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Originally published In Press as doi:10.1074/jbc.M302720200 on September 18, 2003

J. Biol. Chem., Vol. 278, Issue 48, 47946-47959, November 28, 2003
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v-Src Rescues Actin-based Cytoskeletal Architecture and Cell Motility and Induces Enhanced Anchorage Independence during Oncogenic Transformation of Focal Adhesion Kinase-null Fibroblasts*

Konstadinos Moissoglu{ddagger} and Irwin H. Gelman§

From the Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263

Received for publication, March 17, 2003 , and in revised form, September 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Srcinduced pathways, especially as they relate to oncogenic transformation, remain unclear. Here, we investigate the role of FAK in v-Src-induced oncogenic transformation by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK–/– mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induced abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK–/– MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescued the haptotactic, linear directional, and invasive motility defects of FAK–/– cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there was an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK–/– MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK–/–[ts72v-Src] clones exhibited 7–10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK–/–[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Srcinduced anchorage independence. Our data also indicate that the enhanced Pyk2 level found in FAK–/– MEF plays no role in v-Src-induced anchorage independence. Overall, our data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Signals mediated through the interaction of integrins with extracellular matrix (ECM)1 proteins, such as fibronectin, control many significant facets of cell morphology, motility, survival, proliferation, and differentiation (reviewed in Refs. 1–3). The focal adhesion kinase (FAK) is involved intimately in mediating integrin-induced signaling and cytoskeletal pathways through its intrinsic tyrosine kinase activity and through multiple docking functions that recruit signaling and cytoskeletal proteins to sites of activation (reviewed in Refs. 4 and 5). For example, integrin clustering leads to the autophosphorylation of FAK at Tyr397, producing a common docking site for the Src homology 2 domains of Src family tyrosine kinases, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), and the Shc adaptor protein. Binding by Src activates its own kinase domain, which subsequently phosphorylates FAK at multiple sites, leading to enhanced FAK kinase activity (6, 7) and the creation of additional Src homology 2-dependent docking sites (8). Other FAK docking sites bind Crk-associated substrate (p130CAS), the GTPase-activating protein Graf, the signaling adaptor protein Grb2, and the cytoskeletal proteins talin and paxillin, which facilitate binding to {beta}-integrin cytoplasmic domains and to the actin-based cytoskeleton.

Deletion of the FAK gene leads to early embryonic lethality, indicating a critical requirement for FAK in developmental processes (9). However, the presence of increased numbers of focal adhesion complexes in FAK-deficient mouse embryo fibroblasts (MEF) underlines a role for FAK in regulating focal adhesion turnover (9). A confounding issue is that all reported isolates of FAK–/– MEF express higher endogenous levels of the FAK-related kinase, Pyk2 (10–13). Pyk2 overexpression induces some of the cytoskeletal rearrangements and the epithelioid morphology found in FAK–/– MEF (12, 14–16), and at least one study suggests that Pyk2 can partially rescue activation of extracellular signal-regulated kinase (ERK)-2 in FAK–/– MEF.

Some Src-mediated pathways remain active in FAK-deficient cells, such as ERK-2 activation following cell adhesion to fibronectin (10), whereas other pathways, such as motility and cytoskeletal reorganization, are defective (17). The tyrosine phosphorylation of CAS by an activated FAK-Src complex (18) and its subsequent coupling to the Crk adaptor protein have been correlated with increased cell motility (19), possibly as a result of a direct linkage with Rac1 (20, 21), a GTPase involved in cytoskeletal reorganization (22). However, the relatively immotile phenotype of FAK–/– fibroblasts (9), even though these cells exhibit increased levels of CAS tyrosine phosphorylation (23, 24), indicates the dependence on FAK-mediated signals for full motility. Additionally, spatiotemporal constraints seem to be critical because Pyk2 is unable to rescue the immotility of FAK–/– cells as a result of its lack of a focal adhesion targeting protein domain found at the C terminus of FAK (25).

Specific roles for Src family kinases have been difficult to dissect, in part because of the functional redundancy among family members as well as overlapping functions contributed by the FAK/Pyk2 family. One mechanism has been to correlate changes in cell behavior and signaling controls with tyrosine phosphorylation events induced during oncogenic transformation by the viral oncogene for v-Src. The list of potential Src substrates includes structural proteins involved with cytoskeletal architecture and sites of cell-cell and cell-ECM contact (e.g. vinculin, tensin, talin, cortactin, AFAP 110, {beta}1 integrin, {beta}-catenin, {gamma}-catenin, p120CAS), as well as signaling proteins and nuclear transcription factors (e.g. FAK, p85 subunit of PI3K, phospholipase C{gamma}, p120RasGAP, p190RhoGAP, Shc, paxillin, p130CAS, and Stat3) (reviewed in Ref. 26). However, it has been difficult to establish a causative role for these tyrosine phosphorylation events in specific parameters of v-Src-induced oncogenesis such as anchorage-, contact-, and mitogen-independence, metabolic changes, cytoskeletal reorganization, decreases in the number of adhesive sites, and increased motility (27). Only recently, several reports demonstrated the requirement of Stat3 activation for v-Src-induced transformation of mouse fibroblasts (28, 29). In contrast, transformation of chicken fibroblasts involves both the Ras-mitogen-activated protein kinase pathway and the PI3K-mTOR pathway (30, 31).

Although it is assumed that FAK plays a positive role in oncogenesis based on its increased expression and activity in many metastatic tumors (32–36), it is unclear whether specific aspects of v-Src-induced oncogenic transformation require FAK. To address this issue, we used retroviruses to transduce a temperature-sensitive v-Src allele (ts72v-Src) into otherwise genetically matched FAK+/+ and FAK–/– mouse embryo fibroblasts. Our data indicate that FAK is dispensable for v-Src-induced growth factor and anchorage independence, morphological transformation, cell motility, and cytoskeleton reorganization. Data are presented showing an unexpected 7–10-fold enhancement in soft agar colony formation frequency induced by v-Src in the absence of FAK. This phenomenon was attenuated by FAK re-expression but was unaffected by either ts72v-Src or Pyk2 levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines
FAK+/+ and FAK–/– MEF from p53–/– genetic backgrounds (gift of T. Yamamoto and S. Aizawa, University of Tokyo, Tokyo, Japan) and {varphi}NX ecotropic packaging cells (gift of G. Nolan) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Generation of Temperature-sensitive v-Src-expressing Clones
A BamHI/EcoRI fragment from pBluescript/ts72v-Src (37) encoding a temperature sensitive v-Src gene from Rous sarcoma virus strain NY72 (38) was inserted into the retroviral vector pBABE/puro (39). Replication-defective virus was harvested 48 h following transfection of {varphi}NX ecotropic packaging cells (40) with 20 µg of pBABE/puro or pBABE/puro/ts72v-Src DNA/106 cells and then filtered through 0.45-µm pore size low protein-binding filters (Pall/Gelman, Ann Arbor, MI). FAK+/+ and FAK–/– MEF were infected for 24 h in the presence of 5 µg ml–1 Polybrene (Sigma), and 48 h following infection the cultures were split 1:10 into media containing 2 µgml–1 puromycin for colony formation at 35 °C (PT). Clones were selected after growth under soft agar that were morphologically transformed at the PT but untransformed after 1–3 days of growth at 39.5 °C (NPT).

For FAK re-expression, a BamHI fragment from pSR{alpha}-FAK (gift of K. Maruyama) containing the mouse FAK cDNA was inserted into the retroviral vector pBABE/hygro (39). Viruses transducing the empty vector or FAK were produced as described above and used to infect FAK–/–[ts72v-Src] MEF at the PT. Clones of FAK re-expressing MEF were obtained after 2 weeks of hygromycin (300 µg ml–1) selection.

Activation of ts72v-Src
Cells were kept at the NPT for 72 h prior to shift to the PT. Cells were processed for immunofluorescence and protein analysis, as described below, at the indicated time points after the temperature shift.

Protein Analysis
Cells growing at the PT or NPT were washed with cold PBS and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 8% glycerol, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl) with inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM NaF, 5 mM sodium pyrophosphate, and 2 mg ml–1 each of aprotinin, leupeptin, antipain, and pepstatin). Cell lysates were clarified by centrifugation at 4 °C, protein concentration was measured by the Bradford assay (Bio-Rad), and aliquots normalized for protein content were used for immunoprecipitation. 200–500 µg of total protein was incubated for 4 h at 4 °C with the following antibodies: anti-FAK polyclonal antibody A-17 (1.25 µg ml–1; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p130CAS mAb (1.5 µg ml–1; Transduction Laboratories/Pharmingen, San Diego, CA), anti-Pyk2 mAb (1.5 µg ml–1; Transduction Laboratories), and anti-paxillin mAb (1 µg ml–1; Transduction Laboratories). Immune complexes were immobilized on protein A/G PLUS agarose (Santa Cruz Biotechnology), washed three times with RIPA buffer, and boiled in Laemmli buffer. Immunoprecipitates or equal amounts of protein were separated by SDS-PAGE, electrophoretically transferred to PolyScreen polyvinyl difluoride membrane (PerkinElmer Life Sciences) and immunoblotted, as described before (41), with the following primary antibodies: anti-Tyr(P) 4G10 mAb (1:5000; Upstate Biotechnology, Lake Placid, NY), anti-FAK polyclonal antibody (gift of Jun-Lin Guan), anti-v-Src EC10 mAb (1:1000; Ref. 32), anti-actin AC-40 mAb (1:1000; Sigma), anti-p130CAS mAb (1:1000; Transduction Laboratories), anti-paxillin mAb (1:5000; Transduction Laboratories), and anti-Pyk2 mAb (1:1000; Transduction Laboratories). Secondary antibodies were horseradish peroxidase-conjugated antimouse or anti-rabbit Ig (Chemicon, Temecula, CA) followed by ECL substrate (PerkinElmer Life Sciences). Membranes were stripped of antibody as described previously (42).

Proliferation Assay
Cells (104) were seeded onto 35-mm dishes and allowed to grow under high (10% FBS) or low (0.5% FBS) serum conditions. Triplicate dishes were trypsinized and counted every 2 days using a hemacytometer (Fisher Scientific).

Anchorage-independent Growth
Growth in soft agar was assayed in 60-mm dishes prepared with a lower layer of 0.7% agar (Difco Laboratories, Franklin Lakes, NJ) overlaid with top agar (0.4%) containing 104 suspended cells. 15 days after plating, colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma) and counted. For growth in methylcellulose, 2.5–5 x 106 cells were mixed into 50 ml of medium containing 1.3% methylcellulose (Sigma) and allowed to incubate in Petri dishes for 24–48 h. Cells were collected by centrifugation, washed with cold PBS, and lysed as described above.

Stimulation with Fibronectin
Cell stimulation with fibronectin was performed as described previously (43) with the following modifications: cells were serum starved for 18 h in DMEM containing 0.5% FBS and harvested by limited trypsin-EDTA treatment (0.05% trypsin and 2 mM EDTA in PBS). Trypsin was inactivated in DMEM containing 0.5 mg ml–1 soybean trypsin inhibitor (Roche Molecular Biochemicals) and 0.5% BSA (Sigma), and cells were collected by centrifugation, resuspended in 0.5% FBS-containing DMEM and kept in suspension for 1 h at the PT (105 cells ml–1) prior to replating onto fibronectin- or poly-L-lysine-precoated 10-cm dishes (10 µg ml–1 fibronectin or 100 µg ml–1 poly-L-lysine in PBS, incubated overnight at 4 °C). 106 cells were seeded onto the precoated dishes and allowed to attach for 1 h at the PT. Attached cells and cells in suspension were lysed as described above.

Immunofluorescence
Cells grown on 22-mm2 coverslips were fixed in 60% acetone, 3.7% formaldehyde at –20 °C for 20 min or, for v-Src analysis, in 3.7% formaldehyde at 4 °C for 15 min followed by permeabilization with 0.5% Triton X-100 at room temperature, as described previously (43). Cells were incubated with the following primary antibodies: anti-v-Src mAb EC10 (1:100), anti-Tyr(P) PY20 mAb (1:200; Transduction Laboratories), anti-paxillin mAb (1:250), anti-vinculin mAb (clone hVIN-1; 1:250; Sigma). Primary antibodies were detected with fluorescein isothiocyanate- or TRITC-conjugated goat anti-mouse or anti-rabbit IgG, respectively (1:250; Chemicon). Actin was visualized with rhodamine-conjugated phalloidin (Sigma). Images were obtained on an Olympus IX-70 fluorescence microscope fitted with a Sony Catseye digital camera and processed using Photoshop version 4.01 (Adobe).

ts72v-Src Half-life Determination
FAK+/+ and FAK–/– [ts72v-Src] cells grown at the PT for 2 h in methionine- and cysteine-free DMEM (ICN, Costa Mesa, CA) containing dialyzed 10% FBS were pulsed for 2 h with 50 µCi ml–1 Tran35S-label (methionine/cysteine; ICN), followed by one wash with PBS and a chase with DMEM containing 10% FBS plus a 5 M excess of cold methionine and cysteine (Sigma). Cells were lysed in RIPA buffer, and then equal protein aliquots were subjected to v-Src-specific immunoprecipitation as described above. The immunoprecipitations were resolved by SDS-PAGE, and then phosphorimaged (Amersham Biosciences) or immunoblotted for v-Src as described above.

Cell Motility Assays
Linear Motility—Cells plated sparsely (3 x 103 cells on gridded 22-mm CELLocate® coverslips; Eppendorf, Hamburg, Germany) were photographed every 60 min for the first 12 h, and then once at 20 h, for linear, directional motility. At least three independent, fixed loci were monitored to establish the origin (O) points of individual cells (at least 10/field). Directional motility is defined as the linear distance moved (D) from origin (O) to the end point of the cell (E).

Wound Healing—Clones of FAK–/– and FAK+/+ [ts72v-Src] cells were plated at confluence at the PT or NPT on gridded 22-mm coverslips. Monolayer wounds were produced using pipette tips, and then fixed loci were photographed at the times indicated.

Haptotaxis—Assays for haptotaxis toward fibronectin were carried out in modified Boyden chambers containing 8-µm pore size, polyethylene tetraphthalate track-etched membranes (Falcon/Becton Dickinson). Membranes were coated overnight at 4 °C on the underside with human fibronectin (Becton Dickinson; 2.5, 5 and 7.5 µg/ml in PBS), rinsed with PBS, and placed into 24-well plates containing 500 µl of DMEM supplemented with 0.5% FBS and 0.5% BSA. Cells were serum-starved for 18 h in DMEM containing 0.5% FBS, trypsinized and resuspended in DMEM containing 0.5% FBS, 0.5% BSA, and 0.5 mg/ml soybean trypsin inhibitor (Sigma). Cells were then pelleted by centrifugation, resuspended in DMEM containing 0.5% FBS and 0.5% BSA, and counted. 104 cells/300 µl were plated onto the upper side of the membrane, and migration was allowed to proceed for 4 h at the PT. At the end of the assay, cells on the upper surface were removed with a cotton swab and cells that migrated to the underside of the membrane were fixed and stained with Giemsa. Five independent fields of cells within each chamber were monitored under a 10x objective, and stained cells were scored. In control experiments, chambers without fibronectin coating were used for migration assays.

Invasion—The ability of cells to migrate through Matrigel®-coated Boyden chambers has been described previously (41).

ts72v-Src in Vitro Kinase Assays
Equal amounts of protein from RIPA lysates of FAK+/+ and FAK–/– [ts72v-Src] cells grown at the PT were subjected to ts72v-Src immunoprecipitation as described above. Immune complexes were washed twice with Triton-only buffer (RIPA buffer without SDS and deoxycholic acid), twice with HNTG buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol), and once with kinase buffer (150 mM NaCl, 20 mM HEPES, pH 7.4, 10% glycerol, 10 mM MgCl2, 10 mM MnCl2). Immune complexes were then resuspended in kinase buffer containing 5 µg of enolase (Sigma) and 25 µCi of [{gamma}-32P]ATP (NEN), and kinase reactions were allowed to proceed for 15 min at 32 °C. Reactions were terminated by the addition of 2x SDS-PAGE buffer and boiling and resolved by SDS-PAGE. The lower part of the gel containing enolase was stained with Coomassie Brilliant Blue R250, dried, and autoradiographed. The upper part of the gel containing immunoprecipitated ts72v-Src was transferred to polyvinylidene difluoride membrane, followed by immunoblotting for v-Src and autoradiography (autophosphorylation).

Pyk2 RNAi
For RNAi, 29-mer oligonucleotides mapping to the C terminus of murine Pyk2 (ENSEMBL transcript ENSMUST00000022622) were chosen according to the rules described in the Hannon laboratory web site at Cold Spring Harbor Laboratories (www.cshl.org/public/SCI-ENCE/hannon.html). Oligonucleotides (Invitrogen, Carlsbad, CA) consisted of two palindromic 29-mer sequences flanking a 10-nt "bubble" encoding a HindIII site. Complementary oligonucleotides had 5' BamHI and 3' BseRI overhangs. For duplex formation, equimolar amounts of complementary oligonucleotides were boiled in buffer containing 20 mM Tris-HCl, pH 8.4, and 50 mM KCl and allowed to slowly reach room temperature. Duplexes were then phosphorylated with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and ligated into pSHAG (Refs. 44 and 45; gift of G. Hannon, Cold Spring Harbor Laboratory) cut with BseRI and BamHI. Control oligonucleotides were similarly designed to target the pGL3 firefly luciferase transcript (pSHAG-FF; gift of G. Hannon). Recombinant plasmids were identified by HindIII digestion and further verified by sequencing.

Transfections and FACS
Cells were transfected with a 5:1:1 ratio of pCMV-Myc/Pyk2 (Myc tag at the N terminus, gift of W.-C. Xiong; Ref. 14) or pSHAG/Pyk2 RNAi, pEGFP (Clontech/Becton Dickinson), and pBABE/hygro (39) as either CaPO4 precipitates or admixed with LipofectAMINE 2000 (Invitrogen) according to the instructions of the manufacturer, and GFP-expressing cells were sorted under sterile conditions using a FACSVantage SE (Becton Dickinson) with technical help from the Mount Sinai Flow Cytometry Shared Research Facility.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FAK Is Dispensable for Morphological Transformation and Cytoskeletal Rearrangements Induced by v-Src—To evaluate the role of FAK in v-Src-induced transformation, FAK–/– and FAK+/+ MEF were transduced with ecotropic retrovirus encoding ts72v-Src, whose tyrosine kinase is active enzymatically at 35 °C (PT) yet inactive at 39.5 °C (NPT). Independent drugresistant clones were obtained that expressed similar levels of ts72v-Src (Fig. 1B), and multiple clones were examined to exclude clonal idiosyncrasies. This system allowed us to (a) control cellular transformation with temperature, (b) assess the role of FAK in specific parameters of v-Src-induced transformation, and (c) observe the morphological and biochemical properties of the cells at the NPT versus the PT or in a time course following activation of v-Src. Roughly 20% of the puromycin-resistant FAK+/+ colonies displayed robust morphological transformation at the PT, whereas virtually all the FAK–/– colonies were well transformed (data not shown). Although the steady-state level of ts72v-Src protein was roughly 2-fold higher in all FAK–/– clones compared with FAK +/+ controls (Fig. 1, B and D), the relative specific activities of ts72v-Src were similar in FAK–/– and FAK+/+ backgrounds, as assessed by in vitro autophosphorylation and phosphorylation of the exogenous substrate, enolase (Fig. 1D). Additionally, the stability of ectopically expressed ts72v-Src was similar in FAK+/+ and FAK–/– [ts72v-Src] clones (Fig. 1E). These data indicate that the expression levels, specific activities, and stabilities of ts72v-Src are comparable in FAK+/+ and FAK–/– cells.



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  		FIG. 1.
ts72v-Sc-induces abundant tyrosine phosphorylation in FAK–/– MEF. Panel A, total phosphotyrosine levels in clones of FAK+/+ and FAK–/– [ts72v-Src] clones grown at the PT or NPT. Phosphotyrosine substrates whose abundance are increased in the FAK+/+ background at the PT are noted with arrows, whereas those substrates unique to the FAK–/– background are noted with asterisks. Equal protein loading was verified by Amido Black staining (data not shown). Panel B, the blot from panel A was stripped and reprobed with antibodies specific for v-Src and FAK. Panel C, endogenous phosphotyrosine (pTyr) substrates in parental FAK+/+ (+/+) and FAK–/– (–/–) cells grown at the PT or NPT, showing no induction of tyrosine phosphorylation at the PT in the absence of v-Src. Exposure of the chemiluminescence signal was 30 s, compared with the blot in panel A, which was 3 s. Panel D, protein, autokinase, and specific activities of ts72v-Src expressed in FAK+/+ or FAK–/– backgrounds. Equal aliquots of protein from the lysates in panel B were subjected to immunoprecipitation (IP/IB) for v-Src (mAb327) and then analyzed as described under "Materials and Methods" for v-Src levels, autokinase activity, or kinase activity against an exogenous substrate, enolase. Enolase levels were verified by SDS-PAGE followed by Coomassie Blue staining. Autophos., autophosphorylation. Panel E, the half-lives of ts72v-Src proteins in either FAK+/+ or FAK–/– [ts72v-Src] clones were monitored by pulse-labeling cells with Tran35S-label (ICN) for 2 h, followed by a chase with DMEM plus 10% FBS supplemented with a 5 M excess of cold methionine and cysteine. Equal aliquots of protein were subjected to SDS-PAGE, followed by fluorography of dried gels. The half-life of the ts72v-Src proteins from either cell type was roughly 2.7 h. A single representative experiment of three repetitions is shown.

 
v-Src-induced transformation is accompanied by elevated tyrosine phosphorylation of multiple cellular substrates (46). FAK+/+ and FAK–/– [ts72v-Src] clones exhibited elevated levels of v-Src-induced phosphotyrosine substrates (Fig. 1A) compared with vector controls at the PT only (Fig. 1C). Densitometric analysis revealed similar overall levels of in vivo tyrosine phosphorylation in the FAK–/–and FAK+/+ [ts72v-Src] cells at the PT (Fig. 1A). Although the overall pattern of phosphotyrosine-containing substrates was similar, several phosphorylations were unique to or enhanced in the FAK+/+ background (Fig. 1A, arrows), whereas others were enhanced or novel in the absence of FAK (Fig. 1A, asterisks). Although the biological significance of these differences is unclear at present, these data indicate that FAK could manifest both positive and negative regulatory roles on the substrate specificity of v-Src.

FAK+/+ and FAK–/– [ts72v-Src] clones displayed similar transformed phenotypes at the PT, with some cells being fully rounded and others exhibiting a refractile, fusiform morphology (Fig. 2A, panels c and e). This phenomenon was v-Src-specific because the same cells reverted to the parental morphology at the NPT: spindle, fibroblastic morphology for FAK+/+, and polygonal epithelioid morphology for FAK–/– cells (Fig. 2A, panels d and f). Additionally, control cells transduced with empty vector showed no morphological changes at the PT (Fig. 2A, panels a and b).



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  		FIG. 2.
FAK is dispensable for v-Src-induced morphological transformation, cytoskeletal reorganization, and v-Src localization to focal adhesions in mouse embryo fibroblasts. Panel A, phase-contrast microscopy of FAK+/+[puro] or FAK–/–[puro] vector control cells grown at the PT (a and b) or FAK+/+[ts72v-Src] (c and d) or FAK–/–[ts72v-Src] (e and f) cells grown at the PT (c and e) or NPT (d and f). Panel B, immunofluorescence analysis of FAK+/+[ts72v-Src] (g–j) or FAK–/–[ts72v-Src] (k–n) cells grown at the NPT (g, i, k, and m) or 4 h after shift to the PT (h, j, l, and n) stained with anti-v-Src (g, h, k, and l) plus fluorescein isothiocyanate-labeled anti-mouse Ig, and rhodamine-labeled phalloidin (i, j, m, and n). Original magnifications: a–f, x100; g–n, x1000.

 
Activation of v-Src converted the epithelioid FAK–/– cells to a polarized fibroblastic morphology concurrent with the reorganization of F-actin from the cell periphery to longitudinal stress fibers (compare panels m and n in Fig. 2B). This was especially apparent in a time-lapse analysis following temperature shift to the PT (Fig. 3, panel A). v-Src enrichment at the focal adhesions and at the tips of actin stress fibers (Fig. 2B, panels h and l) and reorganization of the actin cytoskeleton (Fig. 2B, panels j and n) were indistinguishable in FAK+/+ and FAK–/– [ts72v-Src] clones at the PT. These results indicate that FAK is dispensable for Src-induced morphological transformation and cytoskeletal reorganization and, as reported by Fincham et al. (47), suggest that FAK-independent mechanisms target v-Src to sites of focal contact.



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  		FIG. 3.
Time-course analysis of cytoskeletal components after activation of v-Src. FAK–/– or FAK+/+ [ts72v-Src] clones grown for 3 days at the NPT were shifted for various times to the PT, then fixed and stained for paxillin, vinculin, F-actin, or phosphotyrosine (P-Tyr). Note the egress from focal complexes of paxillin in FAK–/–[ts72v-Src] cells (top) and of vinculin in FAK+/+[ts72v-Src] cells (bottom) after 20 h. In contrast, vinculin is retained in the FAK–/–[ts72v-Src] cells and paxillin is retained in the FAK+/+[ts72v-Src] cells (arrows; inset and enlargements).

 
v-Src Rescues the Non-polar Phenotype of FAK–/– Fibroblasts—v-Src-induced oncogenic transformation is accompanied by extensive cytoskeletal reorganization and reduction in the number of cellular adhesive sites concurrent with increased parameters of motility (48). FAK-null cells are marked by a lack of cell polarity and by an apparent increase in the number of vinculin-associated adhesion complexes (9). It has been suggested that these structures are "immature" focal adhesions because they are not found at the termini of F-actin fibers, correlating with defects in focal adhesion turnover and motility (49, 50). To address whether v-Src could induce cell polarity and cytoskeletal reorganization in the absence of FAK, FAK–/– and FAK+/+ [ts72v-Src] clones kept at the NPT were shifted to the PT and then analyzed after various durations for cellular localization of paxillin, vinculin, F-actin, and total phosphotyrosine. Fig. 3 shows that by 8 h after temperature shift-down, v-Src could convert the round, epithelioid FAK–/– cells into polarized cells. Additionally, based on monitoring paxillin and vinculin staining, v-Src rapidly converted the morphology of focal adhesions in FAK–/– from multiple minispikes equally dispersed at the cell periphery to larger focal adhesion plaques located at the ends of polar cell formations. This contrasts with FAK+/+[ts72v-Src] cells in which paxillin and vinculin remain enriched in focal adhesion plaques before and after activation of v-Src. Thus, v-Src can compensate for the formation of "mature" focal complexes in the absence of FAK. Twenty hours after v-Src activation, there was a similar decrease in adhesion plaque number in the FAK–/– cells compared with FAK+/+, indicating that v-Src compensates for adhesion plaque turnover in the absence of FAK. One distinction between FAK+/+ and FAK–/– backgrounds is that activation of v-Src for 20 h caused vinculin to egress fully from focal adhesions in FAK+/+ cells, whereas in FAK–/– cells, there was a significant decrease in paxillin enrichment in focal adhesions. Although the significance of this is unclear, it demonstrates a FAK-dependent role in regulating the egress of cytoskeletal proteins from focal contacts. Another distinction is that, in FAK–/– cells, v-Src-induced phosphotyrosine substrates failed to become enriched in focal adhesion sites as was the case in FAK+/+ cells. For example, most of the phosphotyrosine staining in FAK–/– cells after 1–8 h of v-Src activation localized at plasma membrane sites, typified by the halo-like staining at the cell surface, whereas in FAK+/+ cells, phosphotyrosine staining was enriched in discrete focal adhesion plaques over the same period. This suggests that FAK plays a role in regulating the recruitment of specific substrates to focal adhesion complexes following the activation of Src-family kinases. Taken together, these data identify both FAK-dependent and -independent pathways involved in v-Src-induced focal adhesion assembly/turnover and cytoskeletal reorganization.

ts72v-Src Rescues Cell Motility in the Absence of FAK— FAK–/– MEF are deficient in several parameters of cell motility, especially when assayed over the short term (<12 h) (51). Recent studies correlate this deficiency with cell polarity defects tied to overactivation of the Rho GTPase pathway (52, 53). We compared the motility of the FAK+/+ and FAK–/– [ts72v-Src] clones under several conditions including linear motility, haptotactic motility toward extra cellular matrix (fibronectin), or polarized motility into a monolayer wound. FAK has been shown to be critical for these forms of motility, and in the case of directionally linear motility, PI3K and CAS have been shown to be critical mediators (54). Table I shows that, although the FAK–/–[ts72v-Src] clones were relatively immotile at the NPT, no significant differences were noted in haptotaxis and linear motility between FAK+/+ or FAK–/– [ts72v-Src] clones at the PT, and in the case of wound healing, FAK–/–[ts72v-Src] clones exhibited a 2–3-fold enhancement of motility over the FAK+/+[ts72v-Src] clones. However, it should be noted that the precise quantification of wound healing motility may be affected by the ability of highly transformed cells to detach and reattach and/or to move over neighboring cells. Finally, although both FAK+/+ and FAK–/– parental cells were unable to significantly invade Matrigel®-coated transwells, there was equal v-Src-induced invasive motility in the FAK+/+ and FAK–/– backgrounds (data not shown). Thus, v-Src can compensate for FAK in parameters of cell motility, and moreover, v-Src-induced wound healing motility may be attenuated slightly by FAK.


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  		TABLE I
Rescue of motility parameters in FAK -/- MEF by ts72v-Src

 
Tyrosine Phosphorylation of CAS Is Not Sufficient for Cell Motility in the Absence of FAK—Because of the innate ability of FAK-null cells to become motile in the presence of active v-Src (i.e. FAK is not required), we analyzed our clones for the tyrosine phosphorylation of paxillin and CAS, focal adhesion proteins thought to exert negative and positive effects, respectively, on cell motility (55). To analyze CAS phosphorylation, lysates prepared from serum-starved adherent cells kept 3 days at the NPT or PT were analyzed by CAS immunoprecipitation followed by immunoblotting for either CAS or phosphotyrosine. Under these conditions, there is a dearth of tyrosine phosphorylation on FAKY397, CAS, and paxillin.2 Fig. 4A shows a lack of CAS tyrosine phosphorylation at the NPT in the presence of FAK, whereas activation of v-Src can induce phosphorylation without additional adhesion-mediated signals. This is consistent with previous data showing induction of CAS tyrosine phosphorylation by integrin-mediated attachment or in an adhesion-independent manner by v-Src (56). In contrast, the relative level of CAS tyrosine phosphorylation in FAK–/–[ts72v-Src] cells at the NPT is almost as high as in FAK+/+[ts72v-Src] cells at the PT, indicating that FAK normally prevents CAS phosphorylation in the absence of attachment-mediated signals. Moreover, activation of ts72v-Src induced only slightly higher levels of CAS tyrosine phosphorylation in the absence of FAK. The current finding indicates that high levels of CAS phosphorylation are insufficient to induce motility in FAK–/– cells and that v-Src-induced motility in FAK–/– cells does not require super-induction of CAS tyrosine phosphorylation. Taken with the data in Fig. 3, these results suggest that the immotility of FAK–/– cells might be caused by a lack of cell polarity, a phenomenon that seems to be dependent on FAK in untransformed cells, yet which can be overcome by v-Src in the absence of FAK (Figs. 2 and 3).



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  		FIG. 4.
Tyrosine phosphorylation of CAS and paxillin by v-Src in the presence or absence of FAK. Panel A, immunoblot analysis of CAS immunoprecipitates from FAK+/+ or FAK–/– [ts72v-Src] clones grown at the NPT or PT. Membranes were probed with anti-Tyr(P) (pTyr) mAb, stripped, and then reprobed with anti-CAS mAb. Note that the relative phosphotyrosine level of CAS in FAK–/–[ts72v-Src] cells at the NPT is comparable with levels induced by activated v-Src in the FAK+/+ background. IP, immunoprecipitation. Panel B, immunoblot analysis of paxillin immunoprecipitates from FAK+/+ or FAK–/– vector control cells versus FAK–/– or FAK+/+ [ts72v-Src] clones grown for 1 h in suspension (Susp.), or plated for 1 h on either polylysine (PL) or fibronectin (FN). Membranes were probed with anti-Tyr(P) (PTyr) mAb, stripped, and then reprobed with anti-paxillin mAb. Note that activated v-Src does not increase the integrin-mediated tyrosine phosphorylation of paxillin (pax) over the high basal level in parental FAK–/– cells, but does induce slower mobility isoforms (arrow). IP, immunoprecipitation.

 
The integrin-dependent tyrosine phosphorylation of paxillin was significantly higher in FAK–/– compared with FAK+/+ parental cells (Fig. 4B), indicating that FAK normally attenuates the integrin-mediated activation of paxillin kinases (e.g. Src family kinases). In contrast, activation of v-Src in the absence of FAK only increased integrin-independent paxillin phosphorylation; in the presence of FAK, active v-Src seems to induce phosphorylation of paxillin at multiple sites resulting in a slower mobility isoform (Fig. 4B, arrow). These results suggest negative regulatory roles for FAK in the phosphorylation of CAS and paxillin, and positive roles in the establishment of cell polarity and motility, and in actin-based cytoskeletal organization.

FAK Attenuates v-Src-induced Anchorage-independent Growth—v-Src-induced oncogenic transformation leads to anchorage-, contact-, and growth factor-independent proliferation. In high serum conditions (10%), we observed no differences at the PT between the FAK+/+ and FAK–/– [ts72v-Src] clones in regard to contact independence, saturation densities (data not shown), and proliferation rates (Fig. 5A). Importantly, the growth properties of these clones at the NPT resembled those of the empty vector-transduced FAK+/+ and FAK–/– clones at either the PT or NPT, indicating that contact independence and increased proliferation rates were mediated by v-Src activity. Interestingly, FAK–/–[ts72v-Src] clones exhibited 7–10-fold higher colony-forming frequencies in soft agar compared with FAK+/+[ts72v-Src] controls (Fig. 5B). FAK+/+ and FAK–/– clones transduced with empty vector produced similar, low numbers of colonies at the PT, indicating that the anchorage-independent growth resulted from v-Src activity (and not from the incubation temperature). Moreover, FAK–/– cells transduced with wt-v-Src exhibited 6–10-fold higher colony-forming efficiencies at 37 °C than FAK+/+ cells expressing comparable levels of wt-v-Src,3 indicating that our ts72v-Src allele reflects bona fide v-Src activity.



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  		FIG. 5.
Increased v-Src-induced anchorage-independent growth in the absence of FAK. Panel A, proliferation rates (performed in triplicate) of FAK+/+ or FAK–/– [puro] vector control cells, FAK+/+[ts72v-Src], and FAK–/–[ts72v-Src] clones (cl.) grown at the PT in the presence of 10% serum (error bars = S.D.). Panel B, the colony-forming efficiencies of FAK+/+ or FAK–/– [puro] vector control cells, FAK+/+[ts72v-Src], and FAK–/–[ts72v-Src] clones grown at the PT or NPT. Cells (104 cells/6-cm dish) were plated in triplicate, and the number of colonies formed after 2 weeks was scored (error bars = S.D.). Panel C, left side, immunoprecipitation/immunoblot (IP/IB) analysis for FAK re-expression in FAK–/–[ts72v-Src] cells (in aliquots of cells shown in panel D), compared with parental FAK+/+ cells. Note that the FAK re-expression levels never exceed 20% of levels in FAK+/+ cells. Right side, effect of FAK re-expression on the relative colony-forming efficiencies of two FAK–/–[ts72v-Src] clones, in triplicate (error bars = S.D.). Panel D, FAK re-expression does not alter expression levels of ts72v-Src. Following the ectopic expression of FAK (or empty vector) in the two FAK–/–[ts72v-Src] clones (cl.) in panel D, lysates with equal amounts of protein were subjected to immunoblot analyses for FAK or ts72v-Src (v-Src). Panel E, relative protein levels of FAK decrease after activation of ts72v-Src. Following shift to the PT, lysates from FAK+/+[ts72v-Src] were subjected to immunoblot analysis for FAK, paxillin (loading control), and phosphotyrosine (pTyr; control for v-Src activation). A similar analysis was performed on lysates of FAK+/+[puro] cells following temperature shift. Panel F, proliferation rates (performed in triplicate) of FAK+/+ or FAK–/– [puro] vector control cells, FAK+/+[ts72v-Src], and FAK–/–[ts72v-Src] clones grown at the PT in the presence of 0.5% serum (error bars = S.D.).

 

The data above suggest that FAK may encode a negative regulatory role in transformation. This implies that the forced re-expression of FAK in FAK–/– cells should down-modulate the ability of v-Src to induce anchorage independence. To investigate this, we stably transduced FAK–/– [vector] or [ts72v-Src] clones with a retrovirus encoding FAK cDNA (Fig. 5C, left panel). It should be noted that our best attempts to re-express FAK, whether by transfection or by retrovirus transduction, resulted in FAK levels roughly 20% of that in parental FAK+/+ cells (Fig. 5C). Our FAK re-expressor clones proliferated at the same rate at the PT as their parental FAK–/–-[ts72v-Src] cells (Fig. 5F), indicating that, at the level expressed in our clones, constitutive FAK expression is not toxic. Fig. 5C (right panel) shows that the stable re-expression of FAK decreased the colony-forming frequency of the FAK–/–-[ts72v-Src] clones 2–2.5-fold, without affecting the protein (Fig. 5D) and/or activation levels (data not shown) of v-Src.

Consistent with a possible negative role for FAK, previous studies (57, 58) showed that activation of v-Src results in FAK degradation through calpain-mediated proteolysis, a process thought to facilitate transformation-associated focal adhesion turnover. Fig. 5E shows that activation of ts72v-Src resulted in the rapid down-regulation of FAK levels, a phenomenon that may explain our inability to maintain high ectopic FAK reexpression levels in Fig. 5C.

FAK–/–[ts72v-Src] clones also proliferated 2-fold faster (Fig. 5F) and to higher saturation densities (data not shown) under growth factor-independent conditions (0.5% serum) compared with FAK+/+[ts72v-Src] controls. This difference is likely caused by the 2-fold higher v-Src level in FAK–/–[ts72v-Src] cells because re-expression of FAK in the FAK–/–[ts72v-Src] clones did not significantly decrease growth factor-independent proliferation and also had no effect on ts72v-Src levels (i.e. they were still 2-fold higher than in FAK+/+[ts72v-Src] cells). In contrast, the forced overexpression of ts72v-Src in FAK+/+[ts72v-Src] increased growth factor-independent proliferation; the 32 h doubling time of FAK+/+[ts72v-Src] cells was reduced to 19 h following a 3-fold overexpression of ts72v-Src. Interestingly, higher levels of ts72v-Src had no effect on colony-forming frequencies but did increase the size of individual colonies; in contrast, the forced overexpression of an activated PI3K allele did increase the colony-forming frequency of FAK+/+[ts72v-Src] cells.4 Therefore, even though FAK re-expression had no effect on anchorage-dependent proliferation rates, these data strongly suggest a role for FAK in modulating v-Src-induced anchorage-independent growth.

Enhanced Pyk2 Protein and Activation Levels Are Not Responsible for Increased Anchorage Independence of FAK–/–-[ts72v-Src] Cells—Several studies have shown that Pyk2 levels are 2–3-fold higher in FAK–/– MEF than their FAK+/+ siblings (10, 12, 13, 25). Moreover, Pyk2 overexpression in fibroblasts induces an epithelioid-like morphology reminiscent of FAK–/– MEF, marked by the loss of cell polarity, the replacement of typical focal adhesion complexes with more transient focal adhesions, and the deposition of F-actin fibers at the cell periphery (12, 14–16). Our FAK–/– MEF (a gift from T. Yamamoto, University of Tokyo, Tokyo, Japan) were derived from the original early passage stock, and those transduced with ts72v-Src in our lab were of the first passages we established. Indeed, we found a 3–5-fold enhancement in Pyk2 protein level (Fig. 6A) and v-Src-induced phosphotyrosyl levels (Fig. 6B) in the FAK–/– compared with FAK+/+ backgrounds. These data agree with a recent demonstration by Roy et al. (13) of enhanced v-Src-induced Pyk2 phosphorylation levels. These enhanced levels were unaffected by serum growth conditions (10% versus 0% CS) or by growth in adhesive versus anchorage-independent conditions, the latter recapitulated by growth in methylcellulose ("10% on plate" versus "10% MC"). Moreover, the re-expression of FAK in FAK–/–[ts72v-Src] cells, shown in Fig. 5C to diminish enhanced anchorage-independent growth, did not alter Pyk2 protein or tyrosine phosphorylation levels (Fig. 6C).



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  		FIG. 6.
Pyk2 protein and tyrosine phosphorylation levels. Panel A, lysates from adherent (on plate) or FAK+/+ or FAK–/– [ts72v-Src] cells grown in methylcellulose (MC), either in the presence or absence of 10% serum, were subjected to immunoprecipitation (IP) and/or immunoblot (IB) analysis for Pyk2 protein and tyrosine phosphorylation ({alpha}-PTyr) levels. Panel B, analysis of Pyk2 protein and tyrosine phosphorylation (pTyr) levels in FAK+/+ and –/– [ts72v-Src] clones after temperature shift to the PT. Comparisons are shown between longer and shorter exposures to demonstrate v-Src-induced Pyk2 tyrosine phosphorylation in both FAK+/+ and –/– backgrounds. Panel C, re-expression of exogenous FAK in FAK–/–[ts72v-Src] cells does not affect Pyk2 protein or tyrosine phosphorylation (PTyr) levels.

 
To address directly whether increased Pyk2 expression is sufficient to mediate increased colony-forming efficiency, FAK+/+[ts72v-Src] cells were transiently transfected with a vector expressing Myc-tagged Pyk2 (or empty vector) plus vectors expressing enhanced green fluorescent protein (GFP) and a hygromycin resistance cassette (pBABE/hygro). Transfectants were isolated by FACS and then assayed for Pyk2 ectopic expression (Fig. 7A), colony-forming ability, plating efficiency (after selection on hygromycin), and doubling time (proliferation rate). Fig. 7C shows that increased Pyk2 levels failed to induce higher colony-forming frequencies. Indeed, higher Pyk2 levels reduced efficiencies for both plating and anchorage independence, but not for proliferation, suggesting initial toxic effects by Pyk2 on cell survival or adhesion. Interestingly, the toxic effect of Pyk2 overexpression is likely the result of FAK displacement because Pyk2 overexpression in FAK–/–[ts72v-Src] cells did not alter colony-forming efficiencies (Fig. 7C). These data indicate that Pyk2 overexpression is not sufficient for enhanced Src-induced anchorage independence.



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  		FIG. 7.
Pyk2 expression does not correlate with the enhanced anchorage independence of FAK–/–[ts72v-Src] cells. Panel A, immunoblot analysis of the ectopic expression of Myc-Pyk2 (using either anti-myc or -Pyk2) in FAK+/+ and FAK–/– [ts72v-Src] cells. Immunoblotting of paxillin (bottom row) served as a loading control. Panel B, depletion of Pyk2 using RNAi-expressing constructs. FAK–/–[ts72v-Src] cells were transfected by LipofectAMINE 2000 with various pSHAG/Pyk2 RNAi constructs (gh1 and/or ij4), or pSHAG/luciferase as the vector control, along with pEGFP plus pBABE/hygro and 24 h later, sorted for transfectants by FACS, and then analyzed for Pyk2 expression by immunoblotting after additional growth for 3 or 5 days after transfection (d.p.t.). Immunoblotting for actin (bottom row) was used as a loading control. Panel C, the effects of Pyk2 overexpression in FAK+/+[ts72v-Src] or in FAK–/–[ts72v-Src], or RNAi-mediated Pyk2 depletion in FAK–/–[ts72v-Src] cells on relative colony-forming frequencies (as in Fig. 5B), relative plating efficiencies, and proliferation rates (Doubling Time). Plating efficiency was determined by subjecting 104 sorted GFP-expressing cells to selective growth in hygromycin-containing media, followed by the counting of hygromycin-resistant colonies after 2 weeks of incubation. Panel D, morphological effects following Pyk2 depletion by the RNAi-expressing construct gh1. Following sorting of the transfectants in panel C by FACS, cells were plated onto 6-cm dishes and then photographed 1 day later under phase-contrast optics.

 
To determine whether the increased Pyk2 levels in FAK–/– MEF played any role in enhanced Src-induced anchorage independence, FAK–/–[ts72v-Src] clones were transiently transfected with vectors (pSHAG; gift of G. Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) expressing double-stranded Pyk2 RNAi oligomers, plus EGFP vectors, and then subjected to FACS. We developed Pyk2 RNAi vectors based on the rules described in the Hannon laboratory web page (www.cshl.org/public/SCIENCE/hannon.html) that would decrease endogenous Pyk2 expression in FAK–/– MEF only severalfold so as to recapitulate the Pyk2 expression levels found in FAK+/+ MEF. Although constructs gh1 and ij4 suppressed endogenous Pyk2 levels 3–4-fold over at least 6 days (Fig. 7B), no significant change in colony-forming frequency, proliferation rate (Fig. 7C), or attachment efficiency (data not shown) was noted. However, plating of these transfectants showed that loss of Pyk2 correlated with increased cell flattening as well as increased formation of cellular protrusions (Fig. 7D). Taken together, these data indicate no major role for Pyk2 in the enhanced anchorage-independent growth induced by v-Src in the absence of FAK.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We provide evidence that FAK is not necessary for many parameters of morphological transformation of p53–/– mouse fibroblasts by v-Src and, moreover, that several parameters (anchorage-independent growth and wound healing motility) are enhanced in the absence of FAK. These observations are highlighted by the similar transformed morphology exhibited by FAK+/+ and FAK–/–[ts72v-Src] cells, the FAK-independent localization of v-Src in focal adhesion complexes, and the ability of v-Src to promote reorganization of the actin-based cytoskeleton contributing to fibroblastic morphology and cell polarization, and cell motility. All the transformation-related effects could be attributed to v-Src because of the ability to incapacitate Src kinase activity at the NPT, thereby eliminating possible clonal idiosyncrasies or the effects of spontaneous transformation. The innate ability of FAK–/– MEF to be transformed by v-Src is borne out in a recent study by Roy et al. (13) using a tetracycline-regulated re-expression system for FAK. Although fewer v-Src-induced soft agar colonies grew after FAK re-expression in their system, they could not rule out that this was caused by the ability of the tet transactivator to inhibit proliferation. Therefore, the current study provides the first evidence suggesting that FAK may down-modulate some parameters of oncogenic transformation.

Our results show that v-Src can overcome the innate immotility of FAK–/– cells. It should be stressed that, in our hands, FAK–/– cells show defective motility in short term assays (<3 h), whereas in longer term assays (>18 h), these cells exhibit relatively normal motility, indicating that FAK is not required for motility per se but that it may play a role in efficient coordination of early motility mechanisms. The parameters of motility we studied (linear directional motility, haptotaxis, monolayer wound healing, and Matrigel® invasiveness) are known to be influenced by FAK, and in the case of linear directional motility, by PI3K, FAK, and CAS (54). In the all these assay systems, the activation of ts72v-Src rescued the relative immotility of FAK–/– cells, yielding motility levels comparable with cells expressing FAK, whether in the presence or absence of ts72v-Src. Moreover, in the case of wound healing motility, our data indicated an enhancement in the absence of FAK. Interestingly, the motility of FAK+/+[ts72v-Src] cells was lower in some parameters than the parental FAK+/+ cells, but this might be explained by the decreased adhesion of the transformed cells, as has been suggested by others (59). The similar ability of FAK+/+ and –/– [ts72v-Src] clones to invade through Matrigel® in our study conflicts with recent findings of Hauck et al. (59) showing decreased Matrigel® and collagen gel invasiveness of NIH3T3/v-Src cells transfected with a natural FAK antagonist, FRNK. This might be explained by differences in cell type or by the fact that FRNK shares the C-terminal focal adhesion targeting domain and CAS binding sites of FAK, and thus, besides competing with FAK for focal adhesion binding sites, most likely also acts as a sink for limited pools of CAS.

ts72v-Src induced actin-based cytoskeletal reorganization of FAK–/– cells, converting the non-polar morphology to a typical fibroblastic morphology and promoted motility in several assays. However, tyrosine phosphorylation of p130CAS, an agonist of migration (55), was increased in FAK–/–[ts72v-Src] cells at the NPT compared with FAK+/+[ts72v-Src] cells at the PT, supporting the notion that phosphorylated p130CAS is not sufficient on its own to promote migration. Instead, we propose that a proper cytoskeletal architecture and concurrent polarity are also necessary for phospho-p130CAS to exert its pro-migratory function. We also suggest that FAK normally facilitates these changes but that v-Src can substitute in the absence of FAK. This notion is evidenced by the relatively non-polar morphology of FAK–/– cells, including the absence of longitudinal stress fibers and the equal distribution of focal complexes along the periphery of the whole cell. One possible mechanism for v-Src function may include disruption of the actin-rich cell-cell interactions observed in FAK–/– cells or FAK–/–[ts72v-Src] clones at the NPT (data not shown). However, because individual FAK–/– cells are also relatively immotile, FAK and v-Src may facilitate motility by inducing dynamic actomyosin structures associated with cell movement. Additionally, FAK and v-Src, inasmuch as they play roles in focal complex turnover, may induce the generation of PIP2, via activation of phosphoinositol kinases, at sites of actin-based cytoskeletal remodeling such as lamellipodia (60). Ren et al. recently showed (52) that FAK facilitates focal adhesion turnover by suppressing RhoA activation; the relative immotility of FAK–/– cells was explained by the presence of constitutively active RhoA, which could be down-regulated by the transient expression of wt-FAK cDNA. Indeed, the integrin-mediated suppression of RhoA activity in the presence of FAK requires c-Src (61), and this is likely facilitated through the stimulation of p190RhoGAP (62). Further proof of the role of activated RhoA in the immotile, epithelioid morphology of FAK–/– MEF comes from Chen et al. (53), who demonstrated that treatment of FAK–/– MEF with drug inhibitors of ROCK, a downstream mediator of RhoA, and of myosin light chain kinase, a downstream mediator of ROCK, induced cell spreading and a polar, fibroblastic morphology. Arthur and Burridge (62) showed that FAK+/+ cells expressing a dominant-interfering mutant of p190RhoGAP exhibited increased RhoA activity marked by impaired cell spreading and motility, premature assembly of stress fibers, and, most interestingly, an inability to establish polarity toward a monolayer wound.

The 7–10-fold enhancement of anchorage-independent growth in FAK–/– cells is particularly striking and suggests the loss of a modulatory mechanism. In an associated study,4 we show that this effect is both associated with and dependent on the selective super-activation of PI3K by v-Src in the absence of FAK. Indeed, re-expression of FAK in FAK–/–[ts72v-Src] clones, even at levels lower than endogenous FAK, reduced colony-forming frequencies, and presumably, we would have detected greater reduction if FAK could have been re-expressed at higher levels. However, the forced overexpression of ts72v-Src in FAK+/+[ts72v-Src] cells only led to larger soft agar colonies and not increased colony-forming frequency, arguing against the notion that the 2-fold higher ts72v-Src levels in FAK–/–[ts72v-Src] cells could be responsible for the 7–10-fold increased anchorage-independent growth.

In contrast, the enhanced growth factor independence of FAK–/–[ts72v-Src] cells seems to correlate with the 2-fold higher ts72v-Src levels because (i) FAK re-expression in FAK–/–[ts72v-Src] cells failed to reverse the enhancement, (ii) the 2-fold higher ts72v-Src levels were unaffected by FAK re-expression, and (iii) the forced overexpression of ts72v-Src in FAK+/+[ts72v-Src] led to higher growth factor independence. Importantly, the relative specific activities and half-lives of the ts72v-Src proteins in our FAK–/– and FAK+/+ clones are similar, as are the overall levels of Src-induced phosphotyrosine substrates in FAK+/+ and FAK–/– backgrounds. Interestingly, in the absence of FAK, the phosphorylation level of several Src-induced substrates is reduced, whereas several other novel substrates are found. We cannot exclude that these bands arise from changes in substrate expression, FAK-dependent proteolytic cleavages, or changes in protein modifications such as serine/threonine phosphorylations. Nonetheless, our data seem to identify both positive and negative roles for FAK downstream of v-Src.

Our inability to forcibly re-express FAK to more than 20% of endogenous levels parallels previously reported attempts by others (7, 17) and suggests some permanent refractility on the part of the FAK–/– MEF, possibly as a result of small increases in endogenous Pyk2 levels (10). However, stable FAK re-expression in our clones did not affect anchorage-dependent proliferation rates compared with parental FAK–/–[ts72v-Src] cells. This contrasts with a previous report (17) in which higher levels of FAK re-expression (50–75% of endogenous levels versus 20% in our case) slowed proliferation slightly. However, this finding is in conflict with recent data showing that overexpression of FAK in 3T3 cells increases G1 -> S progression (63).

Based on evidence that Pyk2 overexpression in fibroblasts can affect actin-based cytoskeletal architecture and cell motility (12, 14–16), and given that Pyk2 levels are induced 2–3-fold in FAK–/– MEF (10, 12, 13, 25), it is possible that Pyk2 may be responsible for some of the enhanced v-Src-induced parameters we detect. It is yet unclear whether the induction of Pyk2 levels results from the loss of FAK-mediated transcriptional regulation or from pressures during the selection of FAK–/– MEF. However, several lines of evidence strongly suggest that Pyk2 is not responsible for the enhanced anchorage-independent growth of FAK–/–[ts72v-Src] cells: anchorage-independent growth was not altered by either the forced overexpression of Pyk2 in FAK+/+[ts72v-Src] or the depletion of Pyk2 expression in FAK–/–[ts72v-Src] clones using several independent Pyk2 RNAi-expressing constructs. It was interesting to note that activation of ts72v-Src only resulted in the 2–3-fold increase in Pyk2 tyrosine phosphorylation, well shy of the >10-fold induction of FAK phosphorylation following v-Src activation,2 strongly suggesting that Pyk2 is neither a strong substrate nor binding partner of v-Src in vivo. However, in agreement with data that Pyk2 can affect actin-based cytoskeletal architecture and cell polarity, our data indicate morphological changes, namely a conversion to increased cell flattening and formation of cellular protrusions following Pyk2 depletion. This finding is consistent with previous reports showing increased cell rounding after Pyk2 overexpression (12, 15, 16).

Our data show that Pyk2 overexpression inhibited colony formation in the FAK+/+ but not FAK–/– [ts72v-Src] clones. This correlated with a concomitant inhibition of some parameters affecting plating efficiency (e.g. adhesion, apoptosis), strengthening the possibility that the Pyk2 toxic effects result from a direct competition with or displacement of FAK. The overexpression of Pyk2 in rat and mouse fibroblasts was shown previously to induce increased apoptosis (14). Indeed, Roy et al. (13) show a similar ~5-fold increase in v-Src-induced Pyk2 tyrosine phosphorylation as our current study, but additionally, that the re-expression of FAK ablates this increase, again suggesting a direct competition between FAK and Pyk2 for interaction with v-Src. Clearly, a definitive comparison of the relative roles of FAK and Pyk2 will require the generation of FAK/Pyk2-null cells.

Our data suggest a modulatory, not inhibitory role for FAK in Src-induced transformation because v-Src is capable of inducing transformation in many cell types expressing FAK (27). Additionally, a modulatory role must be taken into larger context of data suggesting that FAK plays a positive role in oncogenesis and metastasis. This includes studies that show (i) a correlation between increased FAK activation levels with increased v-Src-induced metastatic potential (34) and metastatic tumor variants in humans (32, 33, 35, 64), (ii) increased FAK tyrosine phosphorylation and association with p130CAS in v-Src-transformed cells (65), and (iii) increased Ras-dependent ERK2 activation and increased FAK/c-Src association following FAK overexpression (66). Indeed, the tetracycline-regulated overexpression of FAK in U-251 MG human astrocytoma cells resulted in increased soft agar colony formation in vitro and increased brain tumor burden in vivo (67). In contrast, recent data (68) suggest that transformation and metastasis may correlate with a turnover of FAK. In this study, Lu et al. demonstrated that the increased invasiveness and metastatic potential following treatment of EGFR-overexpressing carcinoma cells with epidermal growth factor correlates with the dephosphorylation and down-regulation of FAK. Ayaki et al. (69) show significantly reduced FAK protein levels in liver metastases compared with matched primary colonic adenocarcinomas from the same patients. Additionally, activation of v-Src leads to focal adhesion turnover triggered by calpain-mediated cleavage and subsequent degradation of FAK (57, 58), a finding consistent with our current study. Taken together, these data are compatible with varying temporal roles for FAK: a positive role in facilitating the formation of "mature" focal complexes and in the recruitment of signaling proteins, followed by a negative role requiring FAK degradation and focal complex turnover for cell cycle progression and/or cytoskeletal reorganization. Indeed, overexpression of wt-FAK in 3T3 cells was shown to accelerate G1 to S transition, whereas overexpression of FAK mutated in its focal adhesion targeting domain, {Delta}C14, caused G1 phase arrest, presumably as a result of the sequestration of Src family kinases away from focal adhesion complexes (63).

Several groups have attempted to inhibit FAK expression using antisense oligonucleotides or FRNK. Xu et al. (36) showed that antisense FAK oligonucleotides induced detachment and apoptosis in various tumor cells but not normal human fibroblasts. Recently, Hauck et al. (70) showed decreased invasiveness in Matrigel® by A549 adenocarcinoma cells treated with FAK antisense oligonucleotides. In these studies, however, 10–30% of endogenous FAK remained after antisense treatment, and this component was highly phosphorylated on Tyr397 (70), confounding the issue of whether decreased invasiveness resulted from the overall FAK loss or from the hyperactivation of residual FAK.

Until the production of FAK-null fibroblasts, much of FAK-specific signaling was characterized using FRNK, or even just the FAK focal adhesion targeting domain, which presumably inhibit FAK activation by competing for limited focal adhesion binding sites (71). However, not all of the FRNK effects correlate with findings in FAK-null cells. Two circumstances in which they do correlate are (i) integrin-mediated activation of ERK2 and (ii) motility. Specifically, ERK2 activation by integrins was shown to be FAK-independent, inasmuch as FRNK failed to block this pathway (72), and, in agreement, integrin engagement by FAK–/– cells led to normal ERK2 activation levels (10). Similarly, the relative inability of FAK–/– MEF to migrate in response to PDGF (51) correlates with the inhibition of epidermal growth factor- or PDGF-induced migration by FRNK in several FAK+/+ cell types (51, 70, 73). In a recent paper (59), FRNK expression inhibited v-Src-induced Matrigel® invasiveness of 3T3 fibroblasts, but did not affect either haptotactic or chemotactic motility, or the ability to form primary site tumors in immunodeficient mice. Although these data inherently agree with our conclusion that FAK is dispensable for many parameters of v-Src-induced oncogenic growth, the authors show that v-Src-induced tyrosine phosphorylation on FAKY397 is unchanged after the stable expression of FRNK, obscuring the efficacy of FRNK in this system. Interestingly, in this system, FRNK expression inhibited the growth of lung lesions following intravenous tumor cell injection, presumably as a result of a decrease in v-Src-induced metalloproteinase-2 expression.

In contrast, FRNK overexpression does not always correlate with findings in FAK–/– cells. For example, whereas FRNK inhibits epidermal growth factor- and PDGF-induced activation of ERK2 (70, 73), PDGF can induce ERK2 activation in FAK–/– MEF, although only to ~60% of activated levels in FAK re-expressing cells (73). Additionally, there is a worry that the FAK still expressed in FRNK-expressing cells may retain its docking functions (exemplified by the robust v-Src-induced FAKY397 phosphorylation in stable FRNK expressors described by Hauck et al. (Ref. 59)), and thus, may affect some downstream signaling pathways by sequestering specific mediator proteins. Nonetheless, our current work shows clearly that v-Src can induce robust oncogenic transformation in the absence of FAK, indicating that v-Src can compensate fully for FAK-dependent pathways controlling mitogenesis, cytoskeletal remodeling, and migration.

To address the FRNK versus FAK-null controversy in our system, we produced stable FRNK expressors in the FAK+/+-[ts72v-Src] background. We first noted that the plating efficiency of the FRNK transfectants (as monitored by co-transfection with a GFP expression vector) was suppressed ~75% compared with empty vector transfectants, and this correlated with a 70% decrease in colony-forming frequencies (compared with vector alone or the inactivated FRNK mutant, Ser1034) when an initial aliquot of transfectants (isolated by FACS) was plated in soft agar. However, after stable FRNK expressors were isolated, we noted that individual and pooled clones progressively exhibited the same colony-forming frequencies as clones with vector alone or FRNKS1034. Notably, this correlated with high levels of v-Src-induced FAKY397 phosphorylation (data not shown), paralleling the data of Hauck et al. (59). Thus, the initial antagonism by FRNK, which might be interpreted as suggesting a requirement for FAK in anchorage-independent growth, most likely relates to an initial antagonism of growth parameters affecting plating efficiency such as adhesion, cytoskeletal remodeling or apoptosis. Therefore, it is our belief that in the v-Src background, cells are selected that nullify the antagonistic activity of FRNK on FAK, making it impossible to compare long term FRNK expressors with FAK-null cells in regards to anchorage independence.

A central question, therefore, is why FAK acts somewhat antagonistically to v-Src in our system whereas it seems to agonize v-Src in other systems. An important point is that our findings are valid in the context of v-Src-induced transformation and in the absence of p53. It is plausible that human cancers with increased FAK activation levels have other, non-Src-mediated transformation pathways that synergize with FAK-mediated mitogenic and migration pathways. For example, increased FAK expression and signaling correlates with an increase in haptotaxis in PC-3 and DU145 (74), human prostate cancer cell lines that do not exhibit activated c-Src (75). Alternatively, higher levels of FAK might afford a selective advantage after the initial establishment of robust metastatic tumor growth. Nonetheless, our data indicate that FAK may manifest either positive or negative regulatory roles during oncogenic transformation, depending on temporal and spatial constraints as well as the proliferative and cytoskeletal pathways activated in specific tumors. Our data also suggest that inhibition of FAK with specific drug therapies may not necessarily have the desired anti-cancer effect.


   FOOTNOTES
 
* This work was supported by Grants CA65787 and CA94108 from the NCI, National Institutes of Health (to I. H. G.). 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} Current address: Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908. Back

§ To whom correspondence should be addressed: Dept. of Cancer Genetics, Roswell Park Cancer Inst., Elm and Carlton Sts., Buffalo, NY 14263. Tel.: 716-845-7681; Fax: 716-716-1698; E-mail: irwin.gelman{at}roswellpark.org.

1 The abbreviations used are: ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase; PT, permissive temperature; NPT, nonpermissive temperature; FAK, focal adhesion kinase; CAS, Crk-associated substrate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FRNK, focal adhesion kinase-related nonkinase; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; RIPA, radioimmune precipitation assay; mAb, monoclonal antibody; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorting; BSA, bovine serum albumin; MEF, mouse embryo fibroblasts; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; PDGF, platelet-derived growth factor; RNAi, RNA interference. Back

2 K. Moissoglu and I. H. Gelman, unpublished observations. Back

3 K. Moissoglu and I. H. Gelman, manuscript in preparation. Back

4 K. Moissoglu and I. H. Gelman, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank D. Ilic, T. Yamamoto, and S. Aizawa for p53-null FAK+/+ and FAK–/– MEF, G. Nolan for NNX packaging cells, K. Maruyama for the FAK expressing vector, D. Schlaepfer and M. Schaller for FAK cDNAs, G. Hannon for pSHAG and for aid with RNAi development, W.-C. Xiong for the Myc-Pyk2 expression vector, and the Mount Sinai Flow Cytometry Shared Research Facility for technical help with FACS. We are thankful to R. Jove, L.-H. Wang, L. Ossowski, and S. Masur for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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