Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelityapproach to identify constituents of protein complexes

Proteome Science

Julie L Kadrmas¹  Stephen M Pronovost³  Masaaki Yoshigi² 

[1] Department of Oncological Sciences, University of Utah, Salt Lake, UT, 84112,USA;Department of Pediatrics, University of Utah, Salt Lake, UT, 84112, USA;Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 84112,USA

关键词: MudPIT;    DIGE;    2D-GE;    Affinity purification;    Interactome;    PINCH;   

Others  :  816960
DOI  :  10.1186/1477-5956-11-21

 received in 2013-02-26, accepted in 2013-05-08,  发布年份 2013

【 摘 要 】

Background

Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophilamelanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well.

Results

To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry.

Conclusions

The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments.

【 授权许可】

   
2013 Yoshigi et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Rearden A: A new LIM protein containing an autoepitope homologous to “senescent cell antigen”. Biochem Biophys Res Commun 1994, 201:1124-1131.
• [2]Kadrmas JL, Beckerle MC: The LIM domain: from the cytoskeleton to the nucleus. Nat Rev Mol Cell Biol 2004, 5:920-931.
• [3]Braun A, Bordoy R, Stanchi F, Moser M, Kostka GG, Ehler E, Brandau O, Fassler R: PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions. Exp Cell Res 2003, 284:239-250.
• [4]Tu Y, Li F, Goicoechea S, Wu C: The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. Mol Cell Biol 1999, 19:2425-2434.
• [5]Clark KA, McGrail M, Beckerle MC: Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes. Development 2003, 130:2611-2621.
• [6]Kadrmas JL, Smith MA, Clark KA, Pronovost SM, Muster N, Yates JR 3rd, Beckerle MC: The integrin effector PINCH regulates JNK activity and epithelial migration in concert with Ras suppressor 1. J Cell Biol 2004, 167:1019-1024.
• [7]Dougherty GW, Chopp T, Qi SM, Cutler ML: The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions. Exp Cell Res 2005, 306:168-179.
• [8]Elias MC, Pronovost SM, Cahill KJ, Beckerle MC, Kadrmas JL: A critical role for Ras Suppressor-1 (RSU-1) revealed when PINCH-Integrin-linked Kinase (ILK) binding is disrupted. J Cell Sci 2012, 125:3185-3194.
• [9]Washburn MP, Wolters D, Yates JR 3rd: Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001, 19:242-247.
• [10]Banks CA, Kong SE, Washburn MP: Affinity purification of protein complexes for analysis by multidimensional protein identification technology. Protein Expr Purif 2012, 86:105-119.
• [11]Ito S, Takahara Y, Hyodo T, Hasegawa H, Asano E, Hamaguchi M, Senga T: The roles of two distinct regions of PINCH-1 in the regulation of cell attachment and spreading. Mol Biol Cell 2010, 21:4120-4129.
• [12]Montanez E, Karakose E, Tischner D, Villunger A, Fassler R: PINCH-1 promotes Bcl-2-dependent survival signalling and inhibits JNK-mediated apoptosis in the primitive endoderm. J Cell Sci 2012, 125(Pt 21):5233-5240.
• [13]Schieltz DM, Washburn MP, Hays LG: Analysis of complex protein mixtures using nano-LC coupled to MS/MS. CSH Protoc 2006. http://cshprotocols.cshlp.org/content/2006/5/pdb.prot4553 webcite
• [14]Li F, Zhang Y, Wu C: Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats. J Cell Sci 1999, 112(Pt 24):4589-99.
• [15]Tu Y, Huang Y, Zhang Y, Hua Y, Wu C: A new focal adhesion protein that interacts with integrin-linked kinase and regulates cell adhesion and spreading. J Cell Biol 2001, 153:585-98.
• [16]Zhang Y, Chen K, Tu Y, Velyvis A, Yang Y, Qin J, Wu C: Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites. J Cell Sci 2002, 115:4777-86.
• [17]Nikolopoulos SN, Turner CE: Molecular dissection of actopaxin-integrin-linked kinase-paxillin interactions and their role in subcellular localization. J Biol Chem 2002, 277:1568-75.
• [18]Fukuda T, Chen K, Shi X, Wu C: PINCH-1 is an obligate partner of integrin-linked kinase (ILK) functioning in cell shape modulation, motility, and survival. J Biol Chem 2003, 278:51324-33.
• [19]Vakaloglou KM, Chountala M, Zervas CG: Functional analysis of parvin and different modes of IPP-complex assembly at integrin sites during Drosophila development. J Cell Sci 2012, 125:3221-32.
• [20]Stiegler AL, Draheim KM, Li X, Chayen NE, Calderwood DA, Boggon TJ: Structural basis for paxillin binding and focal adhesion targeting of beta-parvin. J Biol Chem 2012, 287:32566-77.
• [21]Timms JF, Cramer R: Difference gel electrophoresis. Proteomics 2008, 8:4886-97.
• [22]Zhang Y, Chen K, Tu Y, Wu C: Distinct roles of two structurally closely related focal adhesion proteins, alpha-parvins and beta-parvins, in regulation of cell morphology and survival. J Biol Chem 2004, 279:41695-705.
• [23]Fu H (Ed): Protein-protein interactions: methods and applications. New Jersey: Humana Press; 2004.
• [24]Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S: Comparison of affinity tags for protein purification. Protein Expr Purif 2005, 41:98-105.
• [25]Kool J, Jonker N, Irth H, Niessen WM: Studying protein-protein affinity and immobilized ligand-protein affinity interactions using MS-based methods. Anal Bioanal Chem 2011, 401:1109-25.
• [26]Dunham WH, Mullin M, Gingras AC: Affinity-purification coupled to mass spectrometry: basic principles and strategies. Proteomics 2012, 12:1576-90.
• [27]Oeffinger M: Two steps forward–one step back: advances in affinity purification mass spectrometry of macromolecular complexes. Proteomics 2012, 12:1591-608.
• [28]Gingras AC, Aebersold R, Raught B: Advances in protein complex analysis using mass spectrometry. J Physiol 2005, 563:11-21.
• [29]Lavallee-Adam M, Cloutier P, Coulombe B, Blanchette M: Modeling contaminants in AP-MS/MS experiments. J Proteome Res 2011, 10:886-95.
• [30]Trinkle-Mulcahy L, Boulon S, Lam YW, Urcia R, Boisvert FM, Vandermoere F, Morrice NA, Swift S, Rothbauer U, Leonhardt H, Lamond A: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol 2008, 183:223-39.
• [31]Rees JS, Lowe N, Armean IM, Roote J, Johnson G, Drummond E, Spriggs H, Ryder E, Russell S, St Johnston D, Lilley KS: In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry. Mol Cell Proteomics 2011, 10:M110.002386.
• [32]Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B: The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 2001, 24:218-29.
• [33]Petriz BA, Gomes CP, Rocha LA, Rezende TM, Franco OL: Proteomics applied to exercise physiology: a cutting-edge technology. J Cell Physiol 2012, 227:885-98.
• [34]Dobreva I, Fielding A, Foster LJ, Dedhar S: Mapping the integrin-linked kinase interactome using SILAC. J Proteome Res 2008, 7:1740-9.
• [35]Fielding AB, Dobreva I, McDonald PC, Foster LJ, Dedhar S: Integrin-linked kinase localizes to the centrosome and regulates mitotic spindle organization. J Cell Biol 2008, 180:681-9.
• [36]Wickstrom SA, Lange A, Hess MW, Polleux J, Spatz JP, Kruger M, Pfaller K, Lambacher A, Bloch W, Mann M, et al.: Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae. Dev Cell 2010, 19:574-88.
• [37]Friedman DB, Hoving S, Westermeier R: Isoelectric focusing and two-dimensional gel electrophoresis. Methods Enzymol 2009, 463:515-40.
• [38]Prout M, Damania Z, Soong J, Fristrom D, Fristrom JW: Autosomal mutations affecting adhesion between wing surfaces in Drosophila melanogaster. Genetics 1997, 146:275-85.
• [39]Cheeseman IM, Anderson S, Jwa M, Green EM, Kang J, Yates JR 3rd, Chan CS, Drubin DG, Barnes G: Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. Cell 2002, 111:163-72.
• [40]Natale M, Maresca B, Abrescia P, Bucci EM: Image Analysis Workflow for 2-D Electrophoresis Gels Based on ImageJ. Proteomics Insights 2011, 4:37-49.
• [41]Dyballa N, Metzger S: Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp 2009, 30:e1431.