Use of Inhibitors in the Study of MAP Kinases
The mitogen-activated protein (MAP) kinases are ubiquitous intracellular signaling proteins that respond to a variety of extracellular signals and regulate most cellular functions including proliferation, apoptosis, migration, differentiation, and secreti
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1. Introduction The mitogen-activated protein (MAP) kinases are ubiquitous regulators of many cellular functions including cell growth, proliferation, differentiation, and inflammatory responses to stress signals (1). The MAP kinase family consists of four major members; the extracellular signal-regulated kinases-1 and 2 (ERK1/2), the c-Jun N-terminal kinases (JNK), p38 MAP kinases, and Big MAP kinase-1 (BMK1) also known as ERK5. Each of the MAP kinases is activated through highly specific interactions with upstream MAP or ERK kinases (MEKs), which phosphorylate Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_6, © Springer Science+Business Media, LLC 2010
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threonine and tyrosine residues within the activation loop. Once activated, MAP kinases, in turn, phosphorylate and regulate a variety of substrates including transcription factors, translation regulators, other kinases, structural proteins, and other signaling proteins. Given the prominent role that constitutive activation of the MAP kinases plays in proliferative diseases like cancer, or inflammatory disorders such as rheumatoid arthritis, a number of pharmacological inhibitors have been developed to block MAP kinase signaling (2–4). These inhibitors target multiple proteins in the signaling cascade starting at the plasma membrane receptors all the way to the specific MAP kinase. The ability to manipulate the MAP kinase signaling cascades have been particularly useful for understanding basic biological mechanisms that regulate cell functions and for clinical therapies to treat disease. Table 1 provides a list of some of the major small molecular weight pharmacological inhibitors and their protein targets within the MAP kinase signaling pathway. Other methods for inhibiting MAP kinase signaling pathways in treating disease include monoclonal antibodies that target extracellular domains or ligands of receptor tyrosine kinases. The use of monoclonal antibodies to block MAP kinase signaling will not be discussed and can be found in other reviews (5). High throughput screening methods have made it feasible to identify potentially target-specific inhibitor compounds with a desired effect from a large pool of chemical compounds. These types of drug discovery projects first develop the appropriate in vitro and cell-based assays to screen large chemical libraries and assess effects on target kinase activity or a cellular response (6). Once active compounds are identified, chemical modifications and refinement of these lead molecules are made to reach greater inhibition in both the in vitro and cell-based models. Drug development efforts also take advantage of the three-dimensional structures of the MAP kinases that have been solved by X-ray crystallography (7). A detailed understanding of the structure– function relationship for MAP kinases allows the design of inhibitor compounds that bind to specific regions on the MAP kinases including the ATP-bin
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