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7639
HTScan® Mst4 Kinase Assay Kit

HTScan® Mst4 Kinase Assay Kit #7639

This product is discontinued

Kinase Assay - Radiometric Image 1

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.

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Kinase Assay - DELFIA Image 2

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.)

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Kinase Assay - DELFIA Image 3

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.)

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Kinase Assay - DELFIA Image 4

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.)

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Kinase Assay - DELFIA Image 5

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.)

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Product Includes Quantity (with Count) Solution Color
Kinase Buffer (10X) 9802 1 x 15 ml
ATP (10 mM) 9804 1 x 1 ml

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 Formula:

Peptide substrate, Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Biotinylated Peptide #1344: 1,958 Daltons, GST-Mst4 Kinase: 76 kDa.

Peptide Core Sequence:

YKT*LR

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 domains of MST kinases are highly similar to the kinase domains of yeast STE20 kinase and other p21-activated kinases (1). The carboxy-terminal regions of MST1 and MST2 contain 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 are critical for activation 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).

  1. Dan, I. et al. (2001) Trends Cell Biol 11, 220-30.
  2. Creasy, C.L. et al. (1996) J Biol Chem 271, 21049-53.
  3. Lee, K.K. and Yonehara, S. (2002) J Biol Chem 277, 12351-8.
  4. Graves, J.D. et al. (2001) J Biol Chem 276, 14909-15.
  5. Lee, K.K. et al. (2001) J Biol Chem 276, 19276-85.
  6. Graves, J.D. et al. (1998) EMBO J 17, 2224-34.
Entrez-Gene Id
51765
Swiss-Prot Acc.
Q9P289
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.

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