Cleavage of Focal Adhesion Kinase by Different Proteases during Src-regulated Transformation and Apoptosis
2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês
10.1074/jbc.m008972200
ISSN1083-351X
AutoresNeil O. Carragher, Valerie J. Fincham, Deborah Riley, Margaret C. Frame,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoIntegrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis. Integrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis. focal adhesion kinase N-acetyl-leucyl-leucyl-methional (calpain inhibitor I), ALLN, N-acetyl-leucyl-leucyl-norleucinal (calpain inhibitor II), CEF, chicken embryo fibroblasts benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (caspase inhibitor I) PBS, phosphate-buffered saline The transforming viral Src gene (v-src) is associated with classic characteristics of oncogenic cell transformation, including deregulated growth control, cell rounding, and substrate detachment resulting from adhesion loss and disruption of the actin cytoskeleton (1Wyke A.W. Frame M.C. Gillespie D.A. Chudleigh A. Wyke J.A. Cell Growth Differ. 1995; 6: 1225-1234PubMed Google Scholar, 2Johnson D. Frame M.C. Wyke J.A. Oncogene. 1998; 16: 2017-2028Crossref PubMed Scopus (48) Google Scholar, 3Jove R. Hanafusa H. Annu. Rev. Cell Biol. 1987; 3: 31-56Crossref PubMed Scopus (299) Google Scholar, 4Kellie S. Bioessays. 1988; 8: 25-30Crossref PubMed Scopus (37) Google Scholar, 5Fincham V.J. Wyke J.A. Frame M.C. Oncogene. 1995; 10: 2247-2252PubMed Google Scholar, 6Fincham V.J. Frame M.C. EMBO J. 1998; 17: 81-92Crossref PubMed Scopus (264) Google Scholar). The stability of the actin cytoskeleton and adhesive properties of cells are mediated, at least in part, by focal adhesion complexes (7Burridge K. Fath K. Kelly T. Nuckolls G. Turner C. Annu. Rev. Cell Biol. 1988; 4: 487-525Crossref PubMed Scopus (1703) Google Scholar, 8Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1663) Google Scholar). Dynamic regulation of these adhesive links through assembly of focal adhesions at the leading edge of cells, coordinated with focal adhesion disassembly at the trailing edge, plays a key role in controlling cell migration (9Huttenlocher A. Sandborg R.R. Horwitz A.F. Curr. Opin. Cell Biol. 1995; 7: 697-706Crossref PubMed Scopus (450) Google Scholar). The mechanisms regulating turnover of focal adhesions are not well understood. However, a recent study suggests that proteolysis of specific components of the focal adhesion complex by the calpain family of proteolytic enzymes promotes disassembly of smooth muscle focal adhesions in response to collagen fragments (10Carragher N.O. Levkau B. Ross R. Raines E.W. J. Cell Biol. 1999; 147: 619-630Crossref PubMed Scopus (211) Google Scholar). Furthermore, calpain activity has been implicated in promoting migration of Chinese hamster ovary cells (11Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. Chem. 1997; 272: 32719-32722Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). The calpains are defined as a well conserved family of intracellular, nonlysosomal calcium-dependent cysteine proteases consisting of two ubiquitously expressed calpain isozymes, μ-calpain (calpain-I) and m-calpain (calpain-II) and several tissue-specific isoforms (12Mellgren R.L. FEBS Lett. 1980; 109: 129-133Crossref PubMed Scopus (220) Google Scholar, 13Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (782) Google Scholar, 14Molinari M. Carafoli E. J. Membr. Biol. 1997; 156: 1-8Crossref PubMed Scopus (138) Google Scholar). Colocalization of calpain II with focal adhesion structures (15Beckerle M.C. Burridge K. DeMartino G.N. Croall D.E. Cell. 1987; 51: 569-577Abstract Full Text PDF PubMed Scopus (212) Google Scholar) and the identification of several focal adhesion proteins as calpain substrates (16Cooray P. Yuan Y. Schoenwaelder S.M. Mitchell C.A. Salem H.H. Jackson S.P. Biochem. J. 1996; 318: 41-47Crossref PubMed Scopus (154) Google Scholar, 17Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar, 18Yamaguchi R. Maki M. Hatanaka M. Sabe H. FEBS Lett. 1994; 356: 114-116Crossref PubMed Scopus (52) Google Scholar, 19Hayashi M. Suzuki H. Kawashima S. Saido T.C. Inomata M. Arch. Biochem. Biophys. 1999; 371: 133-141Crossref PubMed Scopus (56) Google Scholar, 20Selliah N. Brooks W.H. Roszman T.L. J. Immunol. 1996; 156: 3215-3221PubMed Google Scholar, 21Yoshida H. Murachi T. Tsukahara I. FEBS Lett. 1984; 170: 259-262Crossref PubMed Scopus (51) Google Scholar) suggest that calpains are functional at focal adhesion sites. Oncogenic transformation of cells by v-Src is associated with an overall loss in abundance of focal adhesions as well as changes in focal adhesion architecture to smaller more condensed structures (22David-Pfeuty T. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6687-6691Crossref PubMed Scopus (217) Google Scholar). The precise mechanisms by which v-Src promotes the disassembly of focal adhesions have so far not been elucidated. The focal adhesion kinase (FAK)1 is both a substrate of Src kinase activity and a central component of focal adhesions and may be a strong candidate for mediating v-Src-induced disassembly of focal adhesions. Through multiple protein-binding domains, FAK can interact with and phosphorylate several members of the focal adhesion complex (23Hanks S.K. Polte T.R. Bioessays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar). Studies on cells derived from FAK knock-out embryos demonstrate that focal adhesions devoid of FAK are larger and that these cells have a reduced migratory capacity, suggesting that FAK may be required to regulate the turnover of focal adhesion structures (24Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar). To specifically examine the role of calpain-mediated proteolysis in tyrosine kinase-induced focal adhesion disruption, we have used a temperature-dependent mutant of v-Src (ts LA 29 v-Src). Activation of v-Src by shift to the permissive temperature initiates focal adhesion disassembly (5Fincham V.J. Wyke J.A. Frame M.C. Oncogene. 1995; 10: 2247-2252PubMed Google Scholar). Our previous work has indicated that this and consequent morphological transformation are dependent on the catalytic activity of v-Src that induces phosphorylation of focal adhesion components, including FAK, followed by dissociation of the Src-FAK complex and degradation of FAK, events that precede focal adhesion loss and cell rounding (5Fincham V.J. Wyke J.A. Frame M.C. Oncogene. 1995; 10: 2247-2252PubMed Google Scholar, 6Fincham V.J. Frame M.C. EMBO J. 1998; 17: 81-92Crossref PubMed Scopus (264) Google Scholar). Despite this characterization of the sequence of events during the initial stages of v-Src-induced cell transformation, we still lack any understanding of the nature of the process of FAK proteolytic cleavage induced by v-Src, and specifically, the identities of the proteases involved have not been elucidated. As mentioned above, calpains represent good candidates; however, FAK has also been demonstrated to be cleaved by caspases as focal adhesions are lost during apoptosis (25Wen L.P. Fahrni J.A. Troie S. Guan J.L. Orth K. Rosen G.D. J. Biol. Chem. 1997; 272: 26056-26061Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 26Levkau B. Herren B. Koyama H. Ross R. Raines E.W. J. Exp. Med. 1998; 187: 579-586Crossref PubMed Scopus (228) Google Scholar). In this study, we have addressed whether FAK cleavage that is triggered prior to focal adhesion disassembly during cell transformation is mediated by members of either the calpain or caspase family of proteolytic enzymes. Primary chicken embryo fibroblasts (CEF) were subcultured as described previously (5Fincham V.J. Wyke J.A. Frame M.C. Oncogene. 1995; 10: 2247-2252PubMed Google Scholar). Low density cultures were transfected with replication-competent avian retroviral constructs, RCAN-v-src, encoding the temperature-sensitive ts LA 29 v-Src mutant. Transfected CEF were cultured at the permissive temperature of 35 °C until cells were uniformly infected and expressing v-Src protein. For analysis of v-Src-induced transformation, cells were cultured at the restrictive temperature (41 °C) and then examined following shift to the permissive temperature (35 °C). Rat 1 fibroblasts expressingts LA 29 v-Src were maintained in DMEM supplemented with 5% newborn calf serum, 1 mm sodium pyruvate, and 2 mml-glutamine at the permissive temperature (35 °C). Rat 1 cells were serum-starved for 24 h (Dulbecco's modified Eagle's medium supplemented with 0.2% newborn calf serum) prior to a shift to the restrictive temperature (39.5 °C). Calpain inhibitor studies were performed using calpain inhibitor 1 (ALLN; Calbiochem), calpain inhibitor 2 (ALLM), and the cell-permeable sense and scrambled calpastatin peptides (Calbiochem). Caspase inhibitor studies were carried out with caspase inhibitor 1 (ZVAD-FMK; Calbiochem). CEF were preincubated with ALLN, ALLM, or ZVAD-FMK (50–100 μm) for 1 h prior to shift to 35 °C and then subsequently incubated at 35 °C in the presence of ALLN, ALLM, or ZVAD-FMK (50–100 μm) or sense and scrambled calpastatin peptides (50 μm) for the indicated time periods. Serum-starved Rat 1 fibroblasts were preincubated with ZVAD-FMK or ALLN (100 μm) for 1 h prior to shift to restrictive temperature 39.5 °C and then subsequently incubated at this temperature in the presence of the inhibitors. Antibodies for Western blot detection and immunocytochemistry included2–18N pp125FAK and 903–1058C pp125FAK (Santa Cruz Biotechnology, Inc.),354–534N pp125FAK (Transduction Laboratories), paxillin (Transduction Laboratories), and calpain-II (Research Diagnostics, Inc.). Anti-mouse and -rabbit peroxidase-conjugated secondary antibodies were purchased from New England Biolabs, Inc. Nontransformed and transformed ts LA 29 CEF were cultured on permanox plastic chamber slides (Nalge Nunc International). Cells were fixed in 3.7% formaldehyde for 10 min at room temperature, permeabilized in 0.5% Nonidet P-40 in PBS for 10 min at room temperature, and washed serially in PBS, 0.15 mglycine/PBS plus 0.02% NaN3, and PBS. Cells were blocked in 10% fetal calf serum/PBS prior to 1 h incubation at room temperature with primary antibodies, including affinity-purified monoclonal anti-paxillin (Transduction Laboratories) and polyclonal rabbit antiserum generated against the large subunit of m-calpain (calpain II) purified from human placenta (Research Diagnostics Inc.). Primary antibody incubation was followed by several washes in PBS and subsequent incubation with FITC- or Texas Red-conjugated secondary antibodies (Jackson Immunoresearch Laboratories). Cells were also incubated with fluorescein isothiocyanate-labeled phalloidin (Sigma). Immunostaining of cells was analyzed by confocal microscopy. Cells were washed twice with PBS and lysed in low detergent lysis buffer (10 mm Tris, pH 7.4, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.5 mm NaF, 10 mm β−glycerophosphate, 10 mm Na4P2O7, 100 μm NaVO4 with protease inhibitors, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Lysates were clarified by high speed centrifugation at 4 °C, supplemented with SDS-sample buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and immunoblotted with specific antibodies. ts LA 29 v-Src-expressing CEF were cultured on tissue culture plastic substrates at 41 or 35 °C for 18 h in the absence or presence of calpain inhibitor 1 and 2 (ALLN, ALLM; 100 μm), or herbimycin A (5 mm) cells were washed three times with PBS, and those cells remaining attached to the culture substrate were quantified by counting the number of cells per high power field (magnification × 200). Data representing the mean of four high power fields were expressed as the number of cells per high power field remaining attached to the culture substrate. 2 × 105 CEF expressing ts LA 29 v-Src were cultured at 41 °C in 60-mm dishes until 70–80% confluent. The cell monolayers were wounded by scoring with a sterile micropipette tip, and cultures were incubated at 35 °C for a further 18 h in the absence or presence of ALLN (50 μm), ALLM (50 μm), and ZVAD-FMK (100 μm). To analyze the role of the calpain proteolytic enzymes in focal adhesion loss associated with v-Src induced morphological transformation, we examined protein levels and localization of the ubiquitously expressed calpain II in nontransformed (41 °C) and v-Src-transformed CEF (35 °C). Immunoblotting cell lysates prepared at sequential time points following shift to 35 °C demonstrated a consistent rapid increase in levels of calpain II following activation of v-Src, which subsequently declined at 24 h (Fig.1 A). Calpain I protein was detected at extremely low levels in CEF, and levels of calpain I did not vary during the early stages of transformation (results not shown). Messenger RNA levels of calpain II and the endogenous calpain inhibitor calpastatin are not altered in response to v-Src transformation (results not shown), suggesting that elevation of calpain II protein levels and proteolytic activity following v-Src activation are regulated at a post-transcriptional level. Immunostaining of nontransformed CEF (41 °C) with an antibody against calpain II was generally weak and diffuse throughout the nucleus and cytoplasm; however, some calpain II immunoreactivity was evident at peripheral focal adhesion sites colocalizing with the focal adhesion protein paxillin (indicated by arrows) (Fig.1 B). Six hours following shift to the permissive temperature (35 °C), immunostaining of ts v-Src-expressing CEF with anti-calpain II was of greater intensity than that of nontransformed cells (Fig. 1 C), consistent with the elevated protein levels observed by immunoblotting (Fig. 1 A). In v-Src-transformed CEF, calpain II staining often localized at peripheral podosome-like structures (indicated by arrows) that may represent substrate attachment sites consisting of residual focal adhesions (Fig.1 C). Immunostaining of anti-calpain II at these peripheral sites again colocalized with focal adhesion proteins such as paxillin (Fig. 1 C). To determine whether calpain activity was involved in the process of oncogenic transformation, CEF expressing ts v-Src were studied following treatment with pharmacological inhibitors of calpain proteolytic activity as well as a calpastatin peptide highly specific for calpain inhibition. ts v-Src-expressing CEF were shifted from the restrictive temperature (41 °C) to the permissive temperature (35 °C) to induce cell transformation. Characteristic of v-Src transformation, untreated CEF lost focal adhesion structures, exhibited a disorganized actin cytoskeleton, and developed a rounded morphology with many cells having detached from the culture substrate 18 h following shift to 35 °C (Fig.2 B). Treatment of cells cultured at 35 °C with a well characterized pharmacological peptide aldehyde inhibitor of calpain activity, ALLN (100 μm) (27Squier M.K. Miller A.C. Malkinson A.M. Cohen J.J. J. Cell. Physiol. 1994; 159: 229-237Crossref PubMed Scopus (411) Google Scholar, 28Sasaki T. Kishi M. Saito M. Tanaka T. Higuchi N. Kominami E. Katunuma N. Murachi T. J. Enzyme. Inhib. 1990; 3: 195-201Crossref PubMed Scopus (194) Google Scholar, 29Squier M.K. Sehnert A.J. Sellins K.S. Malkinson A.M. Takano E. Cohen J.J. J. Cell. Physiol. 1999; 178: 311-319Crossref PubMed Scopus (116) Google Scholar) suppressed the loss of focal adhesions, cell rounding, and remodeling of the actin cytoskeleton (Fig. 2 C). Calpain inhibitor I-ALLN has also been reported to inhibit the lysosomal proteases cathepsin B, cathepsin L, and the ubiquitin-dependent proteasome complex (27Squier M.K. Miller A.C. Malkinson A.M. Cohen J.J. J. Cell. Physiol. 1994; 159: 229-237Crossref PubMed Scopus (411) Google Scholar, 30Vinitsky A. Michaud C. Powers J.C. Orlowski M. Biochemistry. 1992; 31: 9421-9428Crossref PubMed Scopus (196) Google Scholar, 31Milligan S.A. Owens M.W. Grisham M.B. Arch. Biochem. Biophys. 1996; 335: 388-395Crossref PubMed Scopus (55) Google Scholar). Treatment of ts v-Src-expressing CEF undergoing transformation with ammonium chloride, a reputed inhibitor of lysosomal cathepsins (32Seglen P.O. Grinde B. Solheim A.E. Eur. J. Biochem. 1979; 95: 215-225Crossref PubMed Scopus (355) Google Scholar, 33Ziegler H.K. Unanue E.R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 175-178Crossref PubMed Scopus (478) Google Scholar), or the specific proteasome inhibitor, lactacystin (34Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1504) Google Scholar), had no effect on the characteristics of v-Src transformation (results not shown), indicating that the effects of ALLN were most likely due to calpain inhibition. In addition, treatment ofts v-Src-expressing CEF with ALLM (100 μm), which is inhibitory against calpains, but not the proteasome, can suppress v-Src-induced loss of focal adhesions and remodeling of the actin cytoskeleton and cell morphology (results not shown). A more specific approach to inhibiting intracellular calpain activity can be achieved by exploiting the characteristics of the naturally occurring endogenous inhibitor of calpain, known as calpastatin. Calpastatin is highly specific for calpain I and calpain II and has not been demonstrated to inhibit other proteases (35Suzuki K. Imajoh S. Emori Y. Kawasaki H. Minami Y. Ohno S. FEBS Lett. 1987; 220: 271-277Crossref PubMed Scopus (243) Google Scholar, 36Mohan P.S. Nixon R.A. J. Neurochem. 1995; 64: 859-866Crossref PubMed Scopus (18) Google Scholar). Studies demonstrate that a 27-amino acid synthetic calpastatin peptide corresponding to the minimal inhibitory segment of domain I of calpastatin specifically inhibits activity of calpain both in vitro (37Maki M. Bagci H. Hamaguchi K. Ueda M. Murachi T. Hatanaka M. J. Biol. Chem. 1989; 264: 18866-18869Abstract Full Text PDF PubMed Google Scholar) and in a cell-permeable fashion in vivo (38Eto A. Akita Y. Saido T.C. Suzuki K. Kawashima S. J. Biol. Chem. 1995; 270: 25115-25120Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). ts v-Src-expressing CEF treated with the sense (Fig. 2 D), but not the scrambled (Fig. 2 E), cell-permeable calpastatin peptide demonstrate a reduction in the loss of focal adhesions, disorganization of the actin cytoskeleton, and cell rounding normally associated with v-Src transformation at 35 °C. In contrast, treatment of cells with the broad spectrum caspase inhibitor ZVAD-FMK has no effect on transformation (Fig. 2 F). The results obtained with the above panel of inhibitors indicate that calpains are the proteases involved in promoting disassembly of focal adhesions and the actin cytoskeleton and transition to a less adherent and more rounded morphology during v-Src transformation of CEF. We have previously observed proteolytic degradation of the recognized calpain substrate FAK during v-Src transformation and focal adhesion loss, although the antibody used in these studies did not detect cleavage products (5Fincham V.J. Wyke J.A. Frame M.C. Oncogene. 1995; 10: 2247-2252PubMed Google Scholar). To characterize FAK cleavage in more detail, we have utilized three epitope-specific antibodies raised against defined regions of the FAK protein. Using a low detergent lysis buffer to enrich for proteolytic fragments of FAK present in the cytoplasm (see "Experimental Procedures"), total cell lysates were prepared fromts v-Src-expressing CEF cultured at 41 °C or at sequential time points following v-Src activation by shift to 35 °C. Lysates were subjected to immunoblotting using three antibodies recognizing the amino terminus (N-pp125FAK), residues 354–534 downstream of the amino terminus (354–534N-pp125FAK), and the carboxyl terminus (C-pp125FAK) of FAK as probes (Fig.3). Immunoblotting with all three antibodies indicated that cleavage of native FAK occurred between 6 and 8 h following activation of v-Src. The loss of native FAK was paralleled by the generation of approximately sized 95- and 40-kDa amino-terminal fragments and a 30-kDa C-terminal cleavage product (Fig.3, A and C). Additionally, 55-, 42-, and 30-kDa fragments of FAK derived from the internal region of the protein can be identified by the 354–534N pp125FAK antibody (Fig. 3 B). The initial cleavage of FAK to approximately 95-kDa amino-terminal and 30-kDa C-terminal fragments is consistent with the pattern of calpain-mediated cleavage described previously for other stimuli (16Cooray P. Yuan Y. Schoenwaelder S.M. Mitchell C.A. Salem H.H. Jackson S.P. Biochem. J. 1996; 318: 41-47Crossref PubMed Scopus (154) Google Scholar, 39Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 10Carragher N.O. Levkau B. Ross R. Raines E.W. J. Cell Biol. 1999; 147: 619-630Crossref PubMed Scopus (211) Google Scholar). Pretreatment and subsequent incubation ofts v-Src-expressing CEF at 35 °C in the presence of ALLN at 100 μm significantly inhibited cleavage of FAK and generation of all cleavage products associated with v-Src transformation (Fig. 3, A–C), whereas treatment with a caspase inhibitor (ZVAD-FMK; 100 μm) had no effect (results not shown). To examine the influence calpain inhibition had on the adhesive strength of v-Src-transformed CEF for their culture substrate,ts v-Src-expressing CEF were cultured for 18 h at 41 °C or at 35 °C in the absence or presence of the calpain inhibitors ALLN and ALLM. The number of cells remaining attached to the substrate after extensive washing was quantified and expressed as the number of cells per high power field (Fig.4). These results demonstrated that after 18 h at 35 °C there was a significant loss in the number ofts v-Src-expressing cells remaining strongly adherent to the substrate. Transformation of cells at 35 °C in the presence of the calpain inhibitor ALLN or ALLM (100 μm) resulted in a greater than 2-fold increase in the number of cells remaining attached to the culture substrate. Incubation of CEF at 35 °C in the presence of the general inhibitor of kinase function herbimycin A, which inhibits Src kinase activity, also partially rescued substrate anchorage (Fig. 4). These results indicate that calpain-dependent disassembly of focal adhesions that accompanies v-Src transformation results in a general decrease in the adhesion of transformed CEF to their culture substrate. Thus, treatment of v-Src-transformed CEF with calpain inhibitors stabilized focal adhesion structures and maintained substrate anchorage. Since we have shown that the proteolysis of FAK during transformation by v-Src is most likely calpain-dependent and since we have shown previously that Src-induced focal adhesion disassembly is required for cell migration (6Fincham V.J. Frame M.C. EMBO J. 1998; 17: 81-92Crossref PubMed Scopus (264) Google Scholar), we addressed whether inhibitors of calpain could suppress cell migration into a wounded monolayer of v-Src-transformed cells. After wounding, ts v-Src-expressing CEF cultured at 35 °C for 18 h demonstrated extensive migration into the denuded area (Fig. 5). Incubation of ts v-Src-expressing CEF at 35 °C in the presence of the calpain and proteasome inhibitor (ALLN; 50 μm) inhibited any motility of transformed CEF into the wound (Fig. 5). Treatment of CEF with the inhibitor ALLM (50 μm), which inhibits calpain and not the proteasome complex, also substantially prevented migration. Since ALLN and ALLM have previously been reported to exhibit cathepsin-inhibitory activity (27Squier M.K. Miller A.C. Malkinson A.M. Cohen J.J. J. Cell. Physiol. 1994; 159: 229-237Crossref PubMed Scopus (411) Google Scholar, 30Vinitsky A. Michaud C. Powers J.C. Orlowski M. Biochemistry. 1992; 31: 9421-9428Crossref PubMed Scopus (196) Google Scholar), we also treated ts v-Src-expressing CEF with the reported inhibitor of lysosomal cathepsins, ammonium chloride, as a negative control and found this had no significant influence on migration (results not shown). Treatment of cells with the caspase inhibitor ZVAD-FMK also has no effect on migration of v-Src-transformed CEF (Fig. 5). These results demonstrate that v-Src-transformed CEF are dependent on calpain activity for optimal migration, most likely as a result of its proposed role in FAK cleavage and focal adhesion turnover. Our previous studies demonstrate that Rat-1 fibroblasts expressing thets LA 29 v-Src protein undergo apoptosis when cultured in low serum following attenuation of v-Src activity by culture at the restrictive temperature (39.5 °C). Following shift of serum-starvedts LA 29 Rat-1 cells from permissive (35 °C) to restrictive temperature (39.5 °C), the cells rapidly round up and become less adherent (40Johnson D. Agochiya M. Samejima K. Earnshaw W. Frame M. Wyke J. Death Differ. 2000; 7: 685-696Crossref PubMed Scopus (64) Google Scholar). Proteolytic cleavage of FAK to 85- and 77-kDa N-terminal fragments accompanies these morphological changes (Fig. 6); however, in contrast to v-Src transformation, FAK cleavage in serum-deprived cells switched to the restrictive temperature was not inhibited by ALLN but instead was inhibited by ZVAD-FMK (Fig. 6), indicating that caspases and not calpains are responsible for FAK cleavage that accompanies adhesion loss under these circumstances. Cell adhesion to extracellular matrix substrates regulates many aspects of normal cell physiology, including proliferation, migration, and cell survival (9Huttenlocher A. Sandborg R.R. Horwitz A.F. Curr. Opin. Cell Biol. 1995; 7: 697-706Crossref PubMed Scopus (450) Google Scholar, 41Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (355) Google Scholar, 42Ruoslahti E. Reed J.C. Cell. 1994; 77: 477-478Abstract Full Text PDF PubMed Scopus (972) Google Scholar). An integral role for calpain activity in regulating critical aspects of cell behavior has been suggested by previous studies demonstrating that calpain activity can degrade focal adhesion components (39Yuan Y. Dopheide S.M. Ivanidis C. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 21847-21854Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 10Carragher N.O. Levkau B. Ross R. Raines E.W. J. Cell Biol. 1999; 147: 619-630Crossref PubMed Scopus (211) Google Scholar), mediate substrate detachment at the trailing edge of cells (9Huttenlocher A. Sandborg R.R. Horwitz A.F. Curr. Opin. Cell Biol. 1995; 7: 697-706Crossref PubMed Scopus (450) Google Scholar), and regulate filipodia and lamellipodia formation and forward protrusion at the leading edge of cells (43Potter D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (233) Google Scholar). In addition calpain may also contribute to caspase-mediated apoptotic cell death pathways (44Kato M. Nonaka T. Maki M. Kikuchi H. Imajoh-Ohmi S. J. Biochem. (Tokyo). 2000; 127: 297-305Crossref PubMed Scopus (69) Google Scholar, 45Wang K.K. Trends Neurosci. 2000; 23: 20-26Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, 46Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1044) Google Scholar). However, evidence demonstrating either a physiological or pathological role for calpain-dependent proteolysis of focal adhesion components has not been reported. In this study, we demonstrate that levels of calpain II protein are regulated at the post-transcriptional level following v-Src transformation of CEF. Furthermore, in response to activation of v-Src, calpain activity promotes proteolytic cleavage of FAK, focal adhesion disassembly, and morphological transformation. Using epitope-specific antibodies, we have identified putative cleavage fragments generated by calpain-mediated proteolysis of FAK taking place during v-Src transformation. FAK cleavage data indicate that a 30-kDa C-terminal fragment containing the focal adhesion targeting sequence and a second proline-rich domain dissociates from a 95-kDa amino-terminal fragment containing the kinase domain. Since the C-terminal domains of FAK are important in targeting FAK to focal adhesions (47Hildebrand J.D. Schaller M.D. Parsons J.T. Mol. Biol. Cell. 1995; 6: 637-647Crossref PubMed Scopus (310) Google Scholar), this cleavage event is likely to influence the localization of the kinase domain to focal adhesion sites and may lead to reduced FAK activity at adhesions, something that is known to negatively regulate FAK function and cell motility (48Gilmore A.P. Romer L.H. Mol. Biol. Cell. 1996; 7: 1209-1224Crossref PubMed Scopus (462) Google Scholar). In addition, the C-terminal cleavage fragment is relatively stable following its generation, so it could potentially act in a dominant negative role, competing with functionally intact full-length FAK for focal adhesion substrates. Calpain-dependent cleavage of FAK would also be expected to impair the ability of FAK to act as an adapter protein, thereby compromising the integrity of the focal adhesion complex. Thus, modulation of FAK function through calpain-dependent cleavage is likely to play a significant role in the process of oncogenic transformation. The induction of calpain-dependent proteolytic cleavage following v-Src activation appears to be selective for FAK, since other known calpain substrates within focal adhesions, such as paxillin, talin, or Src itself do not undergo calpain-dependent processing during v-Src transformation (results not shown). Although the detailed regulation of calpain-mediated FAK cleavage induced by v-Src remains to be elucidated, we have found that calpain II protein levels are modulated during transformation. In addition, calpain-induced cleavage of FAK may be linked to specific tyrosine phosphorylation of FAK that leads to complex dissociation, perhaps releasing FAK and permitting its cleavage by calpain. Previous studies demonstrate that Src-mediated tyrosine phosphorylation of cortactin and the NR2 subunits of NMDA receptors influences the ability of these proteins to be cleaved by calpain (49Huang C. Tandon N.N. Greco N.J. Ni Y. Wang T. Zhan X. J. Biol. Chem. 1997; 272: 19248-19252Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 50Bi R. Rong Y. Bernard A. Khrestchatisky M. Baudry M. J. Biol. Chem. 2000; 275: 26477-26483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). It is therefore tempting to speculate that v-Src-dependent phosphorylation of FAK may influence the suitability of FAK to act as a substrate for calpain. We are currently investigating the mechanism by which Src activity controls calpain II protein levels and the influence phosphorylation of specific FAK residues has on its ability to be cleaved by calpain. FAK cleavage mediated by the caspase family of cysteine proteases has previously been demonstrated in T lymphocytes and endothelial cells undergoing apoptosis (25Wen L.P. Fahrni J.A. Troie S. Guan J.L. Orth K. Rosen G.D. J. Biol. Chem. 1997; 272: 26056-26061Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 26Levkau B. Herren B. Koyama H. Ross R. Raines E.W. J. Exp. Med. 1998; 187: 579-586Crossref PubMed Scopus (228) Google Scholar). Treatment of v-Src-expressing CEF with inhibitors against a broad spectrum of caspase family members (ZVAD-FMK) had no effect on FAK cleavage or morphological transformation. In contrast, FAK cleavage in serum-starved Rat-1 fibroblasts induced to detach and undergo apoptosis after switching off v-Src activity is suppressed by caspase inhibitors, whereas calpain inhibitors had no effect. These data lead us to conclude that proteolysis of FAK is a general phenomenon associated with focal adhesion disassembly but can be mediated by distinct proteases under different biological Src-regulated processes: caspases during apoptosis and calpain (most likely calpain II) during tyrosine kinase-induced cell transformation. From the data presented in this report, we propose that calpain- and caspase-mediated proteolysis of FAK are important mechanisms for regulating focal adhesion integrity in both viable and apoptotic cells, respectively. During v-Src transformation of CEF, calpain-dependent cleavage of FAK precedes disassembly of the focal adhesion complex and disorganization of the actin cytoskeleton, leading to a loss of substrate anchorage and increased motility. This implicates calpain as a major effector in the process of v-Src-induced oncogenic transformation and Src-dependent cell migration. A recent study examining the expression of calpain I in human renal cell carcinomas demonstrated significantly higher levels of calpain I expression in tumors that presented evidence of metastases to peripheral lymph nodes relative to low expression levels in tumors that apparently had not metastasized (51Braun C. Engel M. Seifert M. Theisinger B. Seitz G. Zang K.D. Welter C. Int. J. Cancer. 1999; 84: 6-9Crossref PubMed Scopus (64) Google Scholar). This study together with our own data suggests that calpain family proteases could mediate disassembly of focal adhesions and loss of substrate anchorage that may contribute to tumor cell motility and invasion. Targeting the inhibition of calpain activity in transformed cells may provide a useful therapeutic strategy for the prevention of tumor invasion and metastases. We are grateful to John Wyke for critical review of the manuscript.
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