Title: Focal Adhesion Kinase Overexpression Enhances Ras-dependent Integrin Signaling to ERK2/Mitogen-activated Protein Kinase through Interactions with and Activation of c-Src
Abstract: Cell adhesion to extracellular matrix proteins such as fibronectin (FN) triggers a number of intracellular signaling events including the increased tyrosine phosphorylation of the cytoplasmic focal adhesion protein-tyrosine kinase (PTK) and also the stimulation of the mitogen-activated protein kinase ERK2. Focal adhesion kinase (FAK) associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family PTKs and Grb2, respectively. To investigate the mechanisms by which FAK, c-Src, and Grb2 function in FN-stimulated signaling events to ERK2, we expressed wild type and mutant forms of FAK in human 293 epithelial cells by transient transfection. FAK overexpression enhanced FN-stimulated activation of ERK2 ∼4-fold. This was blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 was Ras-dependent. FN-stimulated c-Src PTK activity was enhanced by wild type FAK expression, whereas FN-stimulated activation of ERK2 was blocked by expression of the c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of FAK enhanced activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK did not. Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhanced p130Cas association with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of an FAK·c-Src complex, and several of these can lead to Ras-dependent ERK2 mitogen-activated protein kinase activation. Cell adhesion to extracellular matrix proteins such as fibronectin (FN) triggers a number of intracellular signaling events including the increased tyrosine phosphorylation of the cytoplasmic focal adhesion protein-tyrosine kinase (PTK) and also the stimulation of the mitogen-activated protein kinase ERK2. Focal adhesion kinase (FAK) associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family PTKs and Grb2, respectively. To investigate the mechanisms by which FAK, c-Src, and Grb2 function in FN-stimulated signaling events to ERK2, we expressed wild type and mutant forms of FAK in human 293 epithelial cells by transient transfection. FAK overexpression enhanced FN-stimulated activation of ERK2 ∼4-fold. This was blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 was Ras-dependent. FN-stimulated c-Src PTK activity was enhanced by wild type FAK expression, whereas FN-stimulated activation of ERK2 was blocked by expression of the c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of FAK enhanced activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK did not. Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhanced p130Cas association with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of an FAK·c-Src complex, and several of these can lead to Ras-dependent ERK2 mitogen-activated protein kinase activation. The family of transmembrane integrin receptors mediate cell adhesion to the extracellular matrix and also trigger intracellular signaling events such as the stimulation of the mitogen-activated protein kinase ERK2 (1Chen Q. Kinch M.S. Lin T.H. Burridge K. Juliano R.L. J. Biol. Chem. 1994; 269: 26602-26605Abstract Full Text PDF PubMed Google Scholar, 2Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar, 3Clark E.A. Hynes R.O. J. Biol. Chem. 1996; 271: 14814-14818Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 4Chen Q. Lin T.H. Der C.J. Juliano R.L. J. Biol. Chem. 1996; 271: 18122-18127Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 5Renshaw M.W. Toksoz D. Schwartz M.A. J. Biol. Chem. 1996; 271: 21691-21694Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). 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The non-receptor focal adhesion protein-tyrosine kinase (PTK) 1The abbreviations used are: PTK, protein-tyrosine kinase; RPTK, growth factor receptor PTK; FN, fibronectin; WT, wild type; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CAS, p130Cas; MBP, myelin basic protein; IP, immunoprecipitate; mAb, monoclonal antibody; FAK, focal adhesion kinase; Pipes, 1,4-piperazinediethanesulfonic acid; HA, hemagglutinin; RIPA, radioimmune precipitation buffer; Pyk2, proline-rich tyrosine kinase 2; SH2, Src homology 2; SH3, Src homology 3. 1The abbreviations used are: PTK, protein-tyrosine kinase; RPTK, growth factor receptor PTK; FN, fibronectin; WT, wild type; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CAS, p130Cas; MBP, myelin basic protein; IP, immunoprecipitate; mAb, monoclonal antibody; FAK, focal adhesion kinase; Pipes, 1,4-piperazinediethanesulfonic acid; HA, hemagglutinin; RIPA, radioimmune precipitation buffer; Pyk2, proline-rich tyrosine kinase 2; SH2, Src homology 2; SH3, Src homology 3. localizes with integrins (12Hanks S.K. 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Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar), or calcium-dependent protein-tyrosine kinase) (22Yu H. Li X. Marchetto G.S. Dy R. Hunter D. Calvo B. Dawson T.L. Wilm M. Anderegg R.J. Graves L.M. Earp H.S. J. Biol. Chem. 1996; 271: 29993-29998Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) define a new subfamily of non-receptor PTKs. Both proteins contain a central kinase domain flanked by large N- and C-terminal domains that do not contain Src homology 2 and 3 (SH2 and SH3) domains. Tyr-397 (23Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 24Calalb M. Polte T. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar), the major autophosphorylation site of FAK, serves as a binding site for the SH2 domain of Src family PTKs in vivo (23Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 25Cobb B.S. Schaller M.D. Leu T-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (483) Google Scholar, 26Xing Z. Chen H.C. Nowlen J.K. Taylor S. Shalloway D. Guan J.L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar). Src phosphorylation of FAK in the C-terminal domain at Tyr-925 creates a binding site for the Grb2 SH2 domain (2Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar, 27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). Mutation of FAK Tyr-925 disrupts Grb2 binding, whereas mutation of Tyr-397 disrupts both Grb2 and c-Src binding to FAK in vivo (27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). Since both the c-Src and Grb2 SH2 binding site motifs are conserved at analogous positions in Pyk2, Grb2 binding to FAK (2Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar) and Pyk2 (18Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1243) Google Scholar) have both been proposed as potential signaling pathways to ERK2. Evidence is accumulating for a facilitating role of Src family PTKs in signal transduction events involving both Pyk2 and FAK. In PC12 cells, where Pyk2 is calcium-activated (18Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1243) Google Scholar), calcium also stimulates c-Src kinase activity, and overexpression of kinase-inactive Src blocks calcium-mediated ERK2 activation (28Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (232) Google Scholar). Expression of a c-Src binding site mutant of Pyk2 or enhanced Csk PTK expression have each been shown to inhibit G-protein-initiated signaling to ERK2 (29Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar). In fibroblasts, FN stimulation induces the activation and redistribution of c-Src to focal contact structures (30Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar, 31Kaplan K.B. Swedlow J.R. Morgan D.O. Varmus H.E. Genes Dev. 1995; 9: 1505-1517Crossref PubMed Scopus (295) Google Scholar), where it is localized constitutively in Csk-deficient cells (32Howell B. Cooper J.A. Mol. Cell. Biol. 1994; 14: 5402-5411Crossref PubMed Scopus (122) Google Scholar). In Src-deficient fibroblasts, FAK tyrosine phosphorylation is reduced (33Thomas S.M. Soriano P. Imamaoto A. Nature. 1995; 376: 267-271Crossref PubMed Scopus (303) Google Scholar), and FN-stimulated signaling to ERK2 is 10-fold lower than in Src− cells engineered to re-express normal c-Src (34Schlaepfer D.D. Broome M.A. Hunter T. Mol. Cell. Biol. 1997; 17: 1702-1713Crossref PubMed Scopus (398) Google Scholar). Although integrin-initiated signaling to ERK2 is dependent on the integrity of the cytoskeleton and may also involve the activation of the Rho family of small GTPases (5Renshaw M.W. Toksoz D. Schwartz M.A. J. Biol. Chem. 1996; 271: 21691-21694Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 35Hotchin N.A. Hall A. J. Cell Biol. 1995; 131: 1857-1865Crossref PubMed Scopus (369) Google Scholar), the important signaling proteins and pathways downstream of integrin receptors have not been clearly defined. Ras GTP-loading (3Clark E.A. Hynes R.O. J. Biol. Chem. 1996; 271: 14814-14818Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 36Kapron-Bras C. Fitz-Gibbon L. Jeevaratnam P. Wilkins J. Dedhar S. J. Biol. Chem. 1993; 268: 20701-20704Abstract Full Text PDF PubMed Google Scholar) and both Raf-1 and ERK2/mitogen-activated protein kinases of the Ras cascade are activated by integrin stimulation (4Chen Q. Lin T.H. Der C.J. Juliano R.L. J. Biol. Chem. 1996; 271: 18122-18127Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). However, there are conflicting reports as to whether Ras is essential for ERK/mitogen-activated protein kinase activation. In two studies the dominant negative Asn-17 mutant of Ras was found to block FN-mediated ERK2 activation in NIH3T3 cells (3Clark E.A. Hynes R.O. J. Biol. Chem. 1996; 271: 14814-14818Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 37Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar), whereas it had a minimal effect on integrin signaling to ERK2 in NIH3T3 cells in another study (4Chen Q. Lin T.H. Der C.J. Juliano R.L. J. Biol. Chem. 1996; 271: 18122-18127Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In addition, there may be more than one signaling pathway upstream of Ras, since antibody-mediated clustering of integrins in suspended cells can generate signals to ERK2 in the absence of FAK tyrosine phosphorylation (37Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar), whereas presentation of fibroblasts to an insoluble FN matrix stimulates FAK tyrosine phosphorylation, transient c-Src association, and Grb2 binding in a time course that parallels ERK2 activation. 2D. D. Schlaepfer and T. Hunter, submitted for publication. 2D. D. Schlaepfer and T. Hunter, submitted for publication. In this study we tested the role of FAK in FN-stimulated signaling events to ERK2. In human 293 epithelial cells, FAK overexpression enhanced c-Src kinase activity and FN-stimulated signaling to ERK2, whereas dominant negative Ras expression blocked ERK2 activation without affecting FAK tyrosine phosphorylation or c-Src activity. Expression of Phe-397 FAK did not stimulate c-Src kinase activity and blocked integrin signaling to ERK2. Thus, we find that Src family PTK and Ras activation events are required for maximal signaling to ERK2 after FN stimulation. Further, we provide evidence that FAK·c-Src complexes may be connected to multiple signaling pathways involving p130Cas, Shc, and Grb2. Monoclonal antibodies to c-Src (mAb 2–17) and to the hemagglutinin epitope tag (anti-HA, mAb 12CA5) were kindly provided by J. Meisenhelder (The Salk Institute) as mouse ascites fluid. Polyclonal antisera to p130Cas (anti-Cas2) and to Shc were generously provided by H. Hirai (University of Tokyo) and P. van der Geer (Mount Sinai Hospital, Toronto), respectively. Polyclonal ERK2 antibody (C-14) and polyclonal c-Src antibody (Src-2) were purchased from Santa Cruz Biotechnology. Monoclonal anti-Tyr(P) (4G10) was purchased from Upstate Biotechnology. Polyclonal Grb2 antiserum was generated to a peptide corresponding to the C-terminal 23 residues of human Grb2 as described (27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). The mouse FAK cDNA containing a triple-HA epitope tag at the FAK C terminus was kindly provided by Steve Hanks (Vanderbilt University). The various FAK constructs used were prepared by site-directed mutagenesis and subcloned into the pcDNA3 eukaryotic expression vector as described (27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). The Shc SH2 domain as a glutathione S-transferase fusion protein was obtained from the laboratory of Tony Pawson. HA-tagged p42 ERK2 in pLNC was a generous gift from M. Weber (University of Virginia). Murine Asn-17 Ras was amplified by polymerase chain reaction from pZIPneoAsn-17 Ras using the sense (5′-AAAATCGATATGACAGAATACAAGCTT-3′) and antisense (5′-TTTATCGATTCAGGACAGCACACA-3′) oligonucleotides. The 588-base pair product was digested with ClaI, cloned into pBluescript KS−, and the sequence was verified by dideoxy chain termination sequencing. Asn-17 Ras was expressed by subcloning into theXhoI/BamHI sites of the pCLXSN eukaryotic expression vector (39Naviaux R.K. Costanzi E. Hass M. Verma I.M. J. Virol. 1996; 70: 5701-5705Crossref PubMed Google Scholar). Human kidney epithelial 293 cells attached to plastic cell culture dishes pre-coated with 10 μg/ml poly-l-lysine were transfected by standard calcium phosphate methods using either 5 μg of pcDNA3-FAK constructs, 10 μg of pCLXSN Asn-17Ras, or 1 μg of pLNC ERK2 per 10-cm dish (2 × 106 cells) in Dulbecco's modified Eagle's medium containing 10% calf serum. After 18 h, the precipitate was removed by washing with PBS, and the cells were incubated in Dulbecco's modified Eagle's medium containing 0.5% calf serum for 24 h prior to cell lysis or FN-replating experiments. Human 293 cells were harvested by limited trypsin/EDTA treatment (0.01% trypsin, 2 mm EDTA in PBS). The trypsin was inactivated by soybean trypsin inhibitor addition (0.5 mg/ml), and cells were collected by centrifugation, resuspended in serum-free Dulbecco's modified Eagle's medium, and held in suspension for 30 min at 37 °C. Cell culture dishes (10 cm) were pre-coated with FN purified from bovine plasma (10 μg/ml, Sigma) or poly-l-lysine (100 μg/ml) in PBS overnight at 4 °C and then rinsed with PBS and warmed to 37 °C for 1 h. Suspended cells were distributed onto ligand-coated dishes (1 × 106 cells/dish), and after 30 min at 37 °C, the attached cells were rinsed in PBS (4 °C) and lysed in 0.75 ml of modified RIPA lysis buffer (see below). Total cell protein in lysates was determined through a colorimetric assay (Bio-Rad) and standardized prior to further analyses. Cells were lysed in modified RIPA lysis buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 10% glycerol, 1.5 mmMgCl2, 1 mm EGTA, 1 mm sodium vanadate, 10 mm sodium pyrophosphate, 100 mmNaF, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 μg/ml leupeptin, 10 units/ml aprotinin, and 1 mmphenylmethylsulfonyl fluoride), insoluble material was removed by centrifugation, and antibodies were added to lysates for 3 h at 4 °C. Antibodies were collected with protein A- or protein G-agarose beads, and protein complexes were washed at 4 °C in Triton-only lysis buffer (RIPA lysis buffer without SDS and sodium deoxycholate) followed by washing in HNTG buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 0.1% Triton X-100, 10% glycerol) prior to direct analysis by SDS-polyacrylamide gel electrophoresis (PAGE, 10% acrylamide) or in vitro 32P labeling. Immunoblotting was performed as described (27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). Briefly, blots were incubated with either 1 μg/ml anti-Tyr(P) (4G10) monoclonal or anti-Src (Src-2) polyclonal antibodies, 1:5000 dilution of anti-HA tag (12CA5), and 1:1000 dilutions of either Grb2, Shc, or p130Cas antiserum for 2 h at room temperature. Bound primary antibody was visualized by enhanced chemiluminescent detection. Membranes were stripped of bound antibodies by incubation in 70 mm Tris-Cl, pH 6.8, 1% SDS, 150 mmβ-mercaptoethanol at 65 °C for 30 min. Prior to reprobing with different primary antibodies, stripped membranes were washed extensively in TBST (10 mm Tris, pH 7.6, 150 mmNaCl, 0.05% Tween 20) and placed in blocking buffer (TBST containing 2% bovine serum albumin) overnight. In vitro ERK2 kinase activity was measured after isolation with antibodies to the HA tag from ∼500 μg of total cell protein in RIPA lysis buffer. The immunoprecipitates (IPs) were washed twice in Triton lysis buffer, once in HNTG buffer, once in ERK2 kinase buffer (25 mm Hepes, pH 7.4, 10 mm MgCl2), and 2.5 μg of myelin basic protein (MBP) was added to each IP. Kinase reactions were initiated by the addition of ATP to 20 μm (10–20 μCi/nmol [γ-32P]ATP), incubated at 37 °C for 15 min, and stopped by the addition of 2 × SDS-PAGE sample buffer, and the phosphorylated MBP was resolved on a 15% acrylamide gel. The bands were visualized by autoradiography and cut from the gel, and the amount of 32P incorporated was determined by Cerenkov counting. HA-tagged FAK did not phosphorylate MBP in vitro. To measure c-Src kinase activity, endogenous c-Src was isolated by IP (mAb 2–17 covalently coupled to protein G-agarose as described (27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar)) from ∼500 μg of total 293 cell protein in RIPA lysis buffer. The IPs were washed twice in Triton lysis buffer, once in HNTG buffer, once in enolase kinase buffer (20 mm Pipes, pH 7.0, 10 mm MgCl2, 1 mm dithiothreitol), and 2.5 μg of acid-denatured enolase was added to each IP. Kinase reactions were initiated by the addition of ATP to 20 μm(10 μCi/nmol [γ-32P]ATP), incubated at 30 °C for 10 min, and stopped by the addition of 2 × SDS-PAGE sample buffer, and products were resolved on a 10% acrylamide gel. The enolase band was visualized by autoradiography and cut from the gel, and the amount of 32P incorporated was determined by Cerenkov counting. FAK did not appreciably phosphorylate enolasein vitro. To test the role of FAK in adhesion-mediated signal transduction events to ERK2, site-directed mutagenesis was used to create single-site phenylalanine replacements of FAK tyrosine residues (397, 407, 861, and 925) that have been shown to be phosphorylated in vivo (23Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 24Calalb M. Polte T. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar, 27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). Mutagenesis was also used to produce a kinase-inactive FAK Arg-454 mutant (24Calalb M. Polte T. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar, 26Xing Z. Chen H.C. Nowlen J.K. Taylor S. Shalloway D. Guan J.L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar) and to create an N-terminally truncated form, Δ1–100 FAK (Fig. 1). HA-tagged wild type (WT) or mutant FAK constructs, HA-tagged ERK2, and a dominant negative Asn-17 mutant of Ras were transiently expressed from cytomegalovirus promoter-driven eukaryotic expression vectors in human 293 epithelial cells that were either serum-starved (Fig. 2, lanes 1–6) or serum-starved and FN-stimulated (Fig. 2, lanes 7–12).Figure 2Ras-dependent FN-initiated and FAK-stimulated signaling to ERK2. A, whole 293 cell RIPA lysates resolved by SDS-PAGE (12.5%) and analyzed by immunoblotting with the 12CA5 mAb to the HA epitope tag. The 293 cells transfected with the indicated constructs were either serum-starved (lanes 1–6) or serum-starved and then FN-replated for 30 min (lanes 7–12). B, anti-HA (12CA5 mAb) IPs were made from ∼1 mg of the total 293 cell RIPA lysates shown inpanel A, and ERK2 activity was measured by in vitro kinase (IVK) assays (20 μCi/nmol [γ-32P]ATP) with myelin basic protein (MBP). The 32P-labeled MBP (top panel) was quantitated by Cerenkov counting, and average values from two separate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since both the FAK and ERK2 constructs were HA-tagged, FAK expression and ERK2 gel shifts were monitored simultaneously by immunoblotting whole cell lysates with the 12CA5 mAb (Fig. 2 A), and HA-tagged ERK2 activity was measured in 12CA5 mAb IPs using an in vitro phosphorylation assay (Fig. 2 B). In serum-starved cells, there was a low basal level of ERK2 activity that was stimulated ∼5-fold by expression of the Δ1–100 FAK construct, which is highly tyrosine-phosphorylated and exhibits elevated association with c-Src (27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). WT FAK overexpression enhanced ERK2 activity ∼3-fold (Fig.2 B), and the extent of FAK-induced ERK2 activity was correlated with the extent of FAK Tyr(P) levels (data not shown). Co-expression of Asn-17 Ras had no effect on FAK Tyr(P) levels (data not shown), but its expression inhibited Δ1–100 and WT FAK-induced ERK2 activation in serum-starved cells (Fig. 2, lanes 4 and6). Activation of the endogenous integrin signaling pathways in the transfected 293 cells by FN replating was sufficient to stimulate exogenously expressed ERK2 (Fig. 2, lanes 7 and8), whereas poly-l-lysine control replating did not result in ERK2 activation (data not shown) (2Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar, 27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar). FN stimulation led to an ∼8-fold activation, whereas FN replating coupled with FAK overexpression led to an ∼20-fold stimulation of ERK2 compared with the basal serum-starved activity level (Fig. 2 B). This FAK-enhanced FN stimulation of ERK2 is consistent with FAK activation after FN stimulation (2Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar, 15Guan J.L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (721) Google Scholar). Co-expression of Asn-17 Ras blocked both the endogenous and FAK-stimulated FN-dependent increases in ERK2 activity, whereas it did not affect the FN-mediated increases in FAK Tyr(P) levels as evidenced by the doublet of Δ1–100 FAK bands (Fig. 2 A, lane 10) and confirmed by anti-Tyr(P) blotting of FAK IPs (data not shown). Interestingly, we found that the lower level of transient Asn-17 Ras expression driven by the vector pZIPneo with a long-terminal repeat promoter did not significantly block FN-stimulated ERK2 activation (data not shown). By placing the Asn-17 Ras construct into a vector with a cytomegalovirus promoter (pCLXSN), transient Asn-17 Ras expression was significantly elevated compared with pZIPneoAsn-17 Ras (data not shown) leading to inhibition of FN and FAK-stimulated signaling to ERK2 (Fig. 2). Our studies are consistent with the reported Ras dependence of integrin signaling to ERK2 (3Clark E.A. Hynes R.O. J. Biol. Chem. 1996; 271: 14814-14818Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 37Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar) and may explain why studies using pZIPneoAsn-17 Ras did not detect a dominant negative Ras effect on integrin signaling to ERK2 (4Chen Q. Lin T.H. Der C.J. Juliano R.L. J. Biol. Chem. 1996; 271: 18122-18127Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Since previous studies have shown that integrin-activated FAK transiently associates with Src family PTKs (2Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar, 27Schlaepfer D.D. Hunter T. Mol. Cell. Biol. 1996; 16: 5623-5633Crossref PubMed Scopus (395) Google Scholar, 34Schlaepfer D.D. Broome M.A. Hunter T. Mol. Cell. Biol. 1997; 17: 1702-1713Crossref PubMed Scopus (398) Google Scholar), the effects of FAK overexpression on endogenous 293 cell c-Src kinase activity were evaluated (Fig.3). Src kinase activity as measured by in vitro phosphorylation of acid-denatured enolase was low in lysates from serum-starved 293 cells and was increased 2-fold by the expression of Δ1–100 FAK (Fig. 3, lane 2), and this stimulation was not significantly affected by the expression of Asn-17 Ras (Fig. 3,lane 3). Overexpression of WT FAK in serum-starved 293 cells did not lead