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Rho GTPases pp 131-140 | Cite as

An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK

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Part of the Methods in Molecular Biology book series (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.

Key words

Kinase Rac1 ERK Phosphorylation ATP In vitro [γ-32P]ATP 
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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Medical GeneticsUniversity of AlbertaEdmontonCanada
  2. 2.Faculty of Medicine and Dentistry, Signal Transduction Research GroupUniversity of AlbertaEdmontonCanada
  3. 3.Department of MedicineUniversity of AlbertaEdmontonCanada

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