Abstract
ERK cascade scaffolds serve as docking platforms to coordinate the assembly of multiprotein complexes that contribute to the spatial and temporal control of ERK signaling. Given that protein–protein interactions are essential for scaffold function, determining the full repertoire of scaffold binding partners will likely provide new insight into the regulation and activities of the ERK cascade scaffolds. In this chapter, we describe methods to identify scaffold interacting proteins using a proteomics approach. This protocol is based on the affinity purification of scaffold complexes from tissue culture cells and utilizes mass spectrometry to identify the protein constituents of the complex.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shaul, Y. D., and Seger, R. (2007) The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta 1773, 1213–26.
Yoon, S., and Seger, R. (2006) The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24, 21–44.
Qi, M., and Elion, E. A. (2005) MAP kinase pathways. J Cell Sci 118, 3569–72.
Morrison, D. K., and Davis, R. J. (2003) Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu Rev Cell Dev Biol 19, 91–118.
Kolch, W. (2005) Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat Rev Mol Cell Biol 6, 827–37.
Schaeffer, H. J., Catling, A. D., Eblen, S. T., Collier, L. S., Krauss, A., and Weber, M. J. (1998) MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade. Science 281, 1668–71.
Roy, M., Li, Z., and Sacks, D. B. (2005) IQGAP1 is a scaffold for mitogen-activated protein kinase signaling. Mol Cell Biol 25, 7940–52.
Ishibe, S., Joly, D., Liu, Z. X., and Cantley, L. G. (2004) Paxillin serves as an ERK-regulated Âscaffold for coordinating FAK and Rac activation in epithelial morphogenesis. Mol Cell 16, 257–67.
Torii, S., Kusakabe, M., Yamamoto, T., Maekawa, M., and Nishida, E. (2004) Sef is a spatial regulator for Ras/MAP kinase signaling. Dev Cell 7, 33–44.
Therrien, M., Michaud, N. R., Rubin, G. M., and Morrison, D. K. (1996) KSR modulates signal propagation within the MAPK cascade. Genes Dev 10, 2684–95.
Dougherty, M. K., Ritt, D. A., Zhou, M., Specht, S. I., Monson, D. M., Veenstra, T. D., and Morrison, D. K. (2009) KSR2 is a calcineurin substrate that promotes ERK cascade activation in response to calcium signals. Mol Cell 34, 652–62.
Casar, B., Arozarena, I., Sanz-Moreno, V., Pinto, A., Agudo-Ibanez, L., Marais, R., Lewis, R. E., Berciano, M. T., and Crespo, P. (2009) Ras subcellular localization defines extracellular signal-regulated kinase 1 and 2 substrate specificity through distinct utilization of scaffold proteins. Mol Cell Biol 29, 1338–53.
Muller, J., Ory, S., Copeland, T., Piwnica-Worms, H., and Morrison, D. K. (2001) C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1. Mol Cell 8, 983–93.
Ory, S., Zhou, M., Conrads, T. P., Veenstra, T. D., and Morrison, D. K. (2003) Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr Biol 13, 1356–64.
Ritt, D. A., Zhou, M., Conrads, T. P., Veenstra, T. D., Copeland, T. D., and Morrison, D. K. (2007) CK2 Is a component of the KSR1 scaffold complex that contributes to Raf kinase activation. Curr Biol 17, 179–84.
Schaffhausen, B., Benjamin, T. L., Pike, L., Casnellie, J., and Krebs, E. (1982) Antibody to the nonapeptide Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu is specific for polyoma middle T antigen and inhibits in vitro kinase activity. J Biol Chem 257, 12467–70.
Grussenmeyer, T., Scheidtmann, K. H., Hutchinson, M. A., Eckhart, W., and Walter, G. (1985) Complexes of polyoma virus medium T antigen and cellular proteins. Proc Natl Acad Sci U S A 82, 7952–4.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Meida, LLC
About this protocol
Cite this protocol
McKay, M.M., Morrison, D.K. (2010). Proteomic Analysis of Scaffold Proteins in the ERK Cascade. In: Seger, R. (eds) MAP Kinase Signaling Protocols. Methods in Molecular Biology, vol 661. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-795-2_19
Download citation
DOI: https://doi.org/10.1007/978-1-60761-795-2_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-794-5
Online ISBN: 978-1-60761-795-2
eBook Packages: Springer Protocols