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Rho GTPases pp 131–140Cite as

An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1821))

Abstract

Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that T108 within the 106PNTP109 motif of Rac1 is likely an ERK phosphorylation site and Rac1 also has an ERK docking site 183KKRKRKCLLL192 (D-site) at the C-terminus. Protein phosphorylation could be assayed by many different methods. Here, we describe an in vitro kinase assay we used to assess Rac1 phosphorylation by ERK. Rac1 phosphorylation is detected based on the transfer of a radiolabeled phosphate from ATP to Rac1 by the phosphotransferase activity of the kinase EKR. This in vitro kinase assay uses commercially available purified active ERK. Substrate Rac1 was generated and purified as a glutathione S-transferase (GST) fusion protein. [γ-32P]ATP is used to radiolabel Rac1. Phosphorylation of Rac1 is viewed by autoradiography.

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Authors and Affiliations

  1. Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Daniel Brandwein, Junfeng Tong & Zhixiang Wang

  2. Faculty of Medicine and Dentistry, Signal Transduction Research Group, University of Alberta, Edmonton, AB, Canada

    Daniel Brandwein, Junfeng Tong, Laiji Li, Barbara Ballermann & Zhixiang Wang

  3. Department of Medicine, University of Alberta, Edmonton, AB, Canada

    Laiji Li & Barbara Ballermann

Authors
  1. Daniel Brandwein
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  2. Junfeng Tong
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  3. Laiji Li
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  4. Barbara Ballermann
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  5. Zhixiang Wang
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Corresponding author

Correspondence to Zhixiang Wang .

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Editors and Affiliations

  1. The Hull York Medical School, University of Hull, Hull, United Kingdom

    Francisco Rivero

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Brandwein, D., Tong, J., Li, L., Ballermann, B., Wang, Z. (2018). An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 1821. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8612-5_9

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  • DOI: https://doi.org/10.1007/978-1-4939-8612-5_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8611-8

  • Online ISBN: 978-1-4939-8612-5

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