In Situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from Focal Adhesion Kinase Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells
2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês
10.1074/jbc.m502726200
ISSN1083-351X
AutoresDavid M. Humphrey, Zenon Rajfur, M. Eugenio Vázquez, Danielle M Scheswohl, Michael D. Schaller, Ken Jacobson, Barbara Imperiali,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoFocal adhesion kinase (FAK), a non-receptor tyrosine kinase, mediates integrin-based cell signaling by transferring signals regulating cell migration, adhesion, and survival from the extracellular matrix to the cytoplasm. Following autophosphorylation at tyrosine 397, FAK binds the Src homology 2 domains of Src and phosphoinositide 3-kinase, among several other possible binding partners. To further investigate the role of phosphorylated FAK in cell migration in situ, peptides comprising residues 391–406 of human FAK with caged phosphotyrosine 397 were synthesized. Although the caged phosphopeptides were stable to phosphatase activity, the free phosphopeptides showed a half-life of ∼10–15 min in cell lysates. Migrating NBT-II cells (a rat bladder tumor cell line) were microinjected with the caged FAK peptide and locally photoactivated using a focused laser beam. The photoactivation of caged FAK peptide in 8-μm diameter spots over the cell body led to the temporary arrest of the leading edge migration within ∼1 min of irradiation. In contrast, cell body migration was not inhibited. Microinjection of a non-caged phosphorylated tyrosine 397 FAK peptide into migrating NBT-II cells also led to lamellar arrest; however, this approach lacks the temporal control afforded by the caged phosphopeptides. Photoactivation of related phosphotyrosine peptides with altered sequences did not result in transient lamellar arrest. We hypothesize that the phosphorylated FAK peptide competes with the endogenous FAK for binding to FAK effectors including, but not limited to, Src and phosphoinositide 3-kinase, causing spatiotemporal misregulation and subsequent lamellar arrest. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, mediates integrin-based cell signaling by transferring signals regulating cell migration, adhesion, and survival from the extracellular matrix to the cytoplasm. Following autophosphorylation at tyrosine 397, FAK binds the Src homology 2 domains of Src and phosphoinositide 3-kinase, among several other possible binding partners. To further investigate the role of phosphorylated FAK in cell migration in situ, peptides comprising residues 391–406 of human FAK with caged phosphotyrosine 397 were synthesized. Although the caged phosphopeptides were stable to phosphatase activity, the free phosphopeptides showed a half-life of ∼10–15 min in cell lysates. Migrating NBT-II cells (a rat bladder tumor cell line) were microinjected with the caged FAK peptide and locally photoactivated using a focused laser beam. The photoactivation of caged FAK peptide in 8-μm diameter spots over the cell body led to the temporary arrest of the leading edge migration within ∼1 min of irradiation. In contrast, cell body migration was not inhibited. Microinjection of a non-caged phosphorylated tyrosine 397 FAK peptide into migrating NBT-II cells also led to lamellar arrest; however, this approach lacks the temporal control afforded by the caged phosphopeptides. Photoactivation of related phosphotyrosine peptides with altered sequences did not result in transient lamellar arrest. We hypothesize that the phosphorylated FAK peptide competes with the endogenous FAK for binding to FAK effectors including, but not limited to, Src and phosphoinositide 3-kinase, causing spatiotemporal misregulation and subsequent lamellar arrest. Focal adhesions are cell-surface specializations that connect the extracellular matrix to the actin cytoskeleton. Transmembrane integrins and associated cytoskeletal proteins, including talin, vinculin, α-actinin, and filamin, perform this function within the focal adhesion (1Sastry S.K. Burridge K. Exp. Cell Res. 2000; 261: 25-36Crossref PubMed Scopus (429) Google Scholar, 2Zamir E. Geiger B. J. Cell Sci. 2001; 114: 3583-3590Crossref PubMed Google Scholar). In addition, focal adhesions contain numerous regulatory molecules, including focal adhesion kinase (FAK), 1The abbreviations used are: FAK, focal adhesion kinase; SH2, Src homology 2; PI3K, phosphatidylinositol 3-kinase; pTyr, phosphotyrosine; cpTyr, caged pTyr; HPLC, high performance liquid chromatography; GST, glutathione S-transferase; HBTU, O-benzotriazolyl-N-N-N′-N′-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; MS(ESI), mass spectrometry (electrospray ionization); DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; Ahx, aminohexanoic acid; Fmoc, N-(9-fluorenyl)methoxycarbonyl; Rh, rhodamine. which is a non-receptor tyrosine kinase. Cell binding to the extracellular matrix clusters integrins, which, in turn, stimulates phosphorylation of FAK on tyrosine 397; this creates docking sites where SH2 domain-containing proteins, such as Src, can bind. In this way, FAK links integrin receptors to intracellular signaling events related to cell migration and survival. The importance of FAK in cell migration was demonstrated when FAK null fibroblasts showed reduced migration compared with wild type cells generated from the same stage of mouse embryos (3Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1587) Google Scholar). The migration defect could be rescued by expression of wild type FAK (4Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (341) Google Scholar, 5Sieg D.J. Hauck C.R. Schlaepfer D.D. J. Cell Sci. 1999; 112: 2677-2691Crossref PubMed Google Scholar). Overexpression of FAK in Chinese hamster ovary cells also resulted in increased migration (6Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar). However, neither re-expression of mutated FAK (Y397F) in the FAK null cells nor overexpression of Y397F in Chinese hamster ovary cells enhanced migration. Upon phosphorylation, the region containing tyrosine 397 may bind to the SH2 domains of Src (7Schaller 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 (1117) Google Scholar, 8Xing Z. Chen H.C. Nowlen J.K. Taylor S.J. Shalloway D. Guan J.L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar), phospholipase C-γ1 (9Wells A. Ware M.F. Allen F.D. Lauffenburger D.A. Cell Motil. Cytoskeleton. 1999; 44: 227-233Crossref PubMed Scopus (77) Google Scholar, 10Zhang X. Chattopadhyay A. Ji Q.S. Owen J.D. Ruest P.J. Carpenter G. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9021-9026Crossref PubMed Scopus (158) Google Scholar), phosphatidylinositol 3-kinase (PI3K) (10Zhang X. Chattopadhyay A. Ji Q.S. Owen J.D. Ruest P.J. Carpenter G. 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The defect in the stimulation of migration exhibited by Y397F is presumably because of a resultant inability to recruit one or more of these binding partners. FAK phosphopeptides have been synthesized to test interactions of phosphotyrosine 397 (pTyr397 FAK) with the SH2 domains of Src (17Thomas J.W. Ellis B. Boerner R.J. Knight W.B. White G.C. Schaller M.D. J. Biol. Chem. 1998; 273: 577-583Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) and PI3K, respectively (11Chen H.C. Appeddu P.A. Isoda H. Guan J.L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Chen (11Chen H.C. Appeddu P.A. Isoda H. Guan J.L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar) used a FAK Tyr397 phosphopeptide to disrupt the binding of the p85 subunit of PI3K to full-length FAK in vitro using GST fusion proteins. Peptides containing the pTyr397 FAK region of FAK might therefore be useful for probing FAK function in migrating cells. In the present work, we investigated the role of pTyr397 FAK within FAK by introducing photoactivatable caged phosphopeptides containing Tyr397 and the surrounding sequence into migrating cells. A caged compound includes a photocleavable protecting group that masks an essential functionality. In this context, a peptide or protein is prepared by covalently linking a photolabile protecting group to a limited number of critical functional groups in the biomolecule (18Humphrey D. Rajfur Z. Imperiali B. Marriott G. Roy P. Jacobson K. Spector D.L. Goldman R.D. Live Cell Imaging: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2005: 159-176Google Scholar). The caged peptides can be introduced into the cells by microinjection. Upon removal of the caging moiety by photolysis, the bioactive form of the peptide or protein is produced within the cell (19Kaplan J.H. Forbush III, B. Hoffman J.F. Biochemistry. 1978; 17: 1929-1935Crossref PubMed Scopus (638) Google Scholar, 20Park C-H. Givens R.S. J. Am. Chem. Soc. 1997; 119: 2453-2463Crossref Scopus (196) Google Scholar). Photoactivation of caged proteins/peptides thus offers insights into cellular dynamics not achievable using genetic methods: perturbations that can be controlled temporally and, in some cases, spatially are followed with subsequent observations of altered cell behavior. However, thus far, there are few studies using caged peptides and proteins to study cell migration (21Walker J.W. Gilbert S.H. Drummond R.M. Yamada M. Sreekumar R. Carraway R.E. Ikebe M. Fay F.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1568-1573Crossref PubMed Scopus (68) Google Scholar, 22Roy P. Rajfur Z. Jones D. Marriott G. Loew L. Jacobson K. J. Cell Biol. 2001; 153: 1035-1048Crossref PubMed Scopus (60) Google Scholar, 23Ghosh M. Ichetovkin I. Song X. Condeelis J.S. Lawrence D.S. J. Am. Chem. Soc. 2002; 124: 2440-2441Crossref PubMed Scopus (44) Google Scholar, 24Ghosh M. Song X. Mouneimne G. Sidani M. Lawrence D.S. Condeelis J.S. Science. 2004; 304: 743-746Crossref PubMed Scopus (559) Google Scholar). In this study, we showed that photoactivation of a caged phosphotyrosine peptide (cpTyr397FAK), based on the sequence of FAK from residues Val391 to Thr406 surrounding the autophosphorylation site at Tyr397, alters cell migration by temporarily halting lamellar extension. The effect on migration is specific for the pTyr397FAK sequence because similar peptides containing phosphotyrosine but with altered surrounding residues fail to alter cell migration upon uncaging. The time scale of temporary inhibition of migration is consistent with the time to dephosphorylate the pTyr397FAK peptide in cell lysates. We speculate that the phenotype is related to perturbation of FAK-effector interactions including, but not limited to, Src and PI3K. Peptides containing tyrosine (Tyr), free phosphotyrosine (pTyr), and caged phosphotyrosine (cpTyr) were synthesized using standard Fmoc solid phase peptide synthesis protocols, purified by high performance liquid chromatography (HPLC), and confirmed as the desired products by mass spectrometry. The caged phosphotyrosine residue was incorporated into the peptide sequence using a new building block for solid phase synthesis (25Rothman D.M. Vazquez M.E. Vogel E.M. Imperiali B. J. Org. Chem. 2003; 68: 6795-6798Crossref PubMed Scopus (39) Google Scholar). All peptide synthesis reagents were purchased from Applied Biosystems or Novabiochem, and all other chemicals were purchased from Sigma or Molecular Probes. High performance liquid chromatography was performed using a Waters 600E HPLC fitted with a Waters 600 automated control module and a Waters 2487 dual wavelength absorbance detector recording at 228 and 280 nm. For analytical HPLC a Beckman Ultrasphere C18, 5 μm, 4.6 × 150-mm reverse phase column was used. For preparative separations a YMC-pack, C18, 250 × 20-mm reverse phase column was used. The standard gradient for analytical and preparatory HPLC used was 93:7–0:100 over 35 min (water:acetonitrile, 0.1% trifluoroacetic acid). Electrospray ionization mass spectrometry was performed on a PerSeptive Biosystems Mariner Biospectrometry Workstation (turbo ion source). Peptide Synthesis—Peptide synthesis was made using standard solid phase peptide synthesis protocols on a 0.05-mmol scale using a 0.21 mmol/g loading PAL-PEG-PS solid support. Amino acids were purchased from Novabiochem as protected Fmoc amino acids with the standard side chain protecting scheme: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH. Phosphotyrosine was introduced as the monobenzyl ester Fmoc-Tyr(PO(OBzl)OH)-OH. Amino acids were manually coupled in 4-fold excess using a mixture of O-benzotriazolyl-N-N-N′-N′-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIEA) in DMF as activating agents. Each amino acid was activated for 2 min with the HBTU/HOBt mixture and DIEA in DMF before being added to the resin. Amide coupling reactions were conducted for 1 h and monitored using the 2,4,6-trinitrobenzenesulphonic acid TNBS test (26Hancock W.S. Battersby J.E. Anal. Biochem. 1976; 71: 260-264Crossref PubMed Scopus (261) Google Scholar). Deprotection of the base-labile Fmoc protecting group was accomplished by treating the resin with 20% piperidine in DMF solution for 15 min. Acetylation was accomplished by treating the resin with acetic anhydride and DIEA in DMF. Peptides were cleaved from the resin, and side chain protecting groups were simultaneously removed by treatment with the following cleavage mixture: 50 μl of dichloromethane, 25 μl of triisopropyl silane, 25 μl of water, and 950 μl of trifluoroacetic acid (1 ml of mixture/50 mg of resin) for 2 h at room temperature. All peptides were precipitated with diethyl ether (4 °C) and further purified by HPLC. Operations involving the caged phosphotyrosine peptides were performed in the dark. Detailed Protocol for a Typical Coupling—Piperidine (3 ml, 20% in DMF) was added to 0.05 mmol FmocHN-AEIIDEEDT in solid support, and nitrogen was passed through the mixture for 15 min. The resin was then filtered and washed with DMF (3 × 3 ml × 3 min), and the TNBS test was run with a small resin sample to confirm that the deprotection was successful. In a separate vessel, Fmoc-Tyr(tBu)-OH (79 mg, 0.2 mmol) was dissolved in HOBt/HBTU solution (1 ml 0.2 m HBTU, 0.2 m HOBt in DMF), and DIEA (1.5 ml of 0.195 m solution in DMF) was added. The resulting mixture was activated for 2 min and then added to the resin. Nitrogen was passed through the resin suspension for 1 h, at which time the TNBS test of a small resin sample was negative. The resin was washed with DMF (3 × 3 ml × 3 min) and subjected to the subsequent deprotection/coupling cycles in a similar manner. Acetylation—A 500-μl aliquot of acetic anhydride and DIEA (3 ml, 0.195 m in DMF) were added to the resin-bound free amino-terminal peptide. Nitrogen was passed through the mixture for 15 min. The resin was filtered and washed with DMF (3 × 3 ml × 3 min) and CH2Cl2 (2 × 3 ml × 3 min) and dried under vacuum before cleavage. Cleavage—A 0.05-mmol sample of resin-bound peptide was dried overnight and placed in a 50-ml flask. To this, 5 ml of the cleavage mixture (250 μl of CH2Cl2, 125 μl of water, 125 μl of tri-isopropylsilane, trifluoroacetic acid to 5 ml) was added, and the resulting mixture was shaken for 2 h, the resin was filtered, and the trifluoroacetic acid filtrate was concentrated under an argon stream to a volume of 2 ml and added over ice-cold ethyl ether (40 ml). After 5 min, the peptide was centrifuged and washed again with 40 ml of cold ether. The solid residue was dried under argon, redissolved in acetonitrile/water 1:1 (5 ml), and purified by preparative reverse phase HPLC. The pooled fractions were lyophilized and redissolved in deionized water. In some cases, addition of a small amount of base (0.05% NaHCO3) was necessary to solubilize the peptides. The identity of the final peptide products was confirmed by electrospray ionization mass spectrometry. Coupling with Rhodamine—For rhodamine coupling, a 0.015-mmol aliquot of free amine-containing peptide on solid support was washed with DMF (3 × 3 ml × 3 min) and filtered. The resin was then suspended in DMF (500 μl) and DIEA (50 μl, 0.3 mmol, 20 eq), and to this mixture was added 5 (and 6)-carboxy)-X-rhodamine, succinimidyl ester (12 mg, 0.02 mmol). The resulting mixture was stirred in the dark overnight. The resin was filtered, washed with DMF (3 × 3 ml × 3 min), iPrOH (2 × 3 ml × 3 min), and dichloromethane (3 × 3 ml × 3 min), dried under vacuum, and stored at 4 °C. Peptide Characterization—Peptides were purified by reverse phase HPLC, and concentrations of stock solutions were determined by quantitative amino acid analysis. When bright chromophores were present (rhodamine), then the reported extinction coefficients for those chromophores were used for calculating the concentrations by spectrophotometry. cpTyr397FAK sequence: Ac-VSETDD-cpY-AEIIDEEDT-CONH2, C86H125N18O42P, Mass.: 2112.8, MS(ESI); 1057.9 ([MH2]2+ (100); 720.3, [MHNa2]3+ (8)), ϵ (m–1, cm–1) 5332 (267 nm). pTyr397FAK sequence: Ac-VSETDD-pY-AEIIDEEDT-CONH2, C78H118N17O40P, Mass.: 1963.7, MS(ESI); 1057.9, [MH2]2+ (100). ϵ (m–1, cm–1) 350 (264 nm). Rh-Ahx-cpTyr397FAK sequence: Rh-Ahx-VSETDD-cpY-AEIIDEEDT-CONH2, C123H163N21O46P+, Mass.: 2701.1, MS(ESI): 908.3 [MHNa]3+ (70), 915.6 [MNa]3+ (100), 923 [MNa3-H]3+; ϵ (m–1, cm–1); 80,000 (576 nm). Rh-Ahx-pTyr397FAK sequence: Rh-Ahx-VSETDD-cpY-AEIIDEEDT-CONH2, C115H156N20O44P+, Mass.: 2552.0, MS(ESI): 866.0 [MNa2]3+, (60), 873.6 [MNa3-H]3+, (100) 880.6 [MNa4-2H]3+ (50); ϵ (m–1, cm–1) 80,000 (576 nm). Rh-Ahx-scrambledFAK sequence: Rh-Ahx-DDVETS-Y-AEIIDEEDT-CONH, C115H155N20O41+, Mass.: 2472.0, MS(ESI): 1248.0 [Mna2]2+ (100), 1259.0 [MNa2-H]2+, (40) 1270.0 [MNa3-2H]2+, (25); ϵ (m–1m cm–1) 80,000 (576 nm). Ala395cpTyr397FAK sequence: Ac-VSETAD-cpY-AEIIDEEDT-CONH2, C85H125N18O40P, Mass.: 2068.8, characterized before final acetylation: C83H123N18O39P, Mass.: 2026.8 MS(ESI); 1014.8 [MH2]2+ (100), 1025.8 [MHNa]2+ (20); ϵ (m–1, cm–1) 5332 (267 nm). Ala395pTyr397FAK sequence: Ac-VSETAD-pY-AEIIDEEDT-CONH2, C77H118N17O38P, Mass.: 1919.7, MS(ESI): 982.8 [MNa2]2+ (70), 993.8 [MNa3-H]2+ (100), 1004.8 [MNa4-2H]2+ (60), 1015.7 [MNa5-3H]2+ (30). Ala395Ala396cpTyr397FAK sequence: Ac-VSETAA-cpY-AEIIDEEDT-CONH2, C76H118N17O36P, Mass.: 1875.7, characterized before final acetylation: C82H123N18O37P, Mass.: 1982.8 MS(ESI); 992.8 [MH2]2+ (100), 1003.3 [MHNa]2+ (25). Lifetime of pYFAK after Uncaging—A 25-ml sample of NBT-II cell culture was centrifuged; the pellet was then treated with 250 μl of 1% Triton X-100 in phosphate-buffered saline solution and gently mixed with a micropipette. The resulting mixture was transferred to a 1-ml tube and centrifuged at high speed (13,000 rpm) for 8 min. An 80-μl aliquot of the supernatant was added to 5.6 μl of Rh-Ahx-pYFAK stock solution (30.5 μm) to give a final peptide concentration of 2 μm. The resulting mixture was incubated at 25 °C, and 3-μl aliquots were taken consecutively at 5, 10, 15, 20, and 25 min and diluted in 100 μl of phosphate-buffered saline, pH 7.5. Each sample was kept frozen at –78 °C until injected into the HPLC for analysis. As a control, an 80-μl aliquot of lysate was treated for 5 min with 400 μm pervanadate to inactivate tyrosine phosphatases, and then 5.6 μl of stock Rh-Ahx-pYFAK solution was added to the lysate and the peptide was incubated at room temperature. For analysis, 3-μl aliquots were taken at 10 and 20 min and added to 100 μl of phosphate-buffered saline, pH 7.5. Each sample was kept frozen at –78 °C until injection onto the HPLC for analysis. 160 μl of NBT-II cell lysate (from 20 ml of culture) was incubated with Flu-pYFAK (final concentration 2 μm). GST·Src SH2 and GST·PI3K C-terminal SH2 Expression and Purification—Src and PI3K SH2 domains were expressed and purified as GST fusion proteins essentially following reported procedures (27Smith D.B. Johnson K. Gene. 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). In summary, plasmids encoding the GST fusion proteins were transformed into bacteria Escherichia coli DH5-competent cells; Invitrogen). Bacteria were grown to mid-log phase, induced at 37 °C for 3–4 h with isopropyl-1-thio-β-d-galactopyranoside, and lysed by treatment with lysozyme (1 mg/ml, 30 min) followed by sonication (phosphate-buffered saline buffer, pH 7.4, with 100 mm EDTA, 1% Triton X-100, 10% glycerol, 1 mm dithiothreitol, 0.1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 30 μg/ml leupeptin, and 0.5 μg/ml pepstatin A). The lysates were clarified by centrifugation, and fusion proteins were purified by binding to glutathione-agarose beads (Amersham Biosciences). Proteins were eluted from the beads (50 mm Tris-HCl, 10 mm reduced glutathione, pH 8) and concentrated by centrifugation through a cellulose membrane (10 kDa molecular mass cutoff; Millipore). Proteins were quantified by Micro BCA protein assay kit (Pierce) relative to a bovine serum albumin standard. All fusion proteins were analyzed by Coomassie Blue staining and Western blot analysis with anti-GST antibodies. Kd Values for Peptide Binding to SH2 Domains of Src and PI3K—All experiments were performed using a VP-ITC instrument from Microcal Inc. Peptides and proteins were dialyzed extensively against the same buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm β-mercaptoethanol) using a 500-Da molecular mass cutoff cellulose membrane (Spectrum Laboratories Inc.). Initial experiments using lower pH resulted in precipitation of the protein and/or peptide during the titration. In a typical titration, peptides (1–3 mm concentration) were added over 25 injections (5 μl) to purified GST·SH2 proteins (200–400 μm) that were present in the isothermal calorimeter cell at 25 °C. This temperature was chosen to minimize the contributions of ligand-induced refolding of the protein while maintaining a physiologically relevant temperature of operation. In every titration the concentration of the reactants was sufficient to result in saturation of the titration curve. Cell Culture—For the movement of NBT-II cells, suspension culture Petri dishes (35 mm) were coated by incubating with 10 μg/ml collagen (rat tail type I) for 30 min. NBT-II cells (American Type Culture Collection) were treated with trypsin and resuspended in Dulbecco's modified Eagle's medium/F12 medium containing 10% fetal bovine serum, plated at low density on the dishes, and cultured for 12 h at 37 °C. Competitive Binding Assay in Cell Lysates—As previously described, chicken embryo cells were isolated from day 9 embryos and maintained in Dulbecco's modified Eagle's medium + 4% fetal bovine serum + 1% chick serum (28Reynolds A.B. Roesel D.J. Kanner S.B. Parsons J.T. Mol. Cell Biol. 1989; 9: 629-638Crossref PubMed Scopus (288) Google Scholar). FAK was expressed in chicken embryo cells using the replication-competent, avian retroviral vector called RCAS, and cells were transfected as previously described (28Reynolds A.B. Roesel D.J. Kanner S.B. Parsons J.T. Mol. Cell Biol. 1989; 9: 629-638Crossref PubMed Scopus (288) Google Scholar, 29Schaller M.D. Borgman C.A. Parsons J.T. Mol. Cell Biol. 1993; 13: 785-791Crossref PubMed Scopus (279) Google Scholar). Cells were lysed in modified radioimmune precipitation assay buffer (30Thomas J.W. Cooley M.A. Broome J.M. Salgia R. Griffin J.D. Lombardo C.R. Schaller M.D. J. Biol. Chem. 1999; 274: 36684-36692Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) containing protease and phosphatase inhibitors as described (30Thomas J.W. Cooley M.A. Broome J.M. Salgia R. Griffin J.D. Lombardo C.R. Schaller M.D. J. Biol. Chem. 1999; 274: 36684-36692Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The protein concentration of lysates was determined by using the bicinchonic acid assay (Pierce). The expression of GST fusion proteins (Src or PI3K SH2 domains) was induced by using 0.1 mm isopropyl-1-thio-β-d-galactopyranoside. E. coli were harvested, lysed by sonication, and the fusion proteins purified by using glutathione-agarose beads (Sigma) as previously described (30Thomas J.W. Cooley M.A. Broome J.M. Salgia R. Griffin J.D. Lombardo C.R. Schaller M.D. J. Biol. Chem. 1999; 274: 36684-36692Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). GST pulldowns were performed from 0.2 mg of lysate from chicken embryo cells expressing FAK. Lysates were precleared with 10 μg of GST. The supernatant was then incubated with 10 μg of fusion protein immobilized on glutathione-agarose beads either with or without peptides at increasing concentrations for 1 h at 4 °C. The beads were washed twice with lysis buffer and twice with phosphate-buffered saline. Bound proteins were eluted by boiling in sample buffer (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar) and analyzed by Western blotting with polyclonal antiserum BC4 to recognize FAK. Western blots were incubated with horseradish peroxidaseconjugated secondary antibodies and processed for enhanced chemiluminescence (Amersham Biosciences). Microinjection—Microinjection of peptides into cells was performed with a semiautomatic Eppendorf InjectMan NI 2/Femtojet system. Original Eppendorf Femtotip needles were loaded with 1.5–2 μl of injection solution. The base pressure of the system was adjusted (by visual assessment of the fluorescent marker outflow from the microinjection needle in the 20–100 hectopascal range) to keep a constant low outflow of the loaded solute into cell medium. Similarly, because of the increasing viscosity of the loaded solution, the injection pressure was adjusted to achieve a similar brightness of co-loaded fluorescent marker in microinjected cells (50 to up to 1200 hectopascal in some cases). After setting up the vertical limit position of the microneedle, each cell was automatically injected with peptide solution by InjectMan with preset speed of the microinjection needle movement. The velocity of the microneedle movement varied from 45 to 130 μm/s. The procedure was repeated to obtain the desired number of microinjected cells. Cells microinjected with caged phosphopeptides were allowed to recover in the tissue culture incubator for 30 min. During microinjection and subsequent time lapse microscopy, cells were maintained at 37 °C and CO2 was flushed over the dish. To distinguish between microinjected and non-microinjected cells, a tracer (either rhodamine-labeled peptide or rhodamine-dextran) was co-loaded with the peptides at a final concentration range <10% of that of biological test peptide. We evaluated the consistency of microinjected volumes when different caged peptides were introduced. NBT-II cells, seeded on glass or plastic surface, were microinjected with either cpTyr397FAK or Ala395cpTyr397FAK peptides together with fluorescent marker (rhodamine dextran) as described above. Cells were left in the incubator for 1 h and then placed in an environmental chamber on the microscope stage. From phase contrast and epifluorescence images of cells loaded with rhodamine dextran, the area of cells of interest was manually outlined and the average value of fluorescence of the outlined area was determined using Metamorph imaging software routines. The mean value (arbitrary units) of single cell average fluorescence was 105 ± 27 for cpTyr397FAK (269 cells) and 97 ± 28 for Ala395cpTyr397FAK (143 cells). The results indicated that constant volumes of the solutions containing cpTyr397FAK or Ala395cpTyr397FAK peptides could be microinjected. Photoactivation—For uncaging of caged FAK phosphopeptides, UV light from an Argon ion laser (Spectra Physics), with multiline optics covering a 333.6–363.8-nm range, was focused onto a ∼8-μm-diameter spot on the specimen using a ×20 Nikon objective. For all experiments, the estimated power of the laser beam after passing through the objective was 67 microwatts (measured by a power meter) and the duration of illumination was 100 ms. Concentration Estimates of Microinjected and Uncaged Peptide— Estimates for the concentration of the microinjected peptides as well as uncaged peptide are given in Table I. Using the Eppendorf Injectman, we estimate that 20–200 fl of peptide is microinjected into each cell. If the average cell volume of NBT-II cells is 1.5 pl, an additional 1–10% (32Yeh R.H. Yan X. Cammer M. Bresnick A.R. Lawrence D.S. J. Biol. Chem. 2002; 277: 11527-11532Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) of the total cell volume is added by microinjection. To estimate the concentrations of peptide after uncaging, a quantum efficiency of uncaging for these peptides of 0.3 was used based on characterization of a paxillin peptide with the same caging moiety (33Rothman D.M. Vazquez M.E. Vogel E.M. Imperiali B. Org. Lett. 2002; 4: 2865-2868Crossref PubMed Scopus (35) Google Scholar).Table IEstimated concentration of microinjected and uncaged
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