Rapid effects of EGF on cytoskeletal structures and adhesive properties of highly metastatic rat...

Clin. Exp. Metastasis, 1993, 11, 113-125

Rapid effects of EGF on cytoskeletal structures and adhesive properties of highly metastatic rat mammary adenocarcinoma cells

Rosemarie B. Lichtner*t~:, Marion Wiedernuth*, Christiane Noeske-Jungblut§ and Volker Schirrmacher*

*Department of Immunology and Genetics, German Cancer Research Center, Im Neuenheimer Feld 280, 6900 Heidelberg; and §Research Laboratories of Schering AG, Miillerstrafle 170-178, 1000 Berlin 65, Germany

(Received 6 July 1992; revision received 8 September 1992; accepted 7 October 1992)

In the highly metastatic rat m a m m a r y adenocarcinoma cell clone MTLn3, EGF induced increased adhesion to fibronectin while in the human epidermoid carcinoma cell line A431 EGF induced diminished adhesive properties. Flattening of cells with extensive formation of filopodia was observed in MTLn3 cells within 5 min of EGF addition, while in A431 cells EGF induced rounding up and only occasional formation of filopodia. Immunofluorescent analysis revealed extension of microtubules (MT) into the filopodia and Western blot analysis demonstrated an EGF-induced 2- to 3-fold increase in the amount of assembled tubulin in MTLn3 but not in A431 cells. In MTLn3, but only marginally in A431 cells, EGF treatment resulted in phosphorylation of a 280 kD cytoskeleton-associated protein, which was rapid and dose-dependent. These results suggest differential signal transduction pathways of cytoskeleton-associated EGFRs in highly metastatic MTLn3 as compared with A431 cells.

Keywords: cytoskeleton, EGF metastasis

Introduction

Metastasis formation is a complex phenomenon involving several sequential steps [1, 2]. Important in this process is the transport of tumor cells in the circulation where they can interact with each other, host platelets, lymphocytes and the vascular endothelium [2]. Tumor cells attach relatively slowly to the apical surfaces of endothelial cells, though they generally attach more rapidly and strongly to subendothelial matrix [3-5]. It has been shown that adhesion of tumor cells to the subendothelial matrix in vitro and the formation of experimental metastasis in vivo can be inhibited by

-To whom correspondence should be addressed. ;P resen t address: Research Laborator ies of Schering A G , Miil- lerstraBe 170-178, 1000 Berlin 65, Germany.

substances that affect cytoskeletal structures [3, 6-8]. In fact, the ability of normal and malig- nant cells to initiate cell-cell contacts and adhesion, change cell shape, migrate, secrete enzymes and other soluble components are controlled, in part, by the cytoskeleton. Cellular motility [9-12] as well as the expression of proteolytic enzymes [13, 14] can be modulated by EGF, suggesting EGF receptor-cytoskeleton interactions. In fact, among the early effects of EGF on its target cells are morphological changes, such as rounding up of cells [15, 16], induction of membrane ruffling and extension of filopodia [17]. Furthermore, mitogenic stimulation of cultured cells induced a transi- ent reorganization of cytoskeletal structures such as spectrin [15] or components of the filamentous

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R. B. Lichtner et al.

microtubule (MT) network [18]. Interestingly, pre- ferential localization of EGF receptor (EGFR) in the immediate vicinity of membrane-associated cytoskeletal filaments has been shown recently [19-22]. In cytoskeleton preparations obtained after mild detergent treatment of cells, a subset of EGFRs remains associated with the cytoskeleton and can undergo EGF-stimulated phosphorylation on tyrosine. This has been demonstrated for permeabilized fibroblasts [23], A431 cells [24, 25] and rat mammary adenocarcinoma cells [26].

We had reported a mitogenic response to EGF of the highly metastatic cell clone MTLn3 but not of its low metastatic counterpart MTC [27], both derived from the 13762NF rat mammary adenocarcinoma [28]. MTLn3 cells express 5-6 x 104 EGFRs per cell with a large percentage (20%) of receptors being of the high-affinity class [26]. Furthermore, EGFRs failed to autophosphorylate in intact cells. However, EGFRs associated with the cytoskeleton of permeabilized MTLn3 cells were able to undergo basal and EGF-induced phosphorylation, indicating specifc functions of this subset of EGFRs. Since the low metastatic counterpart clone MTC expresses only residual amounts of EGFR (Lichtner et al., in preparation) we chose human epidermoid A431 cells as control cell line. A431 cells provide the most studied model of action of EGF and the association of EGFRs with the cytoskeleton is well established [19-22]. Here we report on changes in morphology, increase in MT formation, increased adhesion to matrix components and phosphorylation of a 280 kD protein in MTLn3 cells stimulated by EGF. In contrast, in A431 cells a different change in morphology with no obvious effect on MT formation, only marginally altered phosphorylation of a 280 kD protein and decreased adhesive properties upon EGF addition could be demonstrated.

Materials and methods

Material [y-32p]ATP (3000 Ci/mmol), [3H]thymidine (122 Ci/mmol) and the ECL Western blotting detection system were purchased from Amersham Buchler (Braunschweig, Germany). EGF, receptor grade, was supplied by Collaborative Research (Lexington, MA, USA) and prestained molecular weight standards for gel electrophoresis were from Sigma (Munich, Germany). Minimum essential medium (MEM) free of phosphate was obtained from Biochrom (Berlin, Germany). Fibronectin

was isolated from human plasma as described by Ruoslahti et al. [29].

Cell lines and culture conditions The tumor cell clone MTLn3 used in this study is derived from the 13762NF rat mammary adenocarcinoma [28]. MTLn3 cells (passages 15-20 of high spontaneous metastatic potential) were grown as described previously in alpha-modified MEM supplemented with 5% fetal calf serum (FCS; Gibco, Eggenstein, Germany) without antibiotics [30]. Human epidermoid carcinoma cells (A431) were a gift from Dr G. E. Gallick (M. D. Anderson Hospital, Houston, TX, USA) and they were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM; Flow, Meckenheim, Germany) and F12 medium (Gibco) supplemented with 10% FCS. Monolayers of A431 cells were grown in 100 mm culture dishes (Corning). At 60-80% confluency, the tumor cells were harvested using 0.125% trypsin in 2 mM EDTA in calcium-magnesium-free Dulbecco's phosphate-buf- fered saline (PBS; trypsin/EDTA).

Growth studies Growth studies were performed as described previously [30]. Cells were seeded at 2.5 × 104 per 60mm culture dish in 3 ml of medium supplemented with 1% FCS and EGF was added after 1.5 h. After 3 days of culture, cell numbers were estimated with a Coulter counter. All experiments were performed at least three times with triplicate samples and cell numbers were normalized to control cell numbers.

Adhesion assay Plates (24-well) were incubated with 500/tl of fibronectin (10~g/ml) in PBS for l h at room temperature. They were washed twice with PBS and three times subsequently with adhesion medium (DMEM containing 12mM HEPES, 1% bovine serum albumin (BSA), 1 mM NaHCO3, pH7.2). Tumor cells were radiolabeled with 100/zCi of [3H]thymidine per 100 mm culture dish for 24 h in fresh culture medium containing the respective serum concentrations. After labeling, the tumor cells were suspended in adhesion medium at a concentration of 4 x 104 cells/ml. EGF was added at 10 ng/ml final concentration at the start of the adhesion experiment. Controls received the appropriate amount of adhesion medium. The adhesion of radiolabeled tumor cells to fibronectin was investigated over a 60-min period at 37°C as described previously [3, 31].

114 Clinical & Experimental Metastasis Vol 11 No 1

EGF effects on cytoskeletal structures

Fluorescent labeling of cells Cells from exponentially-growing cultures were seeded at 2 x 105 per 60 mm culture dish on glass coverslips (22 mm diameter) in 3 ml of medium supplemented with 1% FCS. After a 18-20 h incubation period E G F was added to intact cells at the indicated concentrations. Cells were fixed at the indicated times with formaldehyde and permeabilized with 0.5% TX-100 as described previously [30, 32]. The extracted cytoskeletons were treated with monoclonal mouse anti-o: tubulin antibody (Amersham) and then with a 1:50 dilution of the second antibody (sheep anti-mouse IgG absorbed with rat serum proteins; Sigma) labeled with fluorescein in order to localize microtubules. The actin-containing microfilaments in the same cytoskeleton preparation were subsequently stained with rhodamine-labeled phalloidin (Molecular Probes, Inc., Eugene, OR, USA). Microscopic observations were performed using a Leitz photo- microscope equipped for fluorescein and rhodamine fluorescence. Kodak Tri-X film was used for photography.

Preparation of detergent-insoluble (cytoskeleton) fractions Tumor cells (1.5 x 106 ) were seeded in 10ml of medium with 1% FCS in 100 mm diameter dishes. After a 18-20 h incubation, 150 ng/mg E G F was added to intact cells for 5 rain. The adherent cells were washed twice with PBS and once with 5 ml of HMCa buffer (80mM HEPES, 2 m M MgCt 2, 1 mM CaC12 or 1 mM E G T A , respectively, 1 mM PMSF, pH 6.9) at room temperature and incubated for 0.5 min with 2 ml of washing buffer (HMCa with 2 m M MnCI~, 0 . 1 m M Na3VO4, 10 ~tl/ml leupeptin, 50 Fd/ml aprotinin) containing 0.15% TX-100. The detergent-soluble fraction was discarded and the detergent-insoluble cytoskeleton fraction was scraped with a rubber policeman in two times concentrated Laemmli buffer plus 16% /3-mercaptoethanol and lysates were passed several times through a 22-gauge needle, followed by heating for 5 min at 95°C. Protein determinations of TCA precipitates were performed according to Lowry et al. [33].

Phosphorylation of detergent-insoluble cytoskeleton fractions with [y-32P]A TP Tumor cells (5 x 105 ) were seeded in 3 m l of medium with 1% FCS in 60 mm diameter dishes. After a 18-20 h incubation E G F was added to intact cells at the concentrations and time periods indicated. Preparation of detergent-insoluble cyto-

skeletal fractions was essentially performed as described above. After mild detergent lysis (for 1 min) of adherent cells, the detergent-soluble fraction was discarded and the insoluble cytoskeleton fraction was rinsed three times with washing buffer. Phosphorylation was initiated by the addition of 0.5 ml of washing buffer containing 10/~M ATP and 10-20/~Ci [•-32p]ATP. After 2-10 rain incubation at room temperature with occasional gentle shaking, the plates were rinsed four times with washing buffer, transferred to ice and the cytoskeletons were scraped with a rubber policeman into 100/A of solubilization buffer (10 mM Tris, pH 7.2, 1 mM MgCI2, 1 mM E G T A , 0.1 mM Na3VO4, 1 mM PMSF, 0.1% SDS) followed by addition of 20/A nuclease solution ( l m g / m l DNase, 0.5 mg/ml RNase, 50 mM MgCI2, 50 mM Tris, pH7.2) . Immediately, 30/~1 of five times concentrated Laemmli buffer was added and the samples heated for 5 min at 95°C. To aliquots (5-10/~1) of 32p-labeled solubilisates, 20/A of BSA (1 mg/ml) and subsequently 0.5 ml of ice-cold 10% TCA containing 20 mM H3PO4 were added and incubated on ice for 30 min. The precipitated proteins were filtered over Millipore filters, washed three times and then measured for radioactivity. Samples were normalized on the basis of TCA precipitable counts and analysed by one-dimensional S D S - P A G E .

Metabolic labeling of cells and immunoprecipitation Tumor cells were allowed to attach for 18-20 h. After two washes with D M E M lacking inorganic phosphate (Pi) the monolayers were incubated for 4 h with 5 ml of medium without Pi and serum, supplemented with 0.1 mCi 32p i. E G F was added during the last 5 min of the 4 h incubation, after which the cells were washed three times with ice-cold PBS. Cells were lysed by scraping in 1 ml of ice-cold lysis buffer (1% TX-100, 5 mM EDTA, 50 mM NaC1, 50 mM NaF, 3 0 m M Na4P207, 0.1 mM Na3VO4, 0.1% BSA, 1 mM PMSF, 10 ~g/ml leupeptin, 50/~g/ml aprotinin, 10 mM Tris, pH 7.6) [34]. Samples were incubated on ice with occasional rigorous vortexing for 20 min, and centrifuged at 15 000 g for 15 min at 4°C to remove insoluble debris. The supernatant was precleared with 50/A of Sepharose 4B beads coupled with human serum albumin for 1 h at 4°C. Antibodies were added at 50/~1 to the lysates and protein A-Sepharose at 20 ~1. After 2 h the precipitates were washed three times with lysis buffer and twice with lysis buffer without BSA. Subsequently, the immunoprecipitates were boiled for 5 min in

Clinical & Experimental Metastasis Vol 11 No 1 115


one times Laemmli buffer containing 50 mM D T T or 8% fl-mercaptoethanol. Samples were analysed by one-dimensional S D S - P A G E on 4-15% gels and autoradiography.

Electrophoretic techniques For identification of phosphorylated proteins 5-10 × 103 TCA-precipitable cpm or for Western blotting 10 #g protein/lane were used and separated by one-dimensional S D S - P A G E on 4-15% gels. Gels were dried under vacuum and exposed with intensifying screens at -70°C. Exposure times varied between 2 -4 days for untreated and 7-14 days for KOH-trea ted gels, respectively.

Western blot techniques Transfer of electrophoretically separated polypep- tides onto nitrocellulose was carried out by the method of Towbin et al. [35] in 25 mM Tris-base, 192 mM glycine (pH 8.3) at 50 V for 24 h at 4°C. The transfer was blocked with 0.2% gelatine in PBS for 2 h at room temperature, then overnight in 5% skimmed milk in 0.2% Tween 20 in PBS (Tween/PBS) and then washed with Tween/PBS. The transfer was probed with anti-a'-tubulin or anti-actin antibody (Amersham) diluted 1:1000 in Tween/PBS plus 1% BSA for l h at room temperature, rinsed with Tween/PBS and reprobed with peroxidase-conjugated second Ab (Dianova) diluted 1:5000 at room temperature for 1 h. After rinsing with Tween/PBS labeled proteins were identified with the ECL Western blotting detection system with exposure times between 10 and 60 s.

Results

A431 cells express high levels of E G F receptors and are growth-inhibited by high concentrations of E G F [36,37], while at very low E G F concentrations growth stimulation has been reported [38]. While A431 cells used in this study were not growth-stimulated by very low E G F concentrations (0.02-1 ng/ml; data not shown) their growth was inhibited by 50% after a 3 day incubation with 10 ng/ml E G F and by 60% with 50 or 150 ng/ml EGF, respectively (Table 1). When tested under the same experimental conditions, MTLn3 cells were maximally growth-stimulated by 10ng/ml E G F in agreement with previous findings [27].

Microscopic observation of tumor cells treated with E G F indicated alterations in morphology which were disparate in MTLn3 and A431 cells (Figure 1). Control MTLn3 cells did not spread out

Table 1. Growth of MTLn3 and A431 ceils in the presence of EGF. Cells (2.5 × 104) were treated for 3 days with various concentrations of EGF in o:MEM (MTLn3) or DMEM/F12 (A431) with 1% serum

EGF (ng/ml) MTLn3 A431

0 100 ~ ± 2 h 100 ± l 10 179 ± 11 52 ± 2 50 179 ± 10 38 ± 1

150 192 ± 22 38 ± 2

"Percentage of control. hS.D. of at least three independen t exper iments with triplicate samples.

very well on plastic substrata in the presence ot low serum (Figure 1A). However, 5 rain after addition of E G F (150 ng/ml) to the growth medium (containing Ca 2+) cells formed extensive ruffles and acquired a very flat appearance (Figure 1B, large arrow). In contrast, incubation of A431 cells with E G F induced rounding up of cells with extensive blebbing (Figure 1D, small arrow). This was a t ime-dependent process with more cells (--40%) being rounded after 1 h (data not shown). Nevertheless, some cells at the margin of a cluster exhibited formation of cell extensions such as filopodia (Figure 1D, large arrow).

Since alterations in cell morphology could indicate an effect of E G F on cytoskeletal structures, we examined the distribution of microfilaments (MF) and microtubules (MT) in cytoskeleton preparations of MTLn3 and A431 cells, by immunofluorescent (Figure 2) and Western blotting techniques (Figure 3). In control MTLn3 cells, parallel MF fibers were visible, crossing over and under one another, terminating close to the cell margin in non-ruffling areas, while in ruffling areas they terminated prior to the ruffle (Figure2A). The majority of MT seemed to be displayed parallel to the cell periphery following the MF distribution (Figure 2B). Treating MTLn3 cells for 5 rain with 150 ng/ml E G F induced spreading of cells but did not induce obvious changes in MF organization (Figure 2C) while MT fibers now extended into the margin of cells (Figure 2D). In control A431 cells very few MF were visible crossing the cells (Figure2E) and addition of E G F induced the concentration of filamentous actin in cell contact areas and at the expanding borders of cells facing the margin of a cluster (Figure 2G, 1). In addition, E G F induced the formation of blebs intensively stained for filamentous actin (Figure 2G). Identifi- cation of MT in control A431 cells resulted in


E G F effects on cytoskeletal structures

Figure 1. EGF-induced morphological alterations in MTLn3 and A431 cells. MTLn3 (A, B) and A431 (C, D) cells were allowed to attach overnight. 150 ng/ml EGF (B, D) or PBS (A, C) were added and after 5 min the cells were fixed with glutaraldehyde. Large arrows: cell extensions, filopodia; small arrows: rounded cells. Bar = 50/~m.

diffuse staining, maybe due to the fact that at low serum concentrat ions (1%) cells do not adhere that well (Figure2F). When E G F induced the format ion of blebs in A431 cells they were not stained for tubulin (Figure 2H) while filopodia with close contact to the substratum were filled with discretely stained MT (Figure 2J).

Besides its effects on the distribution of cytoskeletal structures, E G F could also affect the dy- namic equilibrium between m o n o m e r - p o l y m e r formation. Therefore , we prepared detergent-insoluble (cytoskeletal) fractions and identified cytoskeletal proteins actin (Figure3B) and tubulin (Figure3C) by Western blotting in control and EGF- t rea ted tumor cells. F i gu re3A demonstra tes that equal amounts of protein were used in the respective groups. All manipulat ions were carried out at room tempera ture , since some cytoskeletal structures are labile at low tempera tures and we

wanted to preserve the positional relationship between kinases and the respective substrates. Deter- gent lysis of adherent cells was per formed at very mild conditions (0.15% TX-100 for 0.5 min). Also, since MT are sensitive to Ca 2+, we included 1 mM E G T A into the lysis buffer. F igure3C demonstrates a 2- to 3-fold increase in the amount of tubulin associated with the detergent-resistant cytoskeletal fraction in EGF- t rea ted as compared with control MTLn3 cells, but not in A431 cells as determined in three independent experiments by scanning the autoradiographs (Table2) . Thus, E G F t rea tment of intact MTLn3 cells induced a shift f rom soluble tubulin into the microtubule fraction. Since the experimental conditions were chosen in such a way that MT are stable ( - C a 2 + ) , no difference in the association of actin with the cytoskeleton of either cell line was to be expected (Figure 3B).



Figure 2. Immunofluorescent staining of MF and MT in MTLn3 and A431 cells stimulated with EGF. MTLn3 ( A - D ) and A431 ( E - J ) cells were allowed to attach on glass coverslips overnight. 150 ng/ml E G F (C, D, G - J ) or PBS ( A , B , E , F ) were added and after 5 min cells were processed for staining of MF ( A , C , E , G , I ) and MT (B, D, F, H, J). Bar = 10/~m.



Figure3. Identification of tubulin and actin in cytoskeletal fractions of control and EGF-stimulated cells by Western immunoblot analysis. A431 (lanes 1,2) and MTLn3 (lanes 3, 4) cells were stimulated for 5 rain with 150 ng/ml EGF (lanes 2,4) or PBS (lanes 1,3) and cytoskeletons of adherent cells prepared by mild detergent lysis (0.15% TX-100 for 0.5 min). Ten pg protein was separated on 4-15% SDS-PAGE, transferred to nitrocellulose, probed with anti-or actin (B) or anti-tubulin (C) antibody and identified with the ECL Western blotting detection system. For the Coomassie Blue stained gel (A) 50/~g protein was loaded.

Table2. Association of actin and tubulin with the cytoskeletal fraction

MTLn3 A431

Actin 1.2" +_ 0.2 ~ (0.9-1.4)' 1.1 + 0.1 (1.0-1.2) Tubulin 2.5 _+ 1.3 (1.2-4.4) 0.9 + 0.2 (0.6-1.3)

"Relative amount of protein in EGF-treated vs control group as determined by densitometric scanning of autoradiographs (see Figure 3). ~'S.D. of at least three independent experiments. 'Range.

It is well established that enhancement of tubulin assembly by agents that increase intracellular cAMP results in an increase in cell spreading [39] and cell adhesion avidity [3,32,40]. Thus, we tested if the differential effects of E G F on spreading of MTLn3 and A431 cells find a correlate in their adhesive propert ies to fibronectin. Figure 4 shows that MTLn3 cells bind to immobilized fibronectin at a low rate and that addition of E G F

• % of Cells bound

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40

30

20

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"['--7" f " - , , .,,,,,.

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Figure4. Kinetics of adhesion of radiolabeled MTLn3 (C)) and A431 (O) cells to fibronectin (10/~g/ml) coated dishes in the absence (solid line) or presence (dashed line) of i0 ng/ml EGF.

increased adhesion of MTLn3 cells dramatically, 2- to 3-fold. In contrast, adhesion of A431 cells to fibronectin was rapid, reaching 50% after a 30-min incubation period, and was decreased considerably upon addition of EGF.

Since EGF-induced signal transduction is known to be mediated by phosphorylat ion events, we per formed experiments to determine whether the observed morphological changes were accompa- nied by changes in the phosphorylat ion pat tern of cytoskeleton-associated proteins. Intact, adherent cells were stimulated with E G F for 5 min, lysed by mild detergent t rea tment in the presence (1 mM) or absence of Ca 2+ and activity of kinases initiated by the addition of ATP. Figure 5 demonstra tes an autoradiograph of detergent-resistant material , including the surface lamina, the nucleus, fibrous structural elements and associated proteins [41] of



Figure 5. EGF-induced phosphorylation of proteins associated with the cytoskeleton. MTLn3 (lanes 1, 2) and A431 cells (lanes 3, 4) were incubated for 5 min with 150 ng/ml EGF (lanes 2, 4) or PBS (lanes 1, 3), cytoskeletons of adherent cells prepared by mild detergent lysis (0.15% TX-100 for 1 min) and kinase reactions initiated by the addition of 10/~M ATP plus 10/~Ci [y-32p]ATP. After incubation for 10min at room temperature the reaction was terminated by several washes and subsequent addition of sample buffer. Equal amounts of TCA-precipitable radioactivity were analysed by one-dimensional SDS-PAGE. Autoradiograph of untreated gel.

Ponsting or obtained from Amersham Buchler) failed to precipitate a phosphorylated 280 kD protein from A431 or MTLn3 cells (data not shown). Next we used an antiserum against fodrin (generous gift of Dr W. W. Franke) in immunoprecipitation studies using phosphorylated, EGF-stimulated A431 cells. Figure6 demonstrates clearly a dis- tinctive difference in molecular weight of the precipitated proteins and the 280 kD protein. The identity of the 280 kD protein remains unclear.

EGF-induced phosphorylation of the 280kD protein in MTLn3 cells was readily detectable at 10 ng/ml EGF. In order to test the E G F concentration dependence, intact MTLn3 cells were incubated with 0.1-200 ng/ml E G F for 5 min prior to cytoskeleton preparation. Figure 7 shows SDS-gel analysis of phosphorylated cytoskeletal proteins on a 4 -15% gradient gel. An E G F concentration- dependent increase in phosphorylation of the 280 kD protein is evident (arrow), while no signifi- cant changes in phosphorylation of lower molecular weight proteins were induced by E G F treatment (data not shown). Maximal phosphorylation

MTLn3 and A431 cells, separated by one-dimensional S D S - P A G E . A series of proteins were phosphorylated constitutively in cytoskeletons of both cell lines, while no EGF-dependent phosphorylation of a 170 kD protein (EGFR) could be identified in either cell line (Figure 5, lanes 2 and 4). However , E G F increased phosphorylation of a protein with M r of 280 kD. In A431 cells this 280 kD protein was barely visible in controls and E G F changed its phosphorylation level only marginally, while there was a clear increase in MTLn3 cells (lanes 1 and 2). The phosphorylation patterns described were for both cell lines independent of the presence of Ca 2+ (data not shown).

In order to define the identity of the 280 kD protein and based on the observation that it might be involved in MT dynamics, we performed immunoprecipitation studies using three different antibodies against microtubule-associated proteins (MAPs). However , all three antibodies which were raised against brain MAPs (generous gift of Dr H.

Figure6. Size comparison of phosphorylated 280 kD protein and phosphorylated fodrin. MTLn3 cells (lane 1) were incubated for 5 min with 10 ng/ml EGF. Prepara- tion of cytoskeleton and initiation of kinase reaction were performed as described in Figure 5. A431 cells (lane 2) were prelabeled for 4 h with 32p i and for the last 5 min EGF (i0 ng/ml) was added. Immunoprecipitation was performed with 50/~1 of a fodrin antiserum. Auto- radiograph of untreated gel. Arrows indicate 280 kD protein in lane 1 and 240 kD protein in lane 2.



Figure 7. Concentration dependence of EGF-stimulated phosphorylation of the 280 kD cytoskeletal protein in MTLn3 cells. Tumor cells were incubated for 5 min with various concentrations of EGF (lane 2: 0.1; lane 3: 1; lane 4: 5; lane 5: 10; lane 6:200 ng/ml) or PBS (lane 1). Preparation of cytoskeletons and initiation of kinase reaction were performed as described in Figure 5. Auto- radiograph of proteins separated on a 4-7% untreated gel. Arrow indicates 280 kD protein.

levels were reached between 10 and 150ng/ml EGF, which also induced maximal growth response in MTLn3 cells [27].

Finally, we tested the kinetics of EGF-induced phosphorylat ion in MTLn3 cells. E G F (10 ng/ml) was added to intact cells f rom 5 min to 2 h prior to cytoskeleton preparat ion. F igure8 demonstra tes that maximal phosphorylat ion of the 280 kD protein was reached within 5 min, remained constant for 20 min and declined thereaf ter (within 1-2 h) to control level.

Discussion

The ability to proliferate when presented with E G F correlated with the metastat ic capability in two syngeneic animal models, a rat rhabdomyosarcoma [42] and the 13762NF rat m a m m a r y adenocarcinoma [27]. Further biochemical characteriza-

FigureS. Time course of EGF-induced in situ phosphorylation of the 280 kD cytoskeletal protein in MTLn3 cells. Tumor cells were incubated with 10 ng/ml EGF for various time periods (lane 2:5 rain; lane 3:20 min; lane 4: lh ; lane 5: 2h) or for 5min with PBS (lane 1). Preparation of cytoskeletons and initiation of kinase reaction were performed as described in Figure 5. Auto- radiograph of untreated gel.

tion of highly metastatic subclone MTLn3 derived f rom the 13762NF tumor system revealed properties of E G F R which were disparate compared with the receptor expressed in human A431 cells, which provide the most extensively studied model of E G F action. MTLn3 cells express 30 times fewer E G F R than A431 cells (6 x 104 as compared with 2 x 106 receptors per cell) and 20% are of the high-affinity type [26]. Most importantly, no receptor phosphorylat ion could be identifiec'l in intact cells. However , E G F R s associated with the cytoskeleton of detergent- t reated cells were able to undergo basal and EGF-induced phosphorylat ion in vitro. It is therefore tempting to suggest that the cytoskeleton has an important function in signal transduction. A number of cytoskeletal components, such as fodrin, spectrin, tubulin and MAP2 [43], ezrin [15] and lipocortin 1 [44], have been shown to be phosphoryla ted in vivo and in vitro by E G F R kinase. At present the nature of the interaction between E G F R and the cytoskeleton is unknown. It does not result f rom E G F R auto- phosphorylat ion, kinase activity or high affinity as shown in an intricate study by van Belzen et al. [45] investigating the association of muta ted receptors with the cytoskeleton. There is indirect evidence suggesting that actin is the cytoskeletal ele- ment involved in E G F R binding. In double label



immunofluorescent studies it was shown that the distribution of EGFR on the cell surface coincides with the intracellular distribution of actin [46]. Interestingly, co-alignment between actin filaments at the cell surface and extracellular fibronectin had been demonstrated in fibroblasts [47]. Further- more, the receptor for fibronectin belongs to the family of integrin receptors which have been shown to form a linkage between the extracellular matrix and the cytoskeleton of the cell [48]. Thus, we suggest that the rapid effects of EGF on adhesion of MTLn3 and A431 cells to fibronectin indicate a functional linkage between EGFR, the cytoskeleton and the receptor for fibronectin.

Binding of EGF to its receptor could affect the activity of integrin receptors via affecting the function of cytoskeleton structures. Alternatively, a direct modulation of integrin activity via phosphorylation by EGFR kinase might be possible. However, a recent report by Kornberg et al. [49] does not support such a mechanism. Nevertheless, intracellular signals have been shown to be trig- gered by integrins in synergy with growth factors (for review, see Ref. 50). In immunoprecipitation studies using 0~PTyr antibodies, we observed in MTLn3 but not in A431 cells EGF-induced increase in phosphorylation of a protein with Mr of 120 kD [26]. Both cell lines investigated reacted to the addition of EGF with modulation of their adhesive properties to matrix components, but in opposite directions. This further supports our hypothesis of a functional linkage between EGFR, the cytoskeleton and integrin receptors.

A further difference between MTLn3 and A431 cells regarding their response to EGF was the rapid phosphorylation of a cytoskeleton-associated 280 kD protein with concomitant increase in microtubule formation in rat tumor cells. The first question relates to the nature of the 280 kD protein. Several cytoskeleton-associated high molecular weight proteins have been identified which are involved in regulation of cytoskeleton dynamics. For example, spectrin (240 kD) is phosphorylated in A431 cells upon EGF stimulation [15]. However, we could clearly demonstrate a distinc- tive difference in molecular weight of the 280 kD protein and phosphorylated fodrin/spectrin by using specific antisera. The high molecular weight protein plectin (300 kD) was suggested to cross- link spectrin and intermediate filaments with MAPs [51, 52]. However, so far no information is available as to a possible link between plectin phosphorylation and EGF stimulation of receptive cells. The same applies to gyronemin, a recently

identified 240kD protein which associates with intermediate filaments [53].

Phosphorylation of the 280 kD protein and the 2.5-fold increase in MT formation occur concomi- tantly within minutes of exposing cells to EGF. It could be speculated that this protein might repre- sent a member of the MAPs which control MT initiation, elongation or disassembly [54]. In fact, there is evidence that MAP1 (330-350 kD) and/or MAP2 (270-300 kD) could be involved in growth factor mediated signaling pathways. For example, in PC12 cells, nerve growth factor (NGF) mediates neuronal differentiation, including MT assembly, stability and cross-linking. NGF treatment resulted in increased phosphorylation of a 250 kD cytoskeletally-associated protein in PC12 cells [55]. Also in cultured cells of non-neuronal origin, stimulation by different growth factors or phorbol esters induced phosphorylation of MAP2-1ike high molecular weight proteins [18, 56] and extracts of stimulated cells were able to phosphorylate exo- genous MAP2 on serine [57, 58]. Since MT are present almost exclusively in the cytoplasm, it has been considered that MAPs function in the cytoplasm as part of the microtubular structure [59].

The phosphorylation/association of the 280 kD protein with the cytoskeleton was independent of Ca 2+. Therefore, it seems unlikely that this protein is associated with Ca2+-sensitive cytoplasmic MT. However, MAPs also were found to be present in phosphorylated forms, in the nuclei of actively proliferating mammalian cells in culture [56]. All our attempts to immunoprecipitate and thus characterize the phosphorylated 280 kD protein from EGF-stimulated MTLn3 cells with three monoclonal antibodies recognizing brain MAPs were unsuccessful. Antibodies against rat epithelial MAPs (MAPs exhibit cell type rather than species specificity [60]), plectin and gyronemin were not available to us.

The second question relates to the nature of the kinase phosphorylating the 280 kD protein. There are several kinases known which are activated upon EGF stimulation of cells. For example, a cytosolic, EGF-activated 100kD serine protein kinase has been identified in A431 cells [61]. Furthermore, an ubiquitous serine protein kinase (p42) phosphorylates MAPs and becomes itself transiently phosphorylated on tyrosine by a variety of mitogens including EGF, PDGF, TPA, throm- bin and IGF II [62]. This enzyme is known as MAP kinase (mitogen-activated protein kinase) and has been characterized and partially purified from 3T3-L1 adipocytes [63]. In intact MTLn3



cells a protein could be identified within the same molecular weight range as MAP kinase and with similar time kinetics of tyrosine phosphorylation [26]. However, without specific antibodies the ex- act nature of this protein could not be defined.

In summary, we demonstrated rapid effects of E G F on the adhesive properties of tumor cells. We propose a functional linkage between trans- membrane receptors for E G F and matrix proteins. Thus, tumor cells expressing E G F R and respond- ing upon exposure to the ligand with increased interaction with matrix proteins might improve their chances to lodge in target organs and form distant metastases.

Acknowledgements

We thank Ms M. Schleicher for expert technical assistance, Dr W. W. Franke for the generous gift of antiserum against fodrin, Dr H. Ponstingl for helpful discussions, Dr H. Herrmann and K. Kha- zaie for critical reading of the manuscript, and Ms B. Engelhardt, J. K611ner and R. Ktihnl-Bontzol for help with photographic work. This work was supported by Deutsche Forschungsgemeinschaft and Wilhelm Sander Stiftung.

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