Determination of ERK Activity: Anti-phospho-ERK Antibodies and In Vitro Phosphorylation
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 proliferati
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1. Introduction The mitogen-activated protein kinases (MAPKs) are a family of protein serine/threonine kinases that operate within specific signaling pathways termed MAPK cascades (for reviews see first chapter of this book and references therein). Each MAPK cascade is composed of three core tiers (MAP3K, MAPKK, MAPK), which are occasionally accompanied by additional upstream
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_2, © Springer Science+Business Media, LLC 2010
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(MAP4K) and downstream (MAPKAPK2) tiers. Each of the tiers is composed of several dedicated protein kinases, which are either products of distinct genes (e.g., ERK1 and ERK2) or alternative spliced isoforms (e.g., ERK1a, b, c). Upon stimulation, the kinases in each level phosphorylate and activate some or all of the protein kinases in the next level, thus participating in the facilitation, amplification, and specificity-determination of the transmitted signals. In many cases, enzymes at a given tier of the cascade share a common phosphorylation site, such as the ThrXaa-Tyr motif for MAPKs. Eventually, the transmitted signals are passed on from the MAPK and MAPKAPK tier components to several regulatory proteins that essentially govern all stimulated cellular processes including proliferation, differentiation, response to stress, and others. Five distinct MAPK signaling cascades have been identified so far and these are termed according to the components in the MAPK tier of the cascades. These cascades are: (1) Extracellular signal-regulated kinase 1/2 (ERK1/2, ERKs; (1)); (2) Jun N-terminal kinase (JNK; also termed SAPK1 (2, 3)); (3) p38MAPK (p38; also termed SAPK2–4; (4–6)); and (4) ERK5 (also termed BMK1 (7, 8)). Additional kinases termed ERK7 and ERK8, which also contain the Thr-Xaa-Tyr motif and share a sequence homology with other MAPKs, represent an additional group of MAPKs (9, 10). However, the mechanism of activation of these kinases is not fully understood, but it does not seem to include phosphorylation by upstream protein kinases. Therefore, at this stage the ERK7/8 are not considered a genuine MAPK cascade. Finally, ERK3 and ERK4 that present up to 50% identity to ERK1/2 do not contain a Thr-Xaa-Tyr domain in their activation loop (11), and therefore are not considered to act as genuine MAPKs as well. The different groups of MAPKs seem to differ in their physiological activities; the ERKs usually play a role in proliferation, whereas the other cascades seem to respond mainly to stress and are involved in apoptosis. The amount of transmitted signals via each MAPK cascade is important for studying intracellular signaling. Usually, the activity of one component of the MAPK level of each cascade (ERK1/2, JNK, p38MAPK, etc.) is a sufficient indicator of the transmitted signal. However, for certain studies the activity of additional components within the cascades must be determined in order to understand the actual fate
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