Product Pathways - Kinases
DAPK3 Kinase #7441
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Description
Purified recombinant full-length human DAPK3 (Met1-Arg454) 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 full-length human DAPK3 (Met1-Arg454) (GenBank Accession No. NM_001348) 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 DAPK3 protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. DAPK3 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: MBP 200 ng/μl, and variable amounts of recombinant DAPK3.
Quality Control
The theoretical molecular weight of the GST-DAPK3 protein is 79 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. DAPK3 kinase activity was determined using a radiometric assay [Fig.2].
Background
Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated serine/threonine kinase that participates in a wide range of apoptotic signals including interferon-γ, tumor necrosis factor α, Fas, activated c-Myc, and detachment from the extracellular matrix. In addition to the kinase domain and calmodulin regulatory segment, DAPK also has eight ankyrin repeats, a cytoskeleton binding region, and a conserved death domain (1-3). Deletion of the calmodulin-regulatory domain generates a constitutively active mutant kinase. Ectopic expression of wild-type DAPK induced cell death in HeLa cells. Conversely, expression of a catalytically inactive mutant protected cells from interferon-γ-induced cell death (4). The catalytic domain of DAPK has very high sequence similarity to vertebrate myosin light chain kinase (MLCK) and a RXX(S/T)X motif derived from myosin light chain protein was shown to be phosphorylated in vitro by DAPK (5).
- Kimchi, A. (1999) Ann Rheum Dis. 58, I14-I19.
- Cohen, O. et al. (1999) J Cell Biol 146, 141-148.
- Deiss, L. P. et al. (1995) Genes Dev 9, 15-30.
- Cohen, O. et al. (1997) EMBO J 16, 998-1008.
- Velentza, A. V. et al. (2001) J Biol Chem 276, 38956-38965.
Application References
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