Regulation of MAP Kinase Signaling by Calcium
Mitogen-activated protein kinase (MAPK) signaling influences a variety of cellular responses, ranging from stimulation of cell proliferation to induction of senescence and/or apoptosis. Ca2+ is a ubiquitous intracellular signaling molecule that controls m
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1. Introduction Mitogen-activated protein kinases (MAPKs) are ubiquitously expressed enzymes that regulate a wide variety of functions in virtually all cell types (1). The term “MAPK” usually refers to the terminal kinase in a three-tier cascade, in which MAPKs are phosphorylated and activated by MAPK kinases (MAPKK or MEK), which themselves are phosphorylated and activated by MAPK kinase kinases (MAPKKK or MEKK). Of the major MAPK pathways, the Ras/Raf/MEK/ERK cascade is the most widely studied and is the focus of this chapter. Engagement of cell-surface receptors by extracellular signaling molecules, such as growth factors, results in activation of the intracellular small G-protein Ras. The resultant change in Ras conformation facilitates its direct interaction with Raf isoforms, namely A-Raf, B-Raf, and C-Raf (also termed Raf-1) (2). The Raf proteins are serine/threonine kinases, which phosphorylate and activate MEK1 and MEK2. In turn, MEK1 and MEK2 catalyze the phosphorylation of the extracellular signal-regulated kinases, ERK1 and ERK2. Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_9, © Springer Science+Business Media, LLC 2010
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Once active, ERKs either dimerize and remain in the cytosol where they catalyze the phosphorylation of a variety of substrates, or, as monomers, translocate to the nucleus where they phosphorylate transcription factors (3). MAPK function is influenced by several pathways, including Ca2+ (4). For example, an increase in intracellular free Ca2+ concentration ([Ca2+]i) positively regulates Ras signaling in PC12 cells leading to increased ERK phosphorylation (5). Conversely, treating keratinocytes with Ca2+ inhibits activation of ERK by epidermal growth factor (EGF) (6). The reasons for these discrepant data are not known, but differences between the cell types are likely to contribute. The ability to manipulate [Ca2+]i and accurately measure active MAPK is a welcome addition to the researchers’ toolbox. In this chapter, we describe straightforward assays for evaluating the effect of Ca2+ on growth factor-induced MAPK signaling using Western blotting and confocal immunofluorescence. 1.1. Manipulation of [Ca 2+]i
The protocol described in this chapter represents probably the most widely used methods to manipulate [Ca2+]i. A23187 is a Ca2+ ionophore that causes a rapid and sustained increase in [Ca2+]i by permitting entry into the cell of extracellular Ca2+. BAPTA-AM enters cells where it chelates intracellular Ca2+, markedly reducing [Ca2+]i. These reagents therefore allow the investigator to elucidate the regulatory effect of Ca2+ on intracellular signaling. In order to identify the source of the Ca2+ responsible for a specific effect, pharmacological compounds are available (which selectively modulate individual Ca2+ channels or pumps (Table 1)). Each compound can be broadly characterized as inducing either an “on” or an “off” signal (Fig. 1). On signals increase [Ca2+]i,
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