Activity Assays for Extracellular Signal-Regulated Kinase 5
Extracellular signal-regulated kinase 5 (ERK5) is also known as big MAPK (BMK1) or MAPK7. ERK5 is 115 kDa in mass and therefore larger than the other MAPKs such as ERK1/2, JNK, and p38. Like other MAPKs, ERK5 is ubiquitously expressed in mammalian cells a
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1. Introduction The N-terminus of ERK5 encodes the kinase domain that harbors the activation loop TEY motif that is dually phosphorylated for activation. The C-terminal moiety of ERK5 is unique among MAPKs and plays a transcriptional activation function required for regulation of MEF2C, peroxisome proliferator activated receptor (PPARg1), c-Fos, and Fra1 (1, 2). ERK5 is strongly activated by stress stimuli, including oxidative stress and hyperosmolarity (3). ERK5 is also activated by EGF and is involved in the control of the G1/S transition in the cell cycle (4). Serum and other growth factors such as NGF in specific cell types also activate ERK5. ERK5 also plays a critical role in the survival of endothelial cells and the maintenance of vascular integrity in adult mice (5). In mouse embryos, ERK5 is required for vascular development (6–8).
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_5, © Springer Science+Business Media, LLC 2010
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Nakamura and Johnson
The MAPK kinase, MEK5, which phosphorylates the activation loop TEY motif, encodes a Phox/Bem1 (PB1) domain (3). PB1 domains are used for heterodimerization of proteins, and the MAPK kinase kinases, MEKK2 and MEKK3 also encode PB1 domains (9). The MEK5 PB1 domain heterodimerizes with the PB1 domain of MEKK2 or MEKK3 (3, 10), and MEKK2 or MEKK3 and phosphorylate and activate MEK5 in response to different stimuli (9). The PB1 domain-dependent activation of the ERK5 cascade is unique among the MAPK family of kinases (9, 10). Importantly, MEK5 is inhibited by several MEK1/2 inhibi tors, including U0126 and PB184352 (11, 12), so caution in interpretation of ERK1/2 versus ERK5 function with these inhibitors is warranted. Herein, we define methods used to measure the activation of ERK5 using different biochemical and cellbased assays.
2. Materials 2.1. Cell Culture
1. Mouse embryonic fibroblasts (MEFs) were grown in IMDM (Life Technologies, Inc) with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C with 7% CO2. HEK293, COS7, and mouse embryonic endothelial cells (MEECs) were maintained in DMEM supplemented with 10% FCS. Trypsin (0.25%) and EDTA (1 mM) in PBS (GIBCO) is used for the passage of cells. 2. The monoclonal antibody (12CA5) against the hemagglutinin (HA) epitope (Roche Molecular Biochemicals); Anti-Ras antibody (#Y13-259, Abcam); Anti-ERK5 antibody (#E1523, Sigma); the mouse monoclonal antibody for MEK5 (#610957, BD PharMingen); anti-phospho-ERK5 antibody (#3771, Cell Signaling); HRP-donkey anti-rabbit IgG antibody (#711035-152, Jackson Laboratories); and sheep-anti-mouse IgG antibody (#NA931V, GE Healthcare). 3. Lipofectamine and Plus Reagent (Invitrogen) are used for transfection as per the manufacturer’s instruction. 4. MEF2C luciferase reporter gene (3×-MEF2-luc) was constructed with a thymidine kinase promoter with three copies of a high-affinity MEF2 binding site from the desmin gene (13). The pRL-TK
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