Abstract
Elucidation of biological functions of signaling proteins is facilitated by studying their protein–protein interaction networks. Affinity purification combined with mass spectrometry (AP-MS) has become a favorite method to study protein complexes. Here we describe a procedure for single-step purification of ERK (Rolled) and associated proteins from Drosophila cultured cells. The use of the streptavidin-binding peptide (SBP) tag allows for a highly efficient isolation of native ERK signaling complexes, which are suitable for subsequent analysis by mass spectrometry. Our analysis of the ERK interactome has identified both known and novel signaling components. This method can be easily adapted for SBP-based purification of protein complexes in any expression system.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117–1134
Futran AS, Link AJ, Seger R et al (2013) ERK as a model for systems biology of enzyme kinetics in cells. Curr Biol 23:R972–R979
Newbern J, Zhong J, Wickramasinghe RS et al (2008) Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development. Proc Natl Acad Sci U S A 105:17115–17120
Rauen KA (2013) The RASopathies. Annu Rev Genomics Hum Genet 14:355–369
Li WX (2005) Functions and mechanisms of receptor tyrosine kinase Torso signaling: lessons from Drosophila embryonic terminal development. Dev Dyn 232:656–672
Sopko R, Perrimon N (2013) Receptor tyrosine kinases in Drosophila development. Cold Spring Harb Perspect Biol 5(6):a009050
Shilo BZ (2014) The regulation and functions of MAPK pathways in Drosophila. Methods 68:151–159
Friedman AA, Tucker G, Singh R et al (2011) Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling. Sci Signal 4(196):rs10
von Kriegsheim A, Baiocchi D, Birtwistle M et al (2009) Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 11:1458–1464
Gavin AC, Maeda K, Kühner S (2011) Recent advances in charting protein-protein interaction: mass spectrometry-based approaches. Curr Opin Biotechnol 22:42–49
Veraksa A (2013) Regulation of developmental processes: insights from mass spectrometry-based proteomics. Wiley Interdiscip Rev Dev Biol 2:723–734
Rigaut G, Shevchenko A, Rutz B et al (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032
Veraksa A, Bauer A, Artavanis-Tsakonas S (2005) Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry. Dev Dyn 232:827–834
Burckstummer T, Bennett KL, Preradovic A et al (2006) An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods 3:1013–1019
Kyriakakis P, Tipping M, Abed L et al (2008) Tandem affinity purification in Drosophila: the advantages of the GS-TAP system. Fly (Austin) 2:229–235
Keefe AD, Wilson DS, Seelig B et al (2001) One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein Expr Purif 23:440–446
Gilbert MM, Tipping M, Veraksa A et al (2011) A screen for conditional growth suppressor genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie. Dev Cell 20:700–712
Dent LG, Poon CL, Zhang X et al (2014) The GTPase regulatory proteins pix and git control tissue growth via the Hippo pathway. Curr Biol 25(1):124–130
Zhang C, Robinson BS, Xu W et al (2015) The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue. Dev Cell 34:168–180
Kim JH, Chang TM, Graham AN et al (2010) Streptavidin-binding peptide (SBP)-tagged SMC2 allows single-step affinity fluorescence, blotting or purification of the condensin complex. BMC Biochem 11:50
Choi H, Larsen B, Lin ZY et al (2011) SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods 8:70–73
Kwon Y, Vinayagam A, Sun X et al (2013) The Hippo signaling pathway interactome. Science 342:737–740
Yang L, Veraksa A (2015) SAINT output for a complete ERK-SBP purification dataset. https://xythos.umb.edu/xythoswfs/webui/_xy-e3032387_1-t_4JUTz7Bi. Accessed 10 Dec 2015
Rintelen F, Hafen E, Nairz K (2003) The Drosophila dual-specificity ERK phosphatase DMKP3 cooperates with the ERK tyrosine phosphatase PTP-ER. Development 130:3479–3490
Jimenez G, Shvartsman SY, Paroush Z (2012) The Capicua repressor—a general sensor of RTK signaling in development and disease. J Cell Sci 125:1383–1391
Acknowledgements
This protocol was developed with participation of Manuel Valdes, Marla Tipping, and Wenjian Xu. The authors thank Heya Zhao for helpful comments on the manuscript. A.V. was supported by the NIH grant GM105813. L.Y. was supported by the UMass Boston Sanofi Genzyme Doctoral Fellowship. Mass spectrometry was performed at the Taplin Mass Spectrometry Facility at Harvard Medical School.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Yang, L., Veraksa, A. (2017). Single-Step Affinity Purification of ERK Signaling Complexes Using the Streptavidin-Binding Peptide (SBP) Tag. In: Jimenez, G. (eds) ERK Signaling. Methods in Molecular Biology, vol 1487. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6424-6_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6424-6_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6422-2
Online ISBN: 978-1-4939-6424-6
eBook Packages: Springer Protocols