirmation of cell spreading, the dishes were rinsed with pre-warmed PBS and fixed with paraformaldehyde (3% w/v in PBS) for 10 min at room temperature. All of the following procedures were performed at room temperature. Residual aldehyde groups were blocked by incubation with 50 mM ammonium chloride in PBS for 20 min, and the cells were permeabilized with 1% Triton X-100 in PBS for 10 min. After blocking with 1% normal rabbit serum in PBS, cells were incubated for 2 h with monoclonal antibody against alpha2 (IgG1), alpha3 (IgG1), alpha6 (IgG2a), or anti-alpha actinin (IgM) in 1% normal rabbit serum in PBS. The cells were washed with PBS, and labeled second antibody was added. After 1 h, the cells were washed, semi-dried, and examined under epifluorescence (IIRS, Zeiss, Germany). Because preliminary studies showed no cross-reaction between FITC-conjugated antibody against IgG and phycoerythrin-conjugated antibody against IgM (Kirkegaard & Perry Laboratories, Inc.), these second antibodies were added simultaneously in double staining experiments.


RESULTS

Effect of ECM and Divalent Cations on Cell Spreading Induced by Staurosporin

The ability of staurosporin to induce spreading of Colo 201 cells was examined in non-coated wells (3% BSA blocking prior to the assay only) and in wells coated with collagen type I (5 µg/ml), laminin (5 µg/ml), and fibronectin (5 µg/ml) (Fig. 1, A, C, E, and G, respectively). On collagen and laminin, approximately 95% of the cells were found to have spread after a 4-h treatment with staurosporin, and these showed an epithelia-like morphology with formation of micropodia. On fibronectin, about 20% of the cells were spread after a similar treatment, but micropodia formation was poor. In the absence of staurosporin, cells did not spread on either the non-coated or the coated wells (Fig. 1, B, D, F, and H). Staurosporin did not have any effect on cell viability during the 4-h incubation time. We tested the abilities of untreated and staurosporin-induced cells to spread on several substances, collagen type I, fibronectin, laminin, and polylysine, at various concentrations (Fig. 2A). Unless stated otherwise, cell spreading was evaluated after 4 h of incubation with staurosporin. In the absence of staurosporin, cell spreading was not observed in any of the wells. After incubation with staurosporin, spreading was seen in more than 90% of the cells in wells coated with 5 µg/ml collagen or 2.5 µg/ml laminin. In contrast, spreading on fibronectin was very weak, reaching only 30% even on wells coated with 100 µg/ml fibronectin. Staurosporin did not induce cell spreading on polylysine, which occurs via a non-integrin pathway. The effect of divalent cations on the cell spreading was examined after depletion of pre-existing cations with EDTA (Fig. 2B). In the presence of Mg or Mn, cell spreading started after 2 h of incubation and reached a plateau after 3 h. In the presence of Ca, cell spreading progressed more slowly, and 8 h of incubation was necessary for the spreading to reach a plateau. For this reason, the sensitivity of cell spreading to divalent cations was assayed after an 8-h incubation with staurosporin. The cell spreading showed a high dependence on Mn, with marked induction occurring at Mn concentrations of 0.1 mM or greater. Sensitivity to Mg was relatively low; at 0.1 mM Mg, cell spreading was not induced significantly compared with spreading in the absence of Mg. The reliance on Ca was lower still. At 10 mM Ca, cell spreading was only 27.1% on collagen and 25.3% on laminin. In the absence of staurosporin, cell spreading was not induced at the examined concentrations of the three divalent cations.


Figure 1: Staurosporin-induced cell spreading in Colo 201. Cells were seeded on non-coated (A, B), collagen-coated (C, D), laminin-coated (E, F), and fibronectin-coated (G, H) wells. Cell morphology was observed after a 4-h incubation in the presence (A, C, E, G) or absence (B, D, F, H) of staurosporin. The cells were observed and photographed at 100times.




Figure 2: A, effect of extracellular matrix on spreading. Collagen type I, laminin, fibronectin, and polylysine were diluted to the concentrations indicated and used to coat well plates. The extent of cell spreading in the presence (circle) and absence (bullet) of staurosporin was evaluated and is represented as the mean value of triplicate experiments. B, after treatment with 1 mM EDTA, cells were resuspended in Tris-buffered saline, supplemented separately with Ca, Mg, and Mn at 0.1, 1.0, and 10 mM, and seeded on collagen-coated (circle, bullet) and laminin-coated (box, ) wells. The extent of cell spreading in the presence (circle, box) and absence (bullet, ) of staurosporin is indicated as the mean value of triplicate experiments.



VLA-3 Was Involved in Cell Spreading

The above results on the affinity of Colo 201 cells for ECM proteins and on the dependence of cell spreading on divalent cations suggested that the beta1 family of integrins might be in part responsible for this spreading process. To determine which chains of integrin were involved in cell spreading, we tested the ability of monoclonal antibodies to suppress the spreading (Fig. 3). Cell spreading to collagen (panel A) and laminin (panel B) was equally blocked by antibodies against the alpha3 and beta1 chains of integrin at a concentration of 10 µg/ml. Spreading to collagen and laminin was not inhibited by antibodies against alpha2 and alpha6 at the same concentration. In our preliminary study, cell spreading of HT1080 was decreased from 92.4 to 22.6% on collagen and from 88.6 to 16.5% on laminin by the antibody against alpha2 (Gi9). Similarly, the antibody against alpha6 (GoH3) inhibited spreading of HT1080 cells on laminin from 88.2 to 19.8%. Because VLA-3-mediated binding to fibronectin has been reported to be RGD-dependent(22) , we tested both the inhibitory effect of antibodies and the effect of RGD on staurosporin-induced cell spreading on fibronectin. Cell spreading on fibronectin was not significantly affected by monoclonal antibodies against the alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 chains of integrin or by the synthetic peptide GRGDSP. This indicated that cell spreading on fibronectin was not dependent on the RGD sequence of fibronectin and did not occur in a beta1 integrin-dependent manner. The above inhibition study demonstrated that VLA-3, not VLA-2 or VLA-6, was involved in cell spreading on collagen and laminin. Next, the expression of integrin molecules was analyzed by indirect immunofluorescence (Fig. 4). Flow cytometric analysis indicated that Colo 201 expressed alpha2, alpha3, alpha6, and beta1 chains on the cell surface and that the level of expression was not altered after 4 h of incubation with staurosporin. The alpha4 and alpha5 chains were not expressed on the Colo 201 cells. To evaluate the effects of protein synthesis and mRNA transcription on the staurosporin-induced cell spreading, cells were first incubated on collagen and laminin for 4 h with cycloheximide (20 µg/ml) or actinomycin D (20 µg/ml) and subsequently maintained in cycloheximide or actinomycin D throughout the 4-h spreading assay. Cell spreading was not affected by either cycloheximide or actinomycin D, which indicated that the spreading process was not accompanied by staurosporin-induced protein synthesis or mRNA transcription. Taken together, the results of antibody inhibition studies and flow cytometry revealed that staurosporin-induced spreading on collagen and laminin was mediated by VLA-3 and was not accompanied by quantitative changes in synthesis of the VLA-3 protein or transcript.


Figure 3: Effect of blocking antibody on cell spreading. The effects of monoclonal antibodies to integrin subunits on staurosporin-induced cell spreading were assayed on collagen-coated (10 µg/ml) and laminin-coated (5 µg/ml) well plates (panels A and B, respectively). The following monoclonal antibodies were used at a final concentration of 10 µg/ml: Gi9 (anti-alpha2), P1B5 (anti-alpha3), SG/17 (anti-alpha4), KH/72 (anti-alpha5), GoH3 (anti-alpha6), and SG/19 (anti-beta1). G1CL (anti-human IgG1) was used as the negative control. Panel C, for the inhibition assay in fibronectin-coated (10 µg/ml) wells, the effect of GRGDSP (0.2, 1, and 5 mM) was also examined. Values are the mean of triplicate experiments.




Figure 4: Flow cytometric analysis of alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 expression on Colo 201 cells. Staurosporin-treated and untreated cells were stained with a primary antibody, followed by FITC-conjugated second antibody, as described under ``Experimental Procedures.'' A total of 10,000 cells were analyzed with FACScan. The horizontal and verticalaxes represent fluorescence intensity in a log scale and cell number, respectively.



Effect of Tyrosine Kinase on Staurosporin-induced Cell Spreading

To identify the target of staurosporin in this spreading process, we attempted at first to induce spreading of Colo 201 cells using a combination of inhibitors for specific kinases. Cell spreading was not observed after treatments with protein kinase C-specific inhibitors H-7 (10-10M) and photoactivated calphostin (10-10M)(23) , tyrosine kinase inhibitors herbimycin A (10-10M) and 2,5-MeC (10-10M), cAMP-dependent kinase inhibitors H-89 (10-10M) and KT-5720 (10-10M), cGMP-dependent kinase inhibitor KT5823 (10-10M), and calmodulin/calcium-dependent kinase inhibitor W-7 (10-10M) used alone or in combination. Cell spreading was suppressed when a combination of staurosporin and herbimycin A (2 mM) or 2,5-MeC (2 mM) was given to the cells. No inhibitory effect was observed when cells were incubated with a combination of staurosporin and protein kinase C- or protein kinase A-specific inhibitor. These data suggested that a tyrosine kinase that was not inhibited by staurosporin was involved in the cell spreading. Phosphorylation of tyrosine in whole cell lysates was examined at 0, 30, 60, and 120 min during the incubation with staurosporin. At 30 min using collagen-coated wells, almost all of the cells appeared round and were still easily moved by agitating the plates. Western blot analysis (Fig. 5, panel A) at this point revealed that two bands (88 and 50 kDa) had lower levels of tyrosine phosphorylation than the corresponding bands at 0 min. The tyrosine phosphorylation of other bands was unchanged. At 60 min, cells remained round but adhered the bottom of the collagen-coated chamber, based on the observation that the cells were not moved easily by vigorous agitation. The profile of tyrosine phosphorylation at 60 min showed enhanced phosphorylation of several proteins with bands at 220, 175, 150, 98, 62, 58, and 50 kDa, as indicated at the rightside of panel A (Fig. 5). Three proteins with bands at 72, 68, and 64 kDa (indicated by arrows in panel A in Fig. 5) showed decreased tyrosine phosphorylation compared with the corresponding bands at 0 and 30 min. The proteins at 88 and 50 KDa were found to be re-phosphorylated at 60 min. Western blot analysis at 120 min of staurosporin treatment, when cell spreading was observed in the Colo 201 cells, showed the same tyrosine phosphorylation profile seen at 60 min. The membranes were stripped and reprobed separately with anti-Src and anti-tensin antibodies. Fig. 5, panel B, shows that a 58-kDa protein was recognized by the anti-Src antibody (left), and a 220-kDa protein was recognized by the anti-tensin antibody (right). These were identical to the tyrosine-phosphorylated proteins of the same molecular masses in panel A. The stripped membranes were also probed separately with anti-focal adhesion kinase and anti-paxillin antibodies. No bands were found to be recognized by these antibodies. Panel C shows that Src product and tensin precipitated from whole cell lysates (left and right, respectively) were tyrosine phosphorylated at 30 min and that the level of tyrosine phosphorylation remained elevated throughout the remainder of the 120-min incubation with staurosporin. Use of anti-focal adhesion kinase or anti-paxillin antibody in the immunoprecipitation procedure did not reveal any tyrosine-phosphorylated product. Neither cell adhesion nor spreading was observed during an 8-h incubation when Colo 201 cells in collagen-coated wells were simultaneously treated with staurosporin and herbimycin A (2 mM). Western blot analysis (panel D) showed that tyrosine phosphorylation declined during this treatment. The time course of cell spreading and the profile of tyrosine phosphorylation were similar when Colo 201 cells were seeded in laminin-coated wells and treated as described above. In summary, several proteins including Src product and tensin were tyrosine phosphorylated in association with cell adhesion by a tyrosine kinase that was not sensitive to staurosporin.


Figure 5: Time course of tyrosine phosphorylation during the incubation with staurosporin. Cells seeded on collagen-coated wells were harvested at 0, 30, 60, and 120 min during treatment with staurosporin. Panel A, whole cell lysates were separated by 8% SDS-PAGE, transferred onto nitrocellulose, and probed with anti-tyrosine phosphate antibodies. Panel B, the same membrane was stripped and reprobed with anti-Src antibody or anti-tensin antibody (left and right, respectively), as described under ``Experimental Procedures.'' Panel C, proteins precipitated from whole cell lysates of Colo 201 using anti-Src or anti-tensin antibody (upper left and upper right, respectively) were separated by 8% SDS-PAGE, transferred onto nitrocellulose, and probed with anti-tyrosine phosphate antibodies. The membranes were then stripped and reprobed with anti-Src and anti-tensin antibodies (lower left and lower right, respectively) to show the amounts of precipitated protein loaded. Panel D, Colo 201 cells treated simultaneously with staurosporin (35 nM) and herbimycin A (2 µM) were harvested at 0, 30, 60, and 120 min and probed with anti-tyrosine phosphate antibodies. The profiles of tyrosine phosphorylation in panels A, C, and D were reproducible in three independent experiments. The molecular masses of marker proteins are indicated in kilodaltons.



Analysis of Focal Localization of Integrins by Immunofluorescence Microscopy

To examine the distribution of integrin chains on the cell surface in the presence of staurosporin, indirect immunofluorescence microscopy was performed. Focal adhesion sites were identified by means of immunofluorescence microscopy utilizing a monoclonal antibody against cytoskeleton-associated proteins such as alpha actinin(24) . In the absence of staurosporin treatment, the alpha2, alpha3, and alpha6 chains of integrin were diffusely expressed on the surface of Colo 201 cells as shown in Fig. 6A. After 4 h of staurosporin treatment, alpha3 chain was condensed into focal plaques that coincided precisely with the staining of alpha actinin on the spread Colo 201 cells (Fig. 6B), while alpha2 and alpha6 chains remained homogeneously distributed on the cell surface (Fig. 6C). The same result was obtained whether cells were spread on laminin or on collagen.


Figure 6: Indirect immunofluorescence microscopy of Colo 201 cells seeded on collagen. A, cells in the absence of staurosporin were stained with Gi9 (anti-alpha2, left), P1B5 (anti-alpha3, middle), or GoH3 (anti-alpha6, right). B, after a 4-h incubation with staurosporin, cells were stained with P1B5 (anti-alpha3, left) or anti-alpha actinin (right). C, spread cells after treatment with staurosporin were stained with Gi9 (anti-alpha2, left) or GoH3 (anti-alpha6, right). All images of fluorescence were obtained at 400times.




DISCUSSION

VLA-3 (alpha3beta1) is a member of the beta1 integrin family and mediates interaction of the cell with ECM(25) . A wide range of ECM proteins have been shown to be ligands for VLA-3 by antibody inhibition studies, affinity chromatography, and immunofluorescence microscopy. Antibodies against VLA-3 have been demonstrated to block cell attachment to collagen, laminin, and fibronectin(22, 26, 27) , and the affinity of VLA-3 for these proteins has been used in purification procedures for the integrin(22, 26) . Recently, epiligrin/kallinin (28) , invasin(29) , and entactin (30) have been added to the list of ligands for VLA-3. The expanding list of specific ligands for VLA-3 is controversial because adhesion to VLA-3 may be obscured by other activated integrins coexisting on the cell surface. The standard binding assay, which utilizes immobilized proteins, may not reflect the physiological cell-to-matrix interaction.

In this study, staurosporin induced cell spreading through two different pathways in Colo 201 cells. On collagen and laminin, cell spreading was dependent on VLA-3, as demonstrated by the antibody inhibition study. In contrast, cell spreading on fibronectin was independent of beta1-integrin and the RGD sequence of fibronectin. Furthermore, the morphology of the spread cells on collagen and laminin was different from that of spread cells on fibronectin in that the former developed longer micropodia. Colo 201 cells could not spread on polylysine-coated wells either in the presence or in the absence of staurosporin, which indicated that the staurosporin-induced cell spreading did not involve a non-integrin cell-to-matrix interaction.

Staurosporin-induced cell spreading on collagen and laminin responded differently to different divalent cations. After depletion of pre-existing divalent cations by EDTA, as little as 0.1 mM Mn was enough for the recovery of staurosporin-induced cell spreading in most Colo 201 cells. Recovery progressed more slowly in the presence of Ca. At 10 mM Ca, spreading was observed in only 27% of the cells. Sequence analysis of cDNA for the alpha3 subunit of human VLA-3 has shown at least three possible divalent cation sites on the alpha3 chain(25) . These binding sites appear to have different affinities for the three divalent cations examined in the order Mn > Mg > Ca.

In a study utilizing alpha3-transfected cells(31) , adhesion to collagen type I and laminin was weak, even after addition of a stimulatory antibody against beta1 chain. The ability of the alpha3-transfected cells to bind fibronectin was not precisely evaluated in the presence of VLA-4, although attachment to fibronectin was elevated by the anti-beta1 antibody. On Colo 201 cells, VLA-3 has the ability to attach to collagen and laminin and to cause cell spreading. Neither the VLA-2 nor the VLA-6 expressed on the Colo 201 cells was involved in spreading. The process of adhesion and spreading requires redistribution of integrin molecules to focal adhesion plaques and changes in linkage between integrin and cytoskeleton-associated proteins. VLA-2 and VLA-6 did not form adhesion plaques on the Colo 201 cells. The inability of these integrins to mediate adhesion of Colo 201 cells to collagen and laminin as well as the weak affinity of VLA-3 in alpha3-transfected cells for these ECM proteins (31, 32, 33) may be due to poor connections between integrins and cytoskeletal proteins such as vinculin and alpha actinin.

Regarding VLA-5, several reports have indicated that activation of protein kinase C by phorbol ester causes VLA-5-mediated cell adhesion in NB4 cells, a human promyelocytic leukemia cell line(15) , and Chinese hamster ovary cells(34) . The VLA-5-mediated adhesion was specific to fibronectin, blocked by calphostin, a protein kinase C-specific inhibitor, and accompanied by increased phosphorylation of focal adhesion kinase. This spreading started as early as 10 min into the stimulation and reached a maximum within 1 h, significantly earlier than the VLA-3-mediated spreading induced by staurosporin. Neither spreading process was accompanied by a change in the number of integrin molecules, and the slower spreading process with VLA-3 may reflect the differences in ECM ligand and inducing agent.

Staurosporin has an inhibitory effect on a variety of serine/threonine kinases(35) , including protein kinase C, cAMP-dependent protein kinase, cGMP-dependent protein kinase, myosin light chain kinase, and calcium/calmodulin-dependent protein kinase II, each at a similar IC (50% inhibition concentration). Staurosporin also inhibits the tyrosine kinase activities of p60(36) , epidermal growth factor receptor(37) , platelet-derived growth factor receptor(37) , and insulin receptor(38) . In our study, staurosporin, a broad spectrum kinase inhibitor, induced VLA-3-mediated cell spreading. Addition of tyrosine kinase inhibitor herbimycin A or 2,5-MeC inhibited the staurosporin-induced spreading, implying that a certain tyrosine kinase that was inhibited not by staurosporin but by herbimycin A and 2,5-MeC was involved in the spreading process. In unstimulated cells, this tyrosine kinase might be inhibited by other activated kinases.

Western blot analysis showed that during the treatment with staurosporin, tyrosine phosphorylation was enhanced in seven proteins beginning at the time of cell adhesion. The 220- and 58-kDa proteins were identified as tensin and Src product, respectively. Tensin is localized at the focal adhesion site(39, 40) . It has an SH2 domain by which it likely interacts with phosphotyrosine residues of other proteins(41) . Clinical observation has suggested that in human colon cancer cells and cell lines, Src expression is involved in the pathway of differentiation(42) . In addition, many investigations have shown that expression of Src in cells results in the phosphorylation of a group of proteins at the focal adhesion site, including focal adhesion kinase and paxillin(17) . In our study, however, expression and tyrosine phosphorylation of focal adhesion kinase and paxillin could not be detected.

The kinase activity of Src is regulated in two different ways. Phosphorylation of Tyr results in the elevation of Src kinase activity(43) , while phosphorylation of Tyr inactivates the kinase activity(44) . Indeed, it is unclear whether the kinase activity of a Src product is enhanced or decreased by tyrosine phosphorylation during incubation with staurosporin. Nonetheless, Src appears to play a role in VLA-3-mediated cell spreading as a substrate for the tyrosine kinase involved in the spreading process. Further investigation is needed to characterize the tyrosine kinase responsible for staurosporin-induced cell spreading.


FOOTNOTES

*
This work was supported in part by grants-in-aid for cancer research and scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan and from the Mitsubishi Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-6-879-3421; Fax: 81-6-879-3429.

(^1)
The abbreviations used are: ECM, extracellular matrix; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline with 0.05% Tween 20; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; VLA, very late activation antigen; 2,5-MeC, methyl 2,5-dihydroxycinnamate.


ACKNOWLEDGEMENTS

We thank Dr. K. Sekiguchi and Dr. S. Higashiyama for helpful suggestions and critical reading of this manuscript, Dr. K. Miyake for kindly providing monoclonal antibodies (SG/17, SG/19, and KH/72), and Dr. B. Drucker for providing anti-phosphotyrosine. We also thank S. House for editing the manuscript.


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