Abstract
The ERK signaling cascade is composed of several protein kinases that sequentially activate each other by phosphorylation. This pathway is a central component of a complex signaling network that regulates important cellular processes including proliferation, differentiation, and survival. In most of these cases, the ERK cascade is activated downstream of the small GTPase Ras that, upon activation, recruits and activates the first tier in the cascade, which contains the Raf kinases. Afterward the signal is further transmitted by MEKs, ERKs, and often RSKs in the MAPKK, MAPK, and MAPKAPKs tiers of the cascade, respectively. ERKs and RSKs can further disseminate the signal by phosphorylating and modulating the activity of a large number of regulatory proteins including transcription factors and chromatin modifying enzymes. Understanding the mechanisms of activation and the regulation of the various components of this cascade will enhance our insight into the regulation of the ERK-dependent cellular processes in normal cells or of their malfunctioning in various diseases, including cancer. In this chapter, we describe methods used to determine the activity of ERKs, which upon slight modifications can also be used for the study of other signaling kinases, either within the cascade or in other pathways. These methods have been successfully applied to study the ERK signaling cascades in a variety of tissue-cultured cell lines, homogenized animal organs, and lower organisms. As such, the use of these methods should expand our knowledge on the regulation of many distinct systems and upon induction of various stimulations.
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Acknowledgments
This work was supported by grants from the Mario Negri–Weizmann collaborative fund and from the EU Sixth Framework Program under the SIMAP (IST-2004-027265) and GROWTHSTOP (LSHC CT-2006-037731). RS is an Incumbent of the Yale S. Lewine and Ella Miller Lewine professorial chair for cancer research.
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Procaccia, S., Kraus, S., Seger, R. (2010). Determination of ERK Activity: Anti-phospho-ERK Antibodies and In Vitro Phosphorylation. 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_2
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DOI: https://doi.org/10.1007/978-1-60761-795-2_2
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