Mapping the Integrin-Linked Kinase Interactome Using SILAC Iveta Dobreva,† Andrew Fielding,† Leonard J. Foster,‡,# and Shoukat Dedhar*,†,‡,# Department of Cancer Genetics, British Columbia Cancer Research Centre, 675 West 10th Avenue, V5Z 1L3, Vancouver, BC, Canada, UBC Centre for High-throughput Biology and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada. The last two authors share senior authorship Received December 14, 2007
Protein–protein interactions play an essential role in the regulation of vital biological functions. Through a network of interactions, integrin-linked kinase (ILK) functions downstream of integrin receptors to control cell spreading, migration, growth, survival, and cell cycle progression. Despite many reports on the role of ILK in the regulation of multiple signaling pathways, it is still not understood how ILK integrates and controls complex cellular signals. A more global analysis of ILK-protein complexes will give important insights in the complexity of ILK-dependent signal transduction. Here, we applied a SILAC (stable isotope labeling with amino acids in cell culture)-based proteomics approach to discover novel ILK-interacting proteins. Of 752 proteins identified in ILK immunoprecipitates, 24 proteins had SILAC ratios higher than PINCH, previously identified as direct ILK-binding partner. Some of the newly identified proteins specifically enriched in ILK immunoprecipitates, with potentially interesting roles in ILK biology, include rapamycin-insensitive companion of mTOR (Rictor), R- and β-tubulin, RuvB-like 1 and 2, HS1-associating protein 1 (HAX-1), T-complex protein 1 subunits, and Ras-GTP-ase activatinglike protein 1 (IQ-GAP1). Functional interactions between ILK and several of the new binding partners were confirmed by coimmunoprecipitation/Western blot and colocalization experiments. Detailed analysis showed that when ILK is found in a complex with R-tubulin and RuvB-like 1, R-parvin and PINCH are not present, suggesting that ILK has the ability to form distinct protein complexes throughout the cell. Inhibition of ILK activity with an ILK-kinase inhibitor QLT0267 or downregulation of its expression impaired the ability of RuvB-like 1 to bind to tubulin pointing toward a possible role of ILK in the regulation of RuvB-like 1/tubulin interaction. Using the power of quantitative proteomics to resolve specific from nonspecific protein interactions, we identified several novel ILK-binding proteins, which sheds light on the molecular mechanisms of regulation of ILK-dependent signal transduction. Keywords: signal transduction • protein-protein interactions • mass spectrometry • quatitative proteomics • integrin-linked kinase • integrin signaling • cytoskeleton
Introduction Integrin-linked kinase (ILK) is a major regulator of extracellular matrix- and growth factor-induced signaling downstream of integrin receptors. Activation of ILK results in increased cell migration and invasion, processes that are associated with actin filament rearrangements.1 ILK activity is also crucial for the activation of one of the major survival pathways in cells, the PKB/Akt signaling cascade.2,3 In addition, ILK is required for the activation of Rac and Cdc42, resulting in actin cytoskeleton rearrangement and cell spreading.4 ILK can also promote tumor angiogenesis and Wnt3a-induced stabilization and nuclear accumulation of β-catenin, events playing a crucial role in tumorgenesis.5,6 * To whom correspondence should be addressed. Tel.: 1-604-675-8029. Fax: 1-604-675-8184. E-mail:
[email protected]. † British Columbia Cancer Research Centre. ‡ University of British Columbia. # The last two authors share senior authorship.
1740 Journal of Proteome Research 2008, 7, 1740–1749 Published on Web 03/10/2008
A wealth of experimental data supports the observation that many of the signaling pathways regulated by ILK are controlled by the assembly of the IPP (ILK-PINCH-parvin) complex.7 This heterotrimeric complex in which ILK binds directly to R-parvin (actopaxin) through its kinase domain8 and PINCH (particularly interesting Cys-His-rich protein, LIMS 1) through its N-terminal ankyrin repeat domain9 serves as an adaptor between integrins and the actin cytoskeleton and regulates several signaling pathways.7 Although our understanding of the role of ILK in the regulation of multiple signaling pathways has greatly advanced in recent years, the molecular mechanisms by which ILK affects various cellular processes are still poorly characterized. Recent data indicate that in normal cells ILK is not essential for PKB/ Akt activation, while tumor cells are dependent on ILK to fully activate this pathway,10 suggesting divergent regulation of ILK-dependent signal transduction in different cell types and different cellular contexts. Therefore, a more global analysis 10.1021/pr700852r CCC: $40.75
2008 American Chemical Society
Mapping the ILK Interactome Using SILAC of ILK-protein interactions may shed light on the molecular mechanisms by which ILK can regulate complex cellular signals. As we have previously demonstrated, SILAC (stable isotope labeling with amino acids in cell culture) is a powerful technique for the identification of functional and specific protein–protein interactions.11 The advantages of this method are not only that it allows detection of hundreds of proteins in complex samples such as protein immunoprecipitates, but it also distinguishes between background binding (nonspecific) and functionally important protein–protein interactions.12 We employ SILAC here to show that, in addition to known ILK-binding partners, ILK binds rapamycin-insensitive companion of mTOR (Rictor), R- and β-tubulin, RuvB-like 1 (TIP 49a, pontin 52) and RuvB-like 2 (TIP 49b, reptin 52), HS1associated protein 1 (HAX-1), and Ras-GTP-ase activating-like protein 1 (IQ-GAP1). Interestingly, we found that ILK can form different protein complexes, one with R-parvin and PINCH and another with R-tubulin and RuvB-like 1, which could be related to distinct biological functions of ILK than the ones described so far.
Experimental Procedures Materials. 13C6-arginine and 2H4-lysine were purchased from Cambridge Isotope Laboratories, Andover, MA. Arginine and lysine-free Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Caisson Laboratories, North Logan, UT. Dialyzed fetal bovine serum (FBS) and Geneticin were from Invitrogen. EZ-view Red Protein A beads and EZ-view Red ANTI-FLAG M2 affinity gel were purchased from Sigma (cat N P6486 and cat N F 2426). Sequencing grade modified porcine trypsin was from Promega. The following primary antibodies were used: rabbit anti-ILK and rabbit monoclonal anti-ILK (Cell Signaling Technology, cat N3862 and 3856), mouse anti-PINCH (Sigma, cat N P8996), rabbit anti-parvin alpha (Sigma, cat N A1226), rabbit anti-tubulin alpha/beta (Cell Signaling Technology, cat N 2148), rabbit anti-RuvB-like 1 (ProteinTech Group, Inc., Chicago, IL, cat N 10210-2-AP), rabbit anti-Rictor (Bethyl Laboratories, Inc., Montgomery, TX, cat N A300-458A), mouse anti-IQ-GAP1 (BD Transduction Laboratories, cat N 610612), rabbit anti-chTOG (Abcam, Cambridge, MA, cat N AB18320), and mouse anti-HAX-1 (BD Transduction Laboratories, cat N 610824). Stable Isotope Labeling and Cell Culture. HEK293 cells were transfected with FLAG-tag or FLAG-ILK (wild-type) plasmids, and positive clones were selected in the presence of 200 µg/ mL Geneticin. The cells were cultured in and split at a 1:4 dilution into one of two SILAC media formulations: (1) cells expressing FLAG-tag were cultured in the presence of normal isotopic abundance arginine (42 mg/L) and lysine (73 mg/L); (2) cells expressing FLAG-ILK were cultured in 13C6-arginine (43.5 mg/L) and 2H4-lysine (75 mg/L). All SILAC media was based on arginine and lysine-free DMEM supplemented with 10% dialyzed FBS, 1% L-glutamine, 1% penicillin/streptomycin (ThermoFisher Scientific), and 200 µg/mL Geneticin. Cells were split three more times in the above media at a 1:4 dilution each time prior to use. In our experience, these labeling conditions lead to 100% incorporation in most cell types, but in these 293 cells, we found inconsistent levels of heavy isotope incorporation from experiment to experiment. We speculate that this could be due to some ability of these cells to scavenge amino acids from the serum proteins.
research articles Isolation of Cytoskeleton. Cytoskeleton was extracted as described13 with modifications. Briefly, cells were rinsed with 10 mL of cytoskeleton-stabilizing buffer (CSB, 10 mM PIPES, pH 6.2, 50 mM KCl, 10 mM EGTA, 3 mM MgCl2, and 2 M glycerol). The Triton-soluble protein fraction was extracted with 6 mL of CSB, containing 1% Triton, 1 mM PMSF, 50 µg/mL leupeptin, 50 µg/mL aprotinin, 2 mM NaF, and 1 mM Na3VO4 for exactly 2 min at 37 °C. The remaining cytoskeleton was collected in 1 mL of extraction buffer (EB), containing 20 mM Tris-HCl, pH 7.4, 80 mM KCl, 30 mM MgCl2, 1 mM EGTA, 0.25 M NaCl, 1 mM DTT, 50 µg/mL leupeptin, 1 mM PMSF, and 1% Triton. The cytoskeleton fraction was passed through 25 G syringe to reduce viscosity and sonicated. High salt content was removed by overnight dialysis in 5 L of TBS, pH 8.0 (0.05 M Tris, 0.138 M NaCl, and 0.0027 M KCl; Sigma), supplemented with 1 mM EDTA. Protein precipitate was removed by centrifugation at 9000 rcf for 10 min at 4 °C. Protein concentration was measured by the BCA method (Pierce). Immunoprecipitation for MS Analysis. Five milligrams of cytoskeletal protein from FLAG-expressing 293 cells (nonlabeled) and 5 mg from ILK-expressing 293 cells (labeled) were combined, and the lysates were precleared with 50 µL of EZview Red Protein A beads for 1 h at 4 °C to reduce nonspecific binding. The remaining supernatant was immunoprecipitated overnight at 4 °C with 50 µL of EZ-view Red ANTI-FLAG M2 affinity gel. The beads were collected by centrifugation at 5000 rcf for 1 min at 4 °C and washed three times with 1 mL of washing buffer, containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% Triton X-100, 2 mM NaF, 1 mM Na3VO4, and 1 tablet protease inhibitor per 50 mL (Roche). The beads were then boiled in sample buffer for 5 min, and proteins were resolved on 5–15% SDS-PAGE gels. Sypro Ruby Protein Gel Stain (Molecular Probes) was used to determine presence of differentially expressed bands in FLAG-ILK immunoprecipitates. GeLC/MS and Data Analysis. ILK immune complexes were analyzed by gel-enhanced liquid chromatography/tandem mass spectrometry (GeLC-MS/MS): the entire immune complex was resolved on 5–15% gradient SDS-PAGE gels and stained with blue-silver14 prior to in-gel digestion.15 Extracted peptides were concentrated/desalted/filtered on Stop And Go Extraction tips16 and analyzed by LC-MS/MS on an lineartrapping quadrupole-Orbitrap hybrid mass spectrometer (ThermoFisher Scientific, Bremen, Germany) as described.17 Centroided fragment spectra were extracted with Extract_MSN v3.2 using the default parameters (ThermoFisher), monoisotopic peak assignments were corrected with DTASuperCharge (http:// msquant.sourceforge.net), and the resulting peaklist was searched against the human IPI database (v3.29, 68,161 sequences) using Mascot (v2.1,www.matrixscience.com). The following search parameters were used: trypsin cleavage specificity with up to one missed cleavage, 3 ppm accuracy on the precursor ion, 0.6 Da accuracy on fragment ions, ESI-TRAP fragmentation characteristics, cysteine carbamidomethylation as a fixed modification, 13C6-Arg and 2H4-Lys as variable modifications. MSQuant (http://msquant.sourceforge.net) was used to parse Mascot result files, to recalibrate mass measurements, and to extract quantitative ratios. The final, nonredundant list of proteins was generated using finaList.pl, an in-house script available on our Web site (http://www.proteomics.ubc. ca/foster/software/) that finds the smallest number of protein sequences that can explain all the peptide data. Initially, all peptide hits with scores greater than 25 were accepted, regardJournal of Proteome Research • Vol. 7, No. 4, 2008 1741
research articles less of length. Then the following cutoff criteria for identity were established to give a protein false-discovery rate (FDR) of less than 0.5% based on a search of a reversed human IPI database:18 two or more peptides per protein with a measured mass accuracy
