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J. Biol. Chem., Vol. 280, Issue 23, 22091-22101, June 10, 2005

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In Situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from Focal Adhesion Kinase Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells^*

David Humphrey¶||, Zenon Rajfur¶, M. Eugenio Vazquez¶**, Danielle Scheswohl, Michael D. Schaller, Ken Jacobson, and Barbara Imperiali**¶¶

From the Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 and the Departments of ^**Chemistry and ^¶¶Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received for publication, March 11, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Focal adhesions are cell-surface specializations that connect the extracellular matrix to the actin cytoskeleton. Transmembrane integrins and associated cytoskeletal proteins, including talin, vinculin, -actinin, and filamin, perform this function within the focal adhesion (1, 2). In addition, focal adhesions contain numerous regulatory molecules, including focal adhesion kinase (FAK),¹ 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 (3). The migration defect could be rescued by expression of wild type FAK (4, 5). Overexpression of FAK in Chinese hamster ovary cells also resulted in increased migration (6). 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 (7, 8), phospholipase C-1 (9, 10), phosphatidylinositol 3-kinase (PI3K) (10, 11), Grb7 (12–14), Nck-2 (15), and Shc (16), thus mediating numerous downstream effects. 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 (17) and PI3K, respectively (11). Chen (11) 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 (18). 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 (19, 20). 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 (21–24).

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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
General Chemical Methods
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 (25). 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 x 150-mm reverse phase column was used. For preparative separations a YMC-pack, C₁₈, 250 x 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 (26). 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 x 3 ml x 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 x 3 ml x 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 x 3 ml x 3 min) and CH₂Cl₂ (2 x 3 ml x 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 x 3 ml x 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 x 3 ml x 3 min), iPrOH (2 x 3 ml x 3 min), and dichloromethane (3 x 3 ml x 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)), (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). (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⁺; (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); (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); (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); (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 (27). 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--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.

K_d 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 -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.

Biological Methods
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 (28). FAK was expressed in chicken embryo cells using the replication-competent, avian retroviral vector called RCAS, and cells were transfected as previously described (28, 29). Cells were lysed in modified radioimmune precipitation assay buffer (30) containing protease and phosphatase inhibitors as described (30). 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--D-galactopyranoside. E. coli were harvested, lysed by sonication, and the fusion proteins purified by using glutathione-agarose beads (Sigma) as previously described (30).

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 (31) 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 x20 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% (32) 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 (33).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Characterization of Peptide Properties
Peptides Employed—The series of peptides synthesized for this study is shown in Fig. 1, and the rationale for their design is given under "Discussion." The peptides are based on the sequence of FAK from residues Val³⁹¹ to Thr⁴⁰⁶ surrounding the autophosphorylation site at Tyr³⁹⁷ (VSETDDpY³⁹⁷AEIIDEEDT) (pTyr³⁹⁷FAK); a closely related peptide (VSETDDY³⁹⁷AEIIDEED) has been previously used in studies to inhibit Src SH3-SH2/phosphoprotein interactions in a competition enzyme-linked immunosorbent assay (34). For photoactivation, a caged phosphotyrosine (cpY, or cpTyr) was introduced into the sequence (VSETDDcpY³⁹⁷AEIIDEEDT) designated cpTyr³⁹⁷FAK. In addition, a similar caged phosphotyrosine peptide with an amino acid substitution at position 395 (alanine for aspartic acid) was synthesized (VSETADcpY³⁹⁷AEIIDEEDT) and designated Ala³⁹⁵cpTyr³⁹⁷FAK. A caged phosphotyrosine-containing peptide with 2 amino acid substitutions (D395/396A) (VSETAAcpY³⁹⁷AEIIDEEDT), designated Ala³⁹⁵Ala³⁹⁶cpTyr³⁹⁷FAK, and another non-caged peptide with amino acid sequence N-terminal of the phosphotyrosine shuffled (AcDDVTESpY³⁹⁷AEIIDEEDT), designated partially scrambled pYFAK, were also synthesized.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Peptides employed in this study. See "Results" for description.

Lifetime of pTyr³⁹⁷FAK in Cell Lysates after Uncaging— After microinjection of the pTyr³⁹⁷FAK peptide or uncaging of the cpTyr³⁹⁷FAK peptide, the most likely mode of degradation is via dephosphorylation by endogenous phosphatases. Because this action would deactivate the peptide reagent, it was important to estimate the kinetics of dephosphorylation of the intact phosphopeptide. Using HPLC analysis, the dephosphorylation of the FAK phosphopeptides in NBT-II cell lysates was followed at 25 °C (Fig. 3). Aliquots of NBT-II cell lysates incubated with Rh-pTyr³⁹⁷FAK peptide were taken every 5 min and analyzed by HPLC using rhodamine fluorescence for monitoring. We observed the formation of a new product with retention time that matches that of the Rh-Tyr³⁹⁷FAK sample (Fig. 3A). The resulting chromatograms show a steady decrease in the peak corresponding to the phosphopeptide and a simultaneous increase in the intensity of another fluorescent peak with the same retention time as the unphosphorylated peptide. From the HPLC traces, we conclude that the half-life of the phosphopeptide in the NBT-II cell lysates is 10–15 min, in agreement with reported literature values (34). As expected, incubation of the peptide with pervanadate-treated cell lysates does not show degradation of the peptide during the time of the experiment, suggesting that the change in retention time of the peptide was due to dephosphorylation by tyrosine phosphatases in the lysate (Fig. 3B). Over a similar time frame, the caging group protects the phosphate group from phosphatase-mediated degradation (see Fig. 3C).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Ability of phosphorylated FAK peptides to compete for binding to Src and PI3K SH2 domains. The Src and PI3K SH2 domains were expressed as GST fusion proteins and immobilized to glutathione-agarose beads. FAK-expressing chicken embryo cell lysate was precleared by incubation with GST alone immobilized to glutathione-agarose beads. The cleared lysates were then incubated with GST (lane 2), GST·Src SH2 (lanes 3–9), or GST·PI3K SH2 (lanes 10–16). Pulldowns were also performed in the presence of the wild type pTyr397FAK peptide or the Ala395pTyr397FAK peptide at concentrations of 2 µM (lanes 4, 7, 11, 14), 5 µM (lanes 5, 8, 12, 15), or 45 µM (lanes 6, 9, 13, 16). The beads were washed, and bound FAK was detected by Western blotting. 25 µg of lysate was run as a control (lane 1).

Distribution of lamellar arrest duration in different cells is given in Fig. 5A. In these experiments, 18 of 25 cells displayed transient lamellar arrest after photoactivation of cpTyr³⁹⁷FAK (2 cells showed no effect; 4 cells were difficult to assess; 1 cell slowed but did not stop) (Fig. 5A). Two laser shots were required to achieve the effect, including one over the perinuclear region. A single irradiation focus on the lamellipod was insufficient. This suggests that this migration "phenotype" is a global effect in the sense that sufficient free phosphopeptide must be released for an effect, and this is provided by the larger volume of uncaging when the beam is directed over the perinuclear zone as opposed to positioning the beam over the much thinner lamella area.

Uncaging Other Peptides—The Ala³⁹⁵cpTyr³⁹⁷FAK peptide (Fig. 1) contains an amino acid substitution in position 395 of the FAK sequence (D395A) that causes full-length FAK mutants to bind to Src but not to PI3K (35). NBT-II cells were microinjected following the procedure used for the caged pTyr³⁹⁷FAK peptides. When the Ala³⁹⁵cpTyr³⁹⁷FAK was uncaged, little lamellar arrest was seen (Fig. 5B). Of 20 cells, 14 showed no lamellipodial arrest, 4 temporarily ceased protrusion (2 cells for 15 min, 1 cell for 10 min, and 1 cell for 5 min), and 2 cells were difficult to classify. However, when Ala³⁹⁵pTyr³⁹⁷FAK was directly introduced by microinjection, producing a significantly higher concentration of phosphotyrosine peptide, transient lamellar arrest did occur (see below).

View larger version (15K): [in this window] [in a new window]   FIG. 3. A, HPLC traces of Rh-Ahx-pTyr397FAK (Ahx; 6-aminohexanoic acid linker) incubated with NBT-II cell lysate at 5, 10, 15, 20, and 25 min showing the increase of unphosphorylated peptide. B, superposition of the HPLC traces of the stock of Rh-Ahx-pTyr397FAK and the peptide after 20 min of incubation with pervanadate-treated cell lysate, showing that the peptide is stable. C, stability of caged phosphopeptide (Rh-Ahx-cpTyr397FAK) in cell lysates.

View larger version (113K): [in this window] [in a new window]   FIG. 4. Uncaging of cpTyr397FAK peptide produces transient lamellar arrest. The red line indicates the initial position of the leading edge, the green reference line indicates the initial position of the trailing edge, and the blue circles indicate the regions of irradiation (to scale). A, the top panel shows a single migrating cell loaded with cpTyr397FAK before laser irradiation. B, after photoactivation in spots shown in circles (see "Materials and Methods" for details), the release of the phosphopeptide temporarily halts lamellipodial protrusion for 25 min, after which migration resumes. The posterior cell body continues to move forward after peptide photorelease (see green reference line), and the lamellipodium broadens before migration continues. C, the bottom panel depicts a non-loaded, irradiation control showing normal migration after two 100-ms laser irradiations in the spots shown.

Microinjection of pY³⁹⁷FAK Phosphopeptides Also Produces Transient Lamellar Arrest—Corroborating the contention that uncaging within the lamella cannot release sufficient peptide for an effect is the experiment that directly microinjecting pTyr³⁹⁷FAK peptide produces a similar phenotype (Fig. 6). The pTyr³⁹⁷FAK-loaded NBT-II cell exhibits a temporary inhibition of protrusion before starting to extend small local protrusions between 20–30 min after microinjection and then resumes migration at 45 min. Intermediate concentrations (5–10-fold dilution; 200 µM in the needle) of pTyr³⁹⁷FAK peptide were required to produce the same effect as uncaging cpTyr³⁹⁷FAK. Microinjection of undiluted pTyr³⁹⁷FAK peptide (1 mM in the needle) stops migration, and the cells do not recover for the duration of the experiment (1–2 h). This experiment also indicates that the lamellar arrest phenotype is due to the phosphotyrosine peptide itself and not the side product produced after liberation of the caging group. In the pTyr³⁹⁷FAK experiments, described in Fig. 6, changes in the migration rate could be because of either microinjection or peptide. These two possibilities were distinguished by cell morphology. NBT-II cells that have been damaged by microinjection no longer exhibited the fan-like lamellar morphology of a normally migrating cell. Instead, they became rounded and eventually collapsed. In Fig. 6, the microinjected cell maintained its leading lamella and migration resumed after 45 min, indicating that the altered migration was because of the phosphotyrosine peptide and not the microinjection itself.

Caged and free Ala³⁹⁵pTyr³⁹⁷FAK (Fig. 1) were also microinjected into migrating NBT-II cells. Although photoactivation of the caged version of this peptide did not alter migration (Fig. 4B), direct microinjection of Ala³⁹⁵pTyr³⁹⁷FAK caused temporary lamellar arrest in 10 of 14 cells (3 cells changed direction, 1 cell showed no arrest). However, microinjection of the partially scrambled FAK phosphopeptide (Fig. 1) did not alter cell migration significantly (9 cells exhibited no noticeable changes, 4 cells changed direction but continued migrating). A summary of the peptide effects on cell migration together with their binding constants to Src and PI3K SH2 motifs and estimated intracellular concentrations is given in Table III.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (16K): [in this window] [in a new window]   FIG. 5. Distribution of lamellar arrest durations produced by uncaging cpTyr397FAK (A), Ala395cpTyr397FAK (B), and Ala395Ala396cpTyr397FAK (C).

View larger version (31K): [in this window] [in a new window]   FIG. 6. NBT-II cells after microinjection with pTyr397FAK peptide. The time points refer to time elapsed after microinjection. The red line indicates the initial position of the leading lamellar edge. The cell stops protruding after microinjection. Between 20–30 min, the cell begins to form two small extensions at different regions of the cell. By 45 min, the cell recovers, shows broad lamellipodial extension, and resumes migration.

Selected peptides were caged at the key phosphotyrosine residue using recently developed methodology (25). 1-(2-Nitrophenyl)ethyl (NPE) was chosen as the caging group because it satisfies key requirements of biologically useful caging groups and those of Fmoc-based solid phase peptide synthesis (19, 33). The NPE can be photochemically released with reasonable quantum efficiency at wavelengths around 350 nm (33, 43), and the photo-byproduct, nitrosoacetophenone, is less harmful to cells than the corresponding aldehyde released by photolysis of commonly implemented o-nitrobenzyl caging groups (25).

Properties of the Uncaged Peptides—The phosphorylated peptides have a half-life in cell lysates of 10–15 min, roughly consistent with lamellar arrest times. This half-life is only an estimate of stability in intact cells. Isothermal calorimetry studies determining the affinities for the phosphorylated peptides binding to the SH2 domains of Src and PI3K differ by about an order of magnitude, with binding to Src being tighter. These values approximate those reported previously, and any differences in the values probably arise from slight differences in peptide sequences and different methods used to determine affinities (44). Results from the lysate competitive binding assays (Fig. 2) were qualitatively consistent with the isothermal calorimetry data.

Mechanism of Action of the pYFAK Peptides—These phosphotyrosine peptides intervene in signal transduction-mediated events downstream of FAK by abruptly increasing the concentration of pTyr³⁹⁷FAK either upon photoactivation or microinjection to produce transient lamellar arrest. Indeed, this phenotype is remarkably similar to that seen when keratocytes are treated with function-blocking anti-1 integrin antibodies (45), suggesting that the uncaged peptide intervenes in integrin-mediated signaling pathways. For photoactivation, it appears that in order to liberate sufficient phosphopeptide for an effect, irradiation of the thicker part of the cell was required. Free phosphopeptide then presumably diffuses rapidly throughout the cell and binds to the cognate targets. This view was corroborated by the observation that direct microinjection of intermediate concentrations of phosphopeptides produced a similar transient lamellar arrest phenotype (Fig. 6). Thus, in this case, although temporally defined effects were produced, localized effects could not be created by photoactivation. A similar effect was observed by Walker et al. (21) who demonstrated that local photorelease of caged peptide inhibitors of calmodulin or myosin light chain kinase did not produce arrest of eosinophil migration, but global release over the entire cell (similar to microinjection) did. On the other hand, local photorelease of caged actin-binding proteins did produce dramatic local perturbations in migration (22, 24) presumably because a sufficient number of released proteins quickly found their abundant G- or F-actin targets via diffusional encounters.

Two of the most widely studied FAK-binding proteins are Src and PI3K, and previous work has indicated the importance of these two proteins for migration in many cell types (46–49). Our binding and competitive inhibition studies indicate that uncaged peptide could bind to both of these FAK effectors. The literature cited above indicates that similar peptides can activate both Src and PI3K. Thus, temporally and spatially inappropriate activation produces lamellar arrest until the phosphopeptide loses activity via dephosphorylation, at which time normal migration resumes. This does not appear to be the whole story, however. Comparing the actual uncaging results from cells with the lysate competition results suggests that other FAK effectors such as Shc, PLC-, Grb7, or Nck2 may also play a role. Although uncaging of cpTyr³⁹⁷FAK produced the lamellar arrest phenotype, uncaging of Ala³⁹⁵cpTyr³⁹⁷FAK did not; however, the direct microinjection of Ala³⁹⁵pTyr³⁹⁷FAK did produce arrest. This suggests that at the higher microinjected concentrations, Ala³⁹⁵pTyr³⁹⁷FAK most likely titrates other important FAK-effector interactions that are available to uncaged cpTyr³⁹⁷FAK. Further downstream effects that produce this distinctive phenotype remain to be elucidated. These could include 1) an increase in lamellum adhesion or 2) the abrogation of lamellar protrusion by indirectly inhibiting Rac and/or Rho (50–54).

Advantages of Photoactivation of Phosphopeptides in Cell Biology—Chemical probes, including peptides and proteins that can be activated at will, are powerful tools to study signal transduction in dynamic processes like cell migration. Caged phosphotyrosine peptides are examples of such probes, as they can be introduced into cells in an inactive form and subsequently activated by UV light, producing a temporally and, in some cases, spatially controlled biological response. Utilization of caged phosphopeptides provides several technical advantages in in situ studies of signal transduction. The caged group attached directly to the phosphotyrosine increases the stability of the peptide by protecting it from endogenous protein tyrosine phosphatases. Additionally, the phosphopeptide remains in an "inactive" state until the photoactivation step, allowing precise timing of the perturbation. Because the caged phosphopeptides are inactive, each cell can serve as its own control for microinjection.

Nevertheless, challenges remain. It is clear from these studies that the use of short caged phosphopeptides as cellular probes may introduce ambiguity into the studies, because the limited binding determinants of the peptides may not faithfully reflect the behavior of the intact proteins upon which they are patterned. Thus, recent emphasis has been placed on the development of a general methodology for the incorporation of caged phosphoamino acids into native proteins via the suppressor tRNA methodology (55); in the future, this should expand the scope of the caged phosphoprotein-based strategies for probing signal transduction events in cell migration. An additional interpretational limitation is the determination of the absolute amount of peptide microinjected. This measurement is also important for quantitative modeling studies. However, the absolute amount of peptide loaded is difficult to measure directly because there are no convenient assays apart from the actual bioassay consisting of a changed migratory phenotype. In the future, we will concentrate on characterizing peptides conjugated to a fluorophore or caged fluorophore. The fluorescent signal from such peptides could be used directly in single cell concentration assays or in single cell fluorescence correlation spectroscopy measurements to determine concentration. Such efforts are extremely important, but they will involve characterization of essentially new peptides with different solubility, binding, and photochemical properties. Our studies show that defined intervention by the photoactivation methodology in signal transduction networks is an attractive method to test various hypotheses. However, to make the mechanistic connection between a given phenotype and the signaling network involved, more knowledge of relative functional significance of pathways downstream of the uncaging intervention will be required.

	FOOTNOTES

 
^* This work was supported in part by the Cell Migration Consortium (GM64346). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ These authors contributed equally to this work.

^|| Recipient of a fellowship from the Lineberger Comprehensive Cancer Center.

Recipient of a postdoctoral fellowship from the International Human Frontier Science Program Organization.

To whom correspondence should be addressed. Tel.: 919-966-5703; Fax: 919-966-1856; E-mail: frap{at}med.unc.edu.

¹ The 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Sastry, S. K., and Burridge, K. (2000) Exp. Cell Res. 261, 25–36[CrossRef][Medline] [Order article via Infotrieve]
2. Zamir, E., and Geiger, B. (2001) J. Cell Sci. 114, 3583–3590[Medline] [Order article via Infotrieve]
3. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., Yamamoto, T., and Aizawa, S. (1995) Nature 377, 539–544[CrossRef][Medline] [Order article via Infotrieve]
4. Owen, J. D., Ruest, P. J., Fry, D. W., and Hanks, S. K. (1999) Mol. Cell. Biol. 19, 4806–4818[Abstract/Free Full Text]
5. Sieg, D. J., Hauck, C. R., and Schlaepfer, D. D. (1999) J. Cell Sci. 112, 2677–2691[Abstract]
6. Cary, L. A., Chang, J. F., and Guan, J. L. (1996) J. Cell Sci. 109, 1787–1794[Abstract]
7. Schaller, M. D., Hildebrand, J. D., Shannon, J. D., Fox, J. W., Vines, R. R., and Parsons, J. T. (1994) Mol. Cell. Biol. 14, 1680–1688[Abstract/Free Full Text]
8. Xing, Z., Chen, H. C., Nowlen, J. K., Taylor, S. J., Shalloway, D., and Guan, J. L. (1994) Mol. Biol. Cell 5, 413–421[Abstract]
9. Wells, A., Ware, M. F., Allen, F. D., and Lauffenburger, D. A. (1999) Cell Motil. Cytoskeleton 44, 227–233[CrossRef][Medline] [Order article via Infotrieve]
10. Zhang, X., Chattopadhyay, A., Ji, Q. S., Owen, J. D., Ruest, P. J., Carpenter, G., and Hanks, S. K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9021–9026[Abstract/Free Full Text]
11. Chen, H. C., Appeddu, P. A., Isoda, H., and Guan, J. L. (1996) J. Biol. Chem. 271, 26329–26334[Abstract/Free Full Text]
12. Han, D. C., and Guan, J. L. (1999) J. Biol. Chem. 274, 24425–24430[Abstract/Free Full Text]
13. Han, D. C., Shen, T. L., and Guan, J. L. (2000) J. Biol. Chem. 275, 28911–28917[Abstract/Free Full Text]
14. Shen, T. L., Han, D. C., and Guan, J. L. (2002) J. Biol. Chem. 277, 29069–29077[Abstract/Free Full Text]
15. Goicoechea, S. M., Tu, Y., Hua, Y., Chen, K., Shen, T. L., Guan, J. L., and Wu, C. (2002) Int. J. Biochem. Cell Biol. 34, 791–805[CrossRef][Medline] [Order article via Infotrieve]
16. Schlaepfer, D. D., Jones, K. C., and Hunter, T. (1998) Mol. Cell. Biol. 18, 2571–2585[Abstract/Free Full Text]
17. Thomas, J. W., Ellis, B., Boerner, R. J., Knight, W. B., White, G. C., and Schaller, M. D. (1998) J. Biol. Chem. 273, 577–583[Abstract/Free Full Text]
18. Humphrey, D., Rajfur, Z., Imperiali, B., Marriott, G., Roy, P., and Jacobson, K. (2005) in Live Cell Imaging: A Laboratory Manual (Spector, D. L., and Goldman, R. D., eds) pp. 159–176, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
19. Kaplan, J. H., Forbush, B., III, and Hoffman, J. F. (1978) Biochemistry 17, 1929–1935[CrossRef][Medline] [Order article via Infotrieve]
20. Park, C.-H., and Givens, R. S. (1997) J. Am. Chem. Soc. 119, 2453–2463
21. Walker, J. W., Gilbert, S. H., Drummond, R. M., Yamada, M., Sreekumar, R., Carraway, R. E., Ikebe, M., and Fay, F. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1568–1573[Abstract/Free Full Text]
22. Roy, P., Rajfur, Z., Jones, D., Marriott, G., Loew, L., and Jacobson, K. (2001) J. Cell Biol. 153, 1035–1048[Abstract/Free Full Text]
23. Ghosh, M., Ichetovkin, I., Song, X., Condeelis, J. S., and Lawrence, D. S. (2002) J. Am. Chem. Soc. 124, 2440–2441[CrossRef][Medline] [Order article via Infotrieve]
24. Ghosh, M., Song, X., Mouneimne, G., Sidani, M., Lawrence, D. S., and Condeelis, J. S. (2004) Science 304, 743–746[Abstract/Free Full Text]
25. Rothman, D. M., Vazquez, M. E., Vogel, E. M., and Imperiali, B. (2003) J. Org. Chem. 68, 6795–6798[CrossRef][Medline] [Order article via Infotrieve]
26. Hancock, W. S., and Battersby, J. E. (1976) Anal. Biochem. 71, 260–264[CrossRef][Medline] [Order article via Infotrieve]
27. Smith, D. B., and Johnson, K. (1988) Gene 67, 31–40[CrossRef][Medline] [Order article via Infotrieve]
28. Reynolds, A. B., Roesel, D. J., Kanner, S. B., and Parsons, J. T. (1989) Mol. Cell Biol. 9, 629–638[Abstract/Free Full Text]
29. Schaller, M. D., Borgman, C. A., and Parsons, J. T. (1993) Mol. Cell Biol. 13, 785–791[Abstract/Free Full Text]
30. Thomas, J. W., Cooley, M. A., Broome, J. M., Salgia, R., Griffin, J. D., Lombardo, C. R., and Schaller, M. D. (1999) J. Biol. Chem. 274, 36684–36692[Abstract/Free Full Text]
31. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
32. Yeh, R. H., Yan, X., Cammer, M., Bresnick, A. R., and Lawrence, D. S. (2002) J. Biol. Chem. 277, 11527–11532[Abstract/Free Full Text]
33. Rothman, D. M., Vazquez, M. E., Vogel, E. M., and Imperiali, B. (2002) Org. Lett. 4, 2865–2868[CrossRef][Medline] [Order article via Infotrieve]
34. Gilmer, T., Rodriquez, M., Jordan, S., Crosby, R., Alligood, K., Green, M., Kimery, M., Wagner, C., Kinder, D., Charifson, P., Hassell, A. M., Willard, D., Luther, M., Rusnak, D., Sternbach, D. D., Mehrotra, M., Peel, M., Shampine, L., Davis, R., Robbins, J., Patel, I. R., Kassel, D., Burkhart, W., Moyer, M., Bradshaw, T., and Berman, J. (1994) J. Biol. Chem. 269, 31711–31719[Abstract/Free Full Text]
35. Reiske, H. R., Kao, S. C., Cary, L. A., Guan, J. L., Lai, J. F., and Chen, H. C. (1999) J. Biol. Chem. 274, 12361–12366[Abstract/Free Full Text]
36. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hidesaburo, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767–778[CrossRef][Medline] [Order article via Infotrieve]
37. Waksman, G., Shoelson, S. E., Pant, N., Cowburn, D., and Kuriyan, J. (1993) Cell 72, 779–790[CrossRef][Medline] [Order article via Infotrieve]
38. Songyang, Z., Margolis, B., Chaudhuri, M., Shoelson, S. E., and Cantley, L. C. (1995) J. Biol. Chem. 270, 14863–14866[Abstract/Free Full Text]
39. Liu, X., Brodeur, S. R., Gish, G., Songyang, Z., Cantley, L. C., Laudano, A. P., and Pawson, T. (1993) Oncogene 8, 1119–1126[Medline] [Order article via Infotrieve]
40. Alonso, G., Koegl, M., Mazurenko, N., and Courtneidge, S. A. (1995) J. Biol. Chem. 270, 9840–9848[Abstract/Free Full Text]
41. Boerner, R. J., Kassel, D. B., Barker, S. C., Ellis, B., DeLacy, P., and Knight, W. B. (1996) Biochemistry 35, 9519–9525[CrossRef][Medline] [Order article via Infotrieve]
42. Moarefi, I., LaFevre-Bernt, M., Sicheri, F., Huse, M., Lee, C. H., Kuriyan, J., and Miller, W. T. (1997) Nature 385, 650–653[CrossRef][Medline] [Order article via Infotrieve]
43. Corrie, J. E. T., and Trentham, D. R. (1993) Biological Applications of Photochemical Switches (Morrison, H., ed) pp. 243–305, John Wiley and Sons, New York
44. Ladbury, J. E., Lemmon, M. A., Zhou, M., Green, J., Botfield, M. C., and Schlessinger, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3199–3203[Abstract/Free Full Text]
45. de Beus, E., and Jacobson, K. (1998) Cell Motil. Cytoskeleton 41, 126–137[CrossRef][Medline] [Order article via Infotrieve]
46. Klinghoffer, R. A., Sachsenmaier, C., Cooper, J. A., and Soriano, P. (1999) EMBO J. 18, 2459–2471