Product Pathways - Cytoskeletal Signaling
Mst4 Kinase #7638
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 full length human Mst4 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 Mst4 (Met1-Pro416) (GenBank Accession No. NM-016542) with an amino-terminal GST tag. The protein was then purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the Mst4 fusion protein was analyzed using SDS/PAGE followed by anti-GST Western blot (A) or Coomassie stain (B).
Kinase Assay - Radiometric
Figure 2. Mst4 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 uM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 ug/50 ul PEG20,000, Substrate: Myelin basic protein, 5 ug/50 ul, Recombinant Mst4: 100 ng/50 ul.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Mst4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of substrate peptide (#1344) by Mst4 kinase. In a 50 ul reaction, increasing amounts of Mst4 and 1.5 uM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-Mst4 kinase fusion protein is 76 kDa. The purified kinase fusion protein was quality controlled for purity using SDS-PAGE followed by Coomassie stain and Western blot [Fig.1]. Mst4 kinase activity was determined in radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Mst4 activity using HTScan™ Mst4 Kinase Assay Kit #7639[Fig.3].
Background
Mst kinases, members of the STE20 family of kinases, are upstream activators of MAPK pathways that regulate processes such as apoptosis, morphogenesis and cytoskeletal rearrangements. The amino-terminal kinase domain of Mst is considerably homologous to the kinase domain of yeast STE20 kinase and other p21-activated kinases (1). The carboxy-terminal region of Mst1 and Mst2 contains dimerization and inhibitory domains (1-3). Dimerization and phosphorylation at the activation loop results in translocation of Mst1 from the cytosol to the nucleus (3). Growing evidence indicates that Mst1, Mst2 and Mst3 are activated by apoptotic signals as well as other stress conditions (4-6). Complete activation of Mst1 requires both phosphorylation and caspase-mediated cleavage (4). Sequence alignment of the activation loop of the GCK family indicates that Thr183 of Mst1 and Thr180 of Mst2 are the conserved residues and might be critical for the activity of the kinases. Activated Mst kinases may rely on p38 MAPK and JNK pathways to amplify apoptotic signals (5). Phosphorylation at Ser327 of Mst1, which is close to the caspase-3 recognition site, inhibits caspase-mediated cleavage (4).
- Dan, I. et al. (2001) Trends Cell Biol. 11, 220-230.
- Creasy, C.L. et al. (1996) J. Biol. Chem. 271, 21049-21053.
- Lee, K. and Yonehara, S. (2002) J. Biol. Chem. 277, 12351-12358.
- Graves, J.D. et al. (2001) J. Biol. Chem. 276, 14909-14915.
- Lee, K. et al. (2001) J. Biol. Chem. 276, 19276-19285.
- Graves, J.D. et al. (1998) EMBO J. 17, 2224-2234.
Application References
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Companion Products
- 1344 Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide
- 3141 Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody
- 9802 Kinase Buffer (10X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
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.
