Product Pathways - HTScan Kinase Assay Kits
HTScan® Mst4 Kinase Assay Kit #7639
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.
| Kit Includes | Quantity |
|---|---|
| Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody # 3141 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide # 1344 | 1.25 milliliters |
| Mst4 Kinase # 7638 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant active human Mst4 kinase. It includes active Mst4 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide #1344: 1,958 Daltons, GST-Mst4 Kinase: 76 kDa.
Peptide Core Sequence
YKT*LR
Kinase Assay - Radiometric
Figure 1. Mst4 kinase activity was measured in a radioisotopic filter binding assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: Myelin basic protein, 5 µg/50 µl, recombinant Mst4: 100 ng/50 µl.
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 µl reaction, increasing amounts of Mst4 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of Mst4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of Mst4 substrate peptide (#1344) by Mst4 kinase. In a 50 µl reaction, 50 ng Mst4, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence 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 µl reaction, 50 ng of Mst4 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course of Mst4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of Mst4 substrate peptide (#1344) by Mst4 kinase. In a 50 µl reaction, 50 ng Mst4 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human 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.
Quality Control
The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 was used for detection. The quality of the biotinylated peptides was evaluated by reverse-phase HPLC and by mass spectrometry.The specific activity of the Mst4 kinase was determined using a radiometric assay [Fig.1 ].Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify Mst4 activity using the Mst4 substrate peptide provided in this kit. Mst4 sensitivity to the inhibitor staurosporine was measured using the Mst4 substrate peptide provided in this kit [Fig.5].
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
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 3141 Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody
- 1344 Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide
- 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.
