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ERK Signaling pp 137–149Cite as

Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1487))

Abstract

Extracellular signal-regulated kinase (ERK) regulates various cellular functions through phosphorylation of numerous downstream substrates, which have not yet been fully characterized. To date, several phosphoproteomic approaches have been employed to identify novel substrates for ERK. In this chapter, we describe a method to globally identify ERK substrates by combining immobilized metal affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry. Phosphoprotein enrichment by IMAC enables the subsequent detection of many protein spots with different fluorescence intensities between ERK-inhibited and -activated cells in 2D-DIGE analysis. Furthermore, the advanced sensitivity and resolution of liquid chromatography coupled with tandem mass spectrometry allow for a direct identification of proteins obtained from silver-stained 2D-DIGE gels. Validation experiments such as Phos-tag Western blotting are important steps to further elucidate the functional roles of ERK-mediated phosphorylation of these newly identified substrates.

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Acknowledgments

We thank Megumi Kawano, Mayumi Kajimoto, and Junya Yabuno for experimental assistance, Mayumi Iwata for secretarial assistance, Dr. Maria Tsoumpra for helpful advice, and Dr. Naoki Tani for mass spectrometry analysis. This work was supported by JSPS KAKENHI Grant Numbers 23570231 and 26440101, and the program of the Joint Usage/Research Center for Developmental Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University to H.K.

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Authors and Affiliations

  1. Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan

    Hidetaka Kosako & Kou Motani

Authors
  1. Hidetaka Kosako
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  2. Kou Motani
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Correspondence to Hidetaka Kosako .

Editor information

Editors and Affiliations

  1. Institut de Biologia Molecular de Barcelona-CSIC, Parc Científic de Barcelona, Barcelona, Spain

    Gerardo Jimenez

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Kosako, H., Motani, K. (2017). Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE. 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_10

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  • DOI: https://doi.org/10.1007/978-1-4939-6424-6_10

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  • 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

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