Product Pathways - MAPK Signaling
ASK1 Kinase #7676
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
Description
Purified recombinant human ASK1 (Met649-Val946) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human ASK1 (Met649-Lys946) (GenBank Accession No. NM_005923) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-ASK1 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. ASK1 kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: MBP, 400 ng/μL and recombinant ASK1: variable.
Quality Control
The theoretical molecular weight of the GST-ASK1 fusion protein is 60 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. ASK1 kinase activity was determined using a radiometric assay [Fig.2].
Background
Apoptosis signal-regulating kinase 1 (ASK1), a MAP kinase kinase kinase, plays essential roles in stress-induced apoptosis (1,2). ASK1 is activated in response to a variety of stress-related stimuli through distinct mechanisms and activates MKK4 and MKK3, which in turn activate JNK and p38 (3). Overexpression of ASK1 activates JNK and p38 and induces apoptosis in several cell types through signals involving the mitochondrial cell death pathway. Embryonic fibroblasts or primary neurons derived from ASK1-/- mice are resistant to stress-induced JNK and p38 activation and cell death (4,5). Phosphorylation at Ser967 is essential for ASK1 association with 14-3-3 protein and suppression of cell death (6). Oxidative stress induces dephosphorylation of Ser967 and phosphorylation of Thr845 in the activation loop of ASK1, and both are correlated with ASK1 activity and ASK1-dependent apoptosis (7,8). On the other hand, Akt phosphorylates ASK1 at Ser83, which attenuates ASK1 activity and promotes cell survival (9).
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
