Activation of SAPK/JNKs In Vitro
The stress-activated protein kinase/c-jun N-terminal kinases (SAPK/JNKs) are mitogen-activated protein kinases (MAPKs) that are activated by stressful and inflammatory stimuli and regulate cellular responses such as proliferation, differentiation, and apo
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1. Introduction The stress-activated protein kinase/c-jun N-terminal kinases (SAPK/JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs) that are activated preferentially by stressful and inflammatory stimuli including tumor necrosis factor a (TNFa), interleukin 1 (IL-1) and related cytokines, pathogen-associated molecular patterns (PAMPs), heat shock, osmotic shock, UV-C radiation, endothelin, and anisomycin (Table 1) (1). In response to these stimuli, the SAPK/JNK signaling pathway regulates a range of cellular events that include proliferation, differentiation, apoptosis, and inflammation. SAPK/JNKs are activated by Thr/ Tyr phosphorylation by upstream MAPK kinases (MAP2Ks), MKK4, and MKK7. The MAP2Ks are activated by Ser/Thr
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_3, © Springer Science+Business Media, LLC 2010
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Chadee and Kyriakis
Table 1 Stimuli that activate SAPK/JNK in vivoa Amount and duration Stimulus (references) of treatment Responsive cell lines
Notes
UV-C (9–11)
40 J/m2, 20 per s
All adherent cells
TNF (12)
2–100 ng/ml, 1–60 min
CCD-18 Co, HEK-293, COS, U937, HL-60, Jurkat, L929, HeLa, SKOV3, primary thymocytes or hepatocytes
IL-1 (12)
2–100 ng/ml, 1–60 min
EL-4, HepG2, Jurkat, HL-60, KB, SKOV3
Osmotic stress (Sorbitol) (13)
500 mM
All cells
Anisomycin (11)
1–10 mg/ml, 1–60 min
All cells
Endothelin (14, 15)
100–200 nM, 2–60 min
Endothelial, vascular smooth muscle, cardiomyocyte
Methyl methane sulfonate (MMS) (16)
1 mM, 3 h
All cells
Oxidant stress (H2O2) (17)
2–20 mM, 2–60 min
All cells
Angiotensin II (14)
20–500 nM, 2–60 min
Endothelial, mesangial, pulmonary fibroblast, vascular smooth muscle, cardiomyocyte
Choose a cell type that expresses the Angiotensin II receptor and is known to induce a biologic effect
Ischemia/reperfusion (18)
40 min ischemia, 2–120 min reperfusion
Kidney, heart, brain
SAPK/JNK activated only in reperfusion
cis-platinum or (ara-C) 50–300 mM, (16) 2–6 h
All cells
Heat shock (11, 19)
42°C, 30 min
All cells
TGF-b (20)
2–5 ng/ml, 16 h
HepG2
Bacterial lipopolysaccha- 100 ng/ml, ride (21, 22) 5–30 min
Wash cells with 1× PBS before exposure
Primary macrophage, dendritic, RAW264.7 macrophage-like cells, HL-60 (differentiated to macrophages), Jurkat (continued)
Activation of SAPK/JNKs In Vitro
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Table 1 (continued) Amount and duration Stimulus (references) of treatment Responsive cell lines Bacterial peptidoglycan (21, 22)
100 ng/ml, 5–30 min
Primary macrophage, dendritic, RAW264.7 macrophage-like cells, HL-60 (differentiated to macrophages), Jurkat
CpG-DNA (22)
100 nm, 5–30 min
Primary macrophage, dendritic, RAW264.7 macrophage-like cells, HL-60 (differentiated to macrophages), Jurkat
Notes
This list of stimuli is not inclusive but serves as a guide for stimuli that activate SAPK/JNK
a
phosphorylation by a group of over 20 upstream MAP3Ks (MAPK kinase kinases) (1). Some of the MAP3Ks that activate the SAPK/J
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