An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK

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.