Focal Adhesion Kinase Is Upstream of Phosphatidylinositol 3-Kinase/Akt in Regulating Fibroblast Survival in Response to Contraction of Type I Collagen Matrices via a β1 Integrin Viability Signaling Pathway
2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês
10.1074/jbc.m313265200
ISSN1083-351X
AutoresXia Hong, Richard Nho, Judy Kahm, Jill Kleidon, Craig A. Henke,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe β1 integrin, functioning as a mechanoreceptor, senses a mechanical stimulus generated during collagen matrix contraction and down-regulates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signal triggering apoptosis. The identities of integrin-associated signal molecules in the focal adhesion complex that are responsible for propagating β1 integrin viability signals in response to collagen matrix contraction are not known. Here we show that in response to collagen contraction focal adhesion kinase (FAK) is dephosphorylated. In contrast, enforced activation of β1 integrin by anti-β1 integrin antibody, which protects fibroblasts from apoptosis, preserves FAK phosphorylation. We demonstrate that ligation of β1 integrin by type I collagen or by enforced activation of β1 integrin by antibody promotes phosphorylation of FAK, p85 subunit of PI3K, and serine 473 of Akt. Wortmannin inhibited Akt but not FAK phosphorylation in response to enforced activation of β1 integrin by antibody. Blocking FAK by pharmacologic inhibition or by dominant negative FAK attenuated phosphorylation of p85 subunit of PI3K and Akt. Dominant negative FAK augmented fibroblast apoptosis during collagen contraction, and this was associated with diminished Akt activity. Constitutively active FAK augmented levels of p85 subunit of PI3K and Akt phosphorylation, and fibroblasts were protected from apoptosis. Our data identify a novel role for FAK, functioning upstream of PI3K/Akt, in transducing a β1 integrin viability signal in collagen matrices. The β1 integrin, functioning as a mechanoreceptor, senses a mechanical stimulus generated during collagen matrix contraction and down-regulates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signal triggering apoptosis. The identities of integrin-associated signal molecules in the focal adhesion complex that are responsible for propagating β1 integrin viability signals in response to collagen matrix contraction are not known. Here we show that in response to collagen contraction focal adhesion kinase (FAK) is dephosphorylated. In contrast, enforced activation of β1 integrin by anti-β1 integrin antibody, which protects fibroblasts from apoptosis, preserves FAK phosphorylation. We demonstrate that ligation of β1 integrin by type I collagen or by enforced activation of β1 integrin by antibody promotes phosphorylation of FAK, p85 subunit of PI3K, and serine 473 of Akt. Wortmannin inhibited Akt but not FAK phosphorylation in response to enforced activation of β1 integrin by antibody. Blocking FAK by pharmacologic inhibition or by dominant negative FAK attenuated phosphorylation of p85 subunit of PI3K and Akt. Dominant negative FAK augmented fibroblast apoptosis during collagen contraction, and this was associated with diminished Akt activity. Constitutively active FAK augmented levels of p85 subunit of PI3K and Akt phosphorylation, and fibroblasts were protected from apoptosis. Our data identify a novel role for FAK, functioning upstream of PI3K/Akt, in transducing a β1 integrin viability signal in collagen matrices. Integrins are cell surface adhesion receptors that regulate cell viability in response to cues derived from the extracellular matrix (1Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Google Scholar, 2Ruoslahti E. Tumor Biol. 1996; 17: 117-124Google Scholar). The nature of these extracellular cues that regulate cell viability are diverse. In the case of polarized epithelial and endothelial cells, whose basal surface is attached to the basement membrane, direct loss of integrin-matrix interaction can trigger anoikis (3Meredith Jr., J.E. Fazeli B. Schwartz M.A. Mol. Biol. Cell. 1993; 4: 953-961Google Scholar, 4Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Google Scholar, 5Lee J.W. Juliano R.L. Mol. Biol. Cell. 2000; 11: 1973-1987Google Scholar). In the case of mesenchymal cells, which exist encompassed by the extracellular matrix, matrix-derived mechanical stimuli can regulate viability. In this latter scenario, integrins function as mechanoreceptors that detect mechanical stimuli originating from the extracellular matrix and convert them to chemical signals that regulate cell viability pathways (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar, 7Dimmler S. Assmus B. Hermann C. Haendeler J. Zeiher A.M. Circ. Res. 1998; 83: 334-341Google Scholar, 8Chen K.D. Li Y.S. Kim M. Li S. Yuan S. Chien S. Shyy J.Y. J. Biol. Chem. 1999; 274: 18393-18400Google Scholar, 9Davies P.F. Physiol. Rev. 1995; 75: 519-560Google Scholar, 10Wilson E. Sudhir K. Ives H.E. J. Clin. Investig. 1995; 96: 2364-2372Google Scholar, 11Ishida T. Peterson T.E. Kovach N.L. Berk B.C. Circ. Res. 1996; 79: 310-316Google Scholar, 12MacKenna D.A. Dolfi F. Vuori K. Ruoslahti E. J. Clin. Investig. 1998; 101: 310Google Scholar). For example, fibroblast survival in type I collagen matrices is regulated by integrin-extracellular matrix interactions (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). In response to contraction of type I collagen matrices fibroblasts undergo apoptosis (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar, 13Grinnell F. Zhu M. Carlson M.A. Abrams J.M. Exp. Cell Res. 1999; 248: 608-619Google Scholar, 14Fluck J. Querfeld C. Cremer A. Niland S. Krieg T. Sollberg S. J. Investig. Dermatol. 1998; 110: 153-157Google Scholar). During the process of collagen contraction Akt becomes dephosphorylated (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). This down-regulation of Akt activity during collagen contraction is mediated by β1 integrin. Up-regulation of Akt activity, either by enforced activation of β1 integrin by anti-β1 integrin antibody or by ectopic expression of constitutively active phosphatidylinositol 3-kinase (PI3K), 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; FAK, focal adhesion kinase; SH2, Src homology 2; FRNK, dominant negative FAK; IRES, internal ribosome entry site; GFP, green fluorescent protein; Ad, adenovirus; RV, retrovirus; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CA, constitutively active; PP2, PP3, pyrazolopyrimidine 2 and 3, respectively; PTEN, phosphatase and tensin homologue deleted on chromosome 10. protects fibroblasts from collagen gel contraction-induced apoptosis (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). Therefore, functioning as a mechanoreceptor, the β1 integrin is capable of sensing a matrix-derived mechanical stimulus and regulates fibroblast survival by modulating a PI3K/Akt survival pathway. Matrix-derived mechanical stimuli can be transmitted through the direct or indirect interaction of integrins with associated lipid or protein signaling molecules in the focal adhesion complex (15Plopper G.E. McNamee H.P. Dike L.E. Bojanowski K. Ingber D.E. Mol. Biol. Cell. 1995; 6: 1349-1365Google Scholar, 16Chicurel M.E. Chen C.S. Ingber D.E. Curr. Opin. Cell Biol. 1998; 10: 232-239Google Scholar). Currently the identities of integrin-associated signaling molecules that are responsible for propagating the β1 integrin viability signal via PI3K/Akt in response to collagen matrix-derived mechanical signals are unclear. Focal adhesion kinase (FAK), a potential candidate signaling molecule, has been shown to be capable of regulating integrin-mediated survival signaling (17Frisch S.M. Vuori K. Ruoslahti E Chan-Hui P.Y. J. Cell Biol. 1996; 134: 793-799Google Scholar, 18Crouch D.H. Fincham V.J. Frame M.C. Oncogene. 1996; 12: 2689-2696Google Scholar, 19Hadden H.L. Henke C.A. Am. J. Respir. Crit. Care Med. 2000; 162: 1553-1560Google Scholar, 20Hungerford J.E. Compton M.T. Matter M.L. Hoffstrom B.G. Otey C.A. J. Cell Biol. 1996; 135: 1383-1390Google Scholar, 21Levkau B. Herren B. Koyama H. Ross R. Raines E. J. Exp. Med. 1998; 187: 579-586Google Scholar, 22Chen H.C. Guan J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Google Scholar). However, the downstream signaling pathways that mediate integrin-FAK survival signaling are diverse, and the factors determining which pathway is utilized remain obscure. Downstream signal pathways implicated in FAK viability signaling include a c-Jun N-terminal kinase survival pathway that inactivates the tumor suppressor protein p53-regulated cell death pathway (23Ilic D. Almeida E.A.C. Schlaepfer D.D. Dazin P. Aizawa S. Damsky C.H. J. Cell Biol. 1998; 143: 547-560Google Scholar), death-associated protein kinase (24Wang W.J. Kuo J.C. Yao C.C. Chen R.H. J. Cell Biol. 2002; 159: 169-179Google Scholar), and the PI3K/Akt pathway regulating epithelial and endothelial cell viability (22Chen H.C. Guan J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Google Scholar, 25Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Google Scholar). Data supporting a role for FAK in the β1 integrin/PI3K/Akt viability pathway include the finding that integrin-extracellular matrix interaction recruits FAK to the focal adhesion complex and activates it. Integrin ligation and clustering activates FAK by autophosphorylation of tyrosine 397. This creates a potential binding site for the SH2 domains of the p85 subunit of PI3K (26Schlaepfer D.D. Hunter T. Trends Cell Biol. 1998; 8: 151-157Google Scholar, 27Eliceiri B.P. Puente X.S. Hood J.D. Stupack D.G. Schlaepfer D.D. Huang X.Z. Sheppard D. Cheresh D.A. J. Cell Biol. 2002; 157: 149-159Google Scholar, 28Chen H.C. Appeddu P.A. Isoda H. Guan J.L. J. Biol. Chem. 1996; 271: 26329-26334Google Scholar). Phosphorylation of the p85 subunit of PI3K by FAK may activate the p110 catalytic subunit of PI3K and the PI3K/Akt signal pathway. Although it is plausible that FAK regulates fibroblast survival within a three-dimensional collagen matrix via the β1 integrin/PI3K/Akt signal pathway, the role of FAK in this process has not been examined. In this study we show that in response to collagen matrix contraction FAK is dephosphorylated. In contrast, enforced activation of β1 integrin by anti-β1 integrin antibody, which protects fibroblasts from collagen contraction-induced apoptosis, preserves FAK phosphorylation. We demonstrate that ligation of β1 integrin with type I collagen or enforced activation of β1 integrin by anti-β1 integrin antibody promotes the phosphorylation of FAK, the p85 subunit of PI3K, and serine 473 of Akt. Blocking FAK function by pharmacologic inhibition or by dominant negative FAK inhibits both the phosphorylation of the p85 subunit of PI3K and serine 473 of Akt. Furthermore dominant negative FAK promotes fibroblast apoptosis in both anoikis and collagen gel assays. Conversely constitutively active FAK augments p85 phosphorylation and Akt activity and protects fibroblasts from collagen gel contraction-induced apoptosis. Our data identify a novel role for FAK functioning upstream of PI3K and Akt in mediating β1 integrin viability signaling of fibroblasts in collagen matrices. Cell Culture—Human lung fibroblasts (CCL-210, American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10% heat-inactivated fetal calf serum and used between passages 9 and 11. Antibodies and Reagents—Mouse monoclonal antibody P5D2 (raised against the human β1 integrin subunit) was provided by Dr. Leo Furcht (University of Minnesota). Polyclonal anti-Akt antibody and anti-phosphorylated Akt antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA). Polyclonal anti-FAK was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-FAK antibodies directed to tyrosine 397, 407, 576, 577, 861, or 925 were from BIOSOURCE (Camarillo, CA). Anti-PI3K-p85 subunit antibody and anti-phosphorylated tyrosine antibody Py20 were from BD Transduction Laboratories. Wortmannin was from Sigma, and PP2 and PP3 were from Calbiochem. Dominant Negative FAK cDNA Construct—Dominant negative FAK (FRNK) was generated by amplification of the cDNA FRNK encoding sequence of FAK obtained from human lung fibroblasts by reverse transcription-PCR (29Mortier E. Cornelissen F. van Hove C. Dillen L. Richardson A. Cell. Signal. 2001; 13: 901-909Google Scholar). The primers used to generate dominant negative FAK were: 5′-CAG CAC AAT ATC GAT CAG CAA GAA GAG CAC-3′ and 5′-CGC CAC ATT TTG GAT CCT GGA TT-3′. A ClaI restriction site was introduced at the beginning of 5′-FRNK. Both IRES-GFP (pIRES2-EGFP vector from Clontech) and FRNK were cloned into the pBluescript II KS vector. FRNK-IRES-GFP was then inserted into the SwaI site in vector pAxCAwt (TaKaRa, Tokyo, Japan) to generate recombinant adenovirus. Recombinant adenoviruses with FRNK-GFP and IRES-GFP (Ad-FRNK and Ad-GFP) were made according to the company's instruction. Adenovirus with IRES-GFP only (Ad-GFP) served as a control. Adenovirus titers were analyzed by the agarose plaque method. Constitutively Active FAK Construct—Constitutively active FAK (CD2-FAK) in the pCDM8 vector was a gift from Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA. A unique NotI restriction site was introduced at the beginning of 5′-CD2-FAK in the pCDM8 vector by PCR. The primers used to generate the restriction site were 5′-TCA CAG GTC AGG GTT GTG TT-3′ and 5′-ATC GCG GCC GCT TCT AGA GAT CCC TCG-3′. The CD2-FAK fragment from the pCDM8 vector and IRES-GFP from the pIRES2-GFP vector were subcloned into the NotI-SalI in the pBluescript II KS vector. Next the CD2-FAK-IRES-GFP fragment cut by NotI and SalI from the pBluescript II KS vector was cloned into the BglII and SalI site in the amphotropic retrovirus expression vector MIGR1.str. The resulting plasmid MIGR1-CD2FAKGFP and retrovirus genomic plasmid PCL were cotransfected into H-293 cells using a calcium phosphate precipitation method to produce the replication-defective recombinant retrovirus, retrovirus-CD2-FAKGFP (RV-CD2-FAK). Retrovirus-GFP (RV-GFP) served as a control. In addition, the fragment of CD2-FAK and IRES-GFP was cloned into the SwaI site in the vector pAxCAwt to generate the recombinant adenovirus Ad-CD2-FAK according to the manufacturer's instruction. Constitutively Active p110 Subunit of PI3K Construct—Dr. Julian Downward provided the constitutively active p110 subunit of PI3K. CA-p110 was subcloned into the retroviral vector MIGR1.str containing IRES-GFP, creating the bicistronic construct CA-p110/IRES-GFP as described previously (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). A population of CA-p110/GFP-positive cells was obtained by cell sorting using fluorescence-activated cell sorter analysis. Adenoviral and Retroviral Infection—Human lung fibroblasts were plated in tissue culture dishes and infected with Ad-GFP, Ad-FRNK, and Ad-CD2-FAK at a multiplicity of infection of 1:50 for 12 h or with RV-GFP and RV-CD2FAK for 48 h in Dulbecco's modified Eagle's medium + 10% fetal calf serum. After infection the medium was changed to serum-free Dulbecco's modified Eagle's medium, and the cells were serum-starved for 48 h prior to performing the individual experiments. After 48 h of serum starvation the cells were ready to be used for different treatments with anti-β1 integrin antibody (P5D2, 6 μg/ml) or various reagents (wortmannin, 200 nm; PP2 or PP3, 10 μm). Immunoprecipitation and Western Analysis—Fibroblasts were lysed in lysis buffer containing 20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 50 mm NaF, 0.5% sodium deoxycholate, 1% Nonidet P-40, 2.5 mm Na2P2O7, 1 mm glycerol phosphate, 1 mm Na3VO4, 1× protease inhibitor mixture, and 1 mm phenylmethylsulfonyl fluoride. Lysates were precleared for 60 min at 4 °C with protein G-coupled agarose beads and immunoprecipitated overnight (16 h) at 4 °C with the appropriate primary antibody. Western analysis of human lung fibroblasts was performed as described previously (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). Briefly equal amounts of protein from cell lysates were subjected to 8-10% SDS-PAGE and transferred (45 min, 24 V) to nitrocellulose membrane. Membranes were blocked with 20 mm Tris-HCl (pH 7.6), 137 mm NaCl, and 0.05% Tween 20 containing 6% nonfat dry milk; incubated with the primary antibody; washed; and incubated with horseradish peroxidase-conjugated secondary antibody. The membranes were developed using the ECL method (Amersham Biosciences). Anoikis Assay—Anoikis assays were performed as described by Frisch and Francis (4Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Google Scholar). Tissue culture plates were coated twice with poly-HEME (10 mg/ml in ethanol, Sigma) and rinsed extensively with phosphate-buffered saline. Fibroblasts resuspended in Dulbecco's modified Eagle's medium were plated on the poly-HEME plates. At the indicated times, the cells in suspension were recovered and analyzed by fluorescence TUNEL assay. TUNEL Assay—Fibroblasts recovered from anoikis assays and type I collagen gels were analyzed for apoptosis using an in situ cell death detection kit (fluorescence TUNEL assay, Roche Applied Science) according to the manufacturer's instructions. Briefly recovered cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 60 min at room temperature and permeabilized with 0.1% Triton X-100 for 2 min at 4 °C. The cells were resuspended in TUNEL reaction mixture, incubated for 60 min at 37 °C, washed, and analyzed by fluorescence microscopy (30Gavrieli Y. Sherman Y. Ben-Sasson S.A. J. Cell Biol. 1992; 119: 493-501Google Scholar). Collagen Gel Assay—Collagen gels were prepared as described previously (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). Human lung fibroblasts (1.2 × 105) were resuspended in 2.0 ml of 1.5× Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Vitrogen (3 mg/ml; Cohesion, Palo Alto, CA) was added to the cell suspension to achieve a final concentration of 0.5 mg/ml. The cell/collagen solution was incubated in a water bath for 10 min at 37 °C, poured into 3.5-cm uncoated plastic dishes, and placed in a cell culture incubator at 10% CO2 and 37 °C where the gels polymerized in ∼30-60 min. Statistical Analysis—Data are expressed as the mean ± S.D. Experiments were performed three times. For assessment of the percentage of apoptotic cells within collagen gels or on dishes coated with poly-HEME using the TUNEL assay, microscopic analysis of at least 200 cells/slide was performed. Paired evaluations were made for experimental and control conditions within each experiment, and significance was determined by Student's t test. The significance level was set at p < 0.05. Collagen Matrix Contraction Is Associated with FAK Dephosphorylation—Regulation of fibroblast viability in collagen matrices is mediated by the β1 integrin and involves a PI3K/Akt signaling pathway. In response to type I collagen matrix contraction, Akt becomes dephosphorylated triggering fibroblast apoptosis. Previous studies have implicated FAK with β1 integrin viability signaling; therefore we were interested in determining the effect of collagen matrix contraction on FAK activity. Phosphorylation of tyrosine 397 of FAK has been used as a marker of FAK activity. We examined phosphorylation of tyrosine 397 of FAK as a function of time in contractile collagen gels. During the first 2-4 h after fibroblasts have been incorporated into type I collagen matrices but before contraction begins, the cells attach and spread. During this time period FAK became phosphorylated (Fig. 1). However, as matrix contraction progresses the cells become increasingly round in appearance (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). By 24 h the gels have contracted, and many of the cells are undergoing apoptosis (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). At the 24-h time point we found that FAK was dephosphorylated. Therefore, in conjunction with our previous finding that Akt becomes dephosphorylated during matrix contraction, these data suggested a link between FAK and Akt activity and regulation of fibroblast viability in collagen matrices. We have found previously that enforced activation of β1 integrin by P5D2 anti-β1 integrin antibody protects fibroblasts from collagen contraction-induced apoptosis by augmenting Akt phosphorylation/activity (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). Therefore, we assessed the effect of P5D2 antibody treatment on FAK phosphorylation during collagen matrix contraction. Fibroblasts were incorporated into contractile collagen gels. The cells were allowed to attach and spread for 2 h in the gels after which time P5D2 antibody was added to the collagen gels as described previously (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). The effect on FAK phosphorylation was assessed at various times. At the 4- and 6-h time points, phosphorylation of tyrosine 397 of FAK in contractile collagen gels treated with P5D2 antibody was similar to that found in control contractile gels. It should be noted that at these time points little collagen contraction had occurred. However, at the 24-h time point when the gels have largely completed contraction, the level of FAK phosphorylation was preserved in contractile collagen gels treated with P5D2 antibody compared with control contractile gels (Fig. 1). This suggests that the protection of fibroblasts from collagen contraction-induced apoptosis in response to enforced activation of β1 integrin by antibody involves FAK. FAK Phosphorylation and Akt Activity Increase as a Function of Ligation of β1Integrin with Type I Collagen or by Enforced Activation of β1Integrin by Anti-β1Integrin Antibody—To further examine whether FAK plays a role in integrin-mediated survival signaling in collagen matrices, we evaluated the effect of culturing fibroblasts on collagen-coated plates on FAK phosphorylation and Akt activity. Human lung fibroblasts were serum-starved for 48 h and then plated on tissue culture plastic dishes coated with type I collagen. Phosphorylation of serine 473 of Akt was used to assess Akt activity (31Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Google Scholar). Akt phosphorylation increased as a function of time as the fibroblasts were allowed to adhere and spread on type I collagen (Fig. 2A). To examine whether the increase in Akt activity was dependent upon β1 integrin interaction with type I collagen, we examined the effect of blocking β1 integrin-type I collagen interaction on Akt activity using anti-β1 integrin antibody. When cells in suspension are preincubated with P5D2 antibody prior to plating on type I collagen, the anti-β1 integrin antibody functions as an inhibitor, blocking integrin-collagen interaction (6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar). Preincubation of fibroblasts with antibody prior to plating on type I collagen abrogated the increase in Akt activity (Fig. 2A). We next examined whether the β1 integrin-mediated increase in Akt activity associated with ligation of type I collagen was dependent upon PI3K. Serum-starved fibroblasts were pretreated with wortmannin prior to plating on type I collagen-coated dishes. Inhibition of PI3K by wortmannin attenuated the increase in Akt activity associated with β1 integrin-type I collagen interaction (data not shown). These data indicate that β1 integrin-type I collagen interaction promotes Akt activity in a PI3K-dependent manner. The effect of plating fibroblasts on collagen-coated dishes on FAK phosphorylation was also examined. Human lung fibroblasts were serum-starved for 48 h and then plated on type I collagen-coated dishes. Similar to Akt activity, FAK phosphorylation increased as a function of time as the fibroblasts were allowed to adhere and spread on type I collagen (Fig. 2B). To examine whether the increase in FAK phosphorylation was dependent upon β1 integrin-type I collagen interaction, we examined the effect of preincubating fibroblasts with anti-β1 integrin antibody on FAK phosphorylation. Inhibition of β1 integrin-type I collagen interaction by anti-β1 integrin antibody abrogated the increase in FAK phosphorylation associated with plating the cells on type I collagen (Fig. 2B). We have shown previously that enforced activation of β1 integrin by adding P5D2 β1 integrin monoclonal antibody to adherent fibroblasts increases Akt activity in a PI3K-dependent fashion (Ref. 6Tian B. Lessan K. Kahm J. Kleidon J. Henke C. J. Biol. Chem. 2002; 277: 24667-24675Google Scholar; see also Fig. 3C). Under these culture conditions, the P5D2 anti-β1 integrin antibody functions in an agonist fashion. Therefore, to perform these experiments fibroblasts were plated on tissue culture dishes and serum-starved prior to addition of anti-β1 integrin antibody. Consistent with the results obtained when integrin was ligated by type I collagen, we found that enforced activation of β1 integrin by P5D2 antibody also increased FAK phosphorylation in serum-starved fibroblasts adhered to tissue culture dishes (Fig. 3A). We next assessed which FAK phosphorylation sites were phosphorylated upon ligation of β1 integrin with P5D2 antibody by immunoblotting lysates of P5D2 antibody-treated cells with a panel of phosphotyrosine-specific antibodies. These antibodies recognize the tyrosine-phosphorylated state of amino acid 397, 407, 576, 577, 861, or 925 of FAK. Ligation of β1 integrin with P5D2 antibody increased the phosphorylation of tyrosines 397 and 407 in a time-dependent manner (Fig. 3A). Phosphorylation of the other tyrosine residues within FAK were either only slightly increased or not increased at all. Because ligation of β1 integrin with type I collagen or P5D2 anti-β1 integrin antibody phosphorylates FAK, we sought to determine whether ligation of β1 integrin promotes the association of FAK with β1 integrin. Lysates of serum-starved fibroblasts treated with P5D2 antibody were immunoprecipitated using anti-β1 integrin antibody and probed for the presence of FAK or vice versa. A direct physical association between integrin and FAK could not be demonstrated in response to enforced activation of β1 integrin by anti-β1 integrin antibody (data not shown). Phosphorylation of tyrosine 397 of FAK may provide a binding site for the Src homology 2 domains of the p85 subunit of PI3K (31Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Google Scholar). Since enforced activation of β1 integrin by anti-β1 integrin antibody promotes the phosphorylation of tyrosine 397 of FAK, we sought to determine whether ligation of β1 integrin by antibody also promoted the association of FAK with PI3K. Immunoprecipitation of FAK and immunoblotting for the p85 subunit of PI3K or vice versa did not demonstrate a direct physical association of FAK with the p85 subunit of PI3K. However, we found that enforced activation of β1 integrin with anti-β1 integrin antibody promoted phosphorylation of the p85 subunit of PI3K in a time-dependent manner (Fig. 3B). For these experiments, serum-starved adherent fibroblasts were treated with P5D2 antibody for various times. Cell lysates were immunoprecipitated with anti-PI3K-p85 antibody. Phosphorylation of p85 subunit was assessed using an anti-phosphotyrosine antibody and Western analysis. Although we were unable to demonstrate a direct physical association of integrin with FAK or FAK with PI3K, our results suggest a link between ligation of β1 integrin and activation of FAK, PI3K, and Akt. These data support the concept that FAK may play a role in β1 integrin/PI3K/Akt survival signaling on collagen matrices. The above data indicate that ligation of β1 integrin by either type I collagen or by enforced activation of β1 integrin by antibody increases FAK, PI3K, and Akt activity. We were also interested in determining what effect blocking integrin-mediated FAK phosphorylation would have on PI3K and Akt activity. To begin to approach this issue we used PP2, a selective Src kinase family inhibitor. By virtue of its ability to inhibit Src kinases, PP2 has been shown to block integrin-induced FAK phosphorylation (32Salazar E.P. Rozengurt E. J. Biol. Chem. 2001; 276: 17788-17795Google Scholar). We found that PP2 effectively abolished the phosphorylation of tyrosine 397 of FAK, the p85 subunit of PI3K, and serine 473 of Akt (Fig. 4) that occurs upon enforced activation of β1 integrin by anti-β1 integrin antibody. In contrast, PP3, an inactive analog of PP2, had no effect. These results suggest a possible link between integrin-induced FAK phosphorylation and activation of PI3K and Akt. Down-regulation of FAK Activity Inhibits the Phosphorylation of FAK, the p85 Subunit of PI3K, and Akt—These studies strongly suggested to us that FAK activity might be involved in regulating PI3K/Akt survival signaling in response to collagen matrix contraction. To investigate this further, we examined the effect of FRNK on phosphorylation of Akt to determine whether FAK is required for the activation of Akt in response to ligation of β1 integrin by collagen and anti-β1 integrin antibody. We first examined whether dominant negative FAK would inhibit FAK phosphorylation induced by β1 integrin-type I collagen interaction and by enforced activation of β1 integrin by P5D2 antibody. Human lung fibroblasts were infected with either Ad-FRNK or Ad-GFP (empty vector control). The cells were then serum-starved for 48 h and then either plated on tissue culture dishes coated with
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