Product Pathways - HTScan Kinase Assay Kits
HTScan® PAK4 Kinase Assay Kit #7651
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-PLK (Ser137) Antibody # 5070 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| PLK (Ser137) Biotinylated Peptide # 1300 | 1.25 milliliters |
| PAK4 Kinase # 7650 | 2 x 5 micrograms |
Description
The kit provides a means of performing enzymatic assays with recombinant human PAK4 kinase. It includes active PAK4 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, biotin-PLK (Ser137): 1945 Daltons, GST-PAK4: 94 kDa.
Peptide Core Sequence
RRS*LL
Kinase Assay - Radiometric
Figure 1. PAK4 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: tetra (LRRWSLG), 2.5 μg/50 μl, recombinant PAK4: 100 ng/50 μL.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of PAK4 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by PAK4 kinase. In a 50 μl reaction, increasing amounts of PAK4 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 PAK4 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of PAK4 substrate peptide (#1300) by PAK4 kinase. In a 50 μl reaction, 100 ng PAK4, 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 PAK4 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by PAK4 kinase. In a 50 μl reaction, 100 ng of PAK4 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 PAK4 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of PAK4 substrate peptide (#1300) by PAK4 kinase. In a 50 μl reaction, 100 ng PAK4 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 PAK4 (Met1-Arg591) (GenBank Accession No. NM_005884) with an amino-terminal GST tag. The protein was 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-PLK (Ser137) Antibody #5070 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified PAK4 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. PAK4 kinase specific activity 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 PAK4 activity using the PAK4 substrate peptide provided in this kit. PAK4 sensitivity to the inhibitor staurosporine was measured using the PAK4 substrate peptide provided in this kit [Fig.5].
Background
The p21-activated kinase (PAK) family of serine/threonine kinases is engaged in multiple cellular processes, including cytoskeletal reorganization, MAPK signaling, apoptotic signaling, control of phagocyte NADPH oxidase and growth factor-induced neurite outgrowth (1,2). Several mechanisms that induce PAK activity have been reported. Binding of Rac/cdc42 to the CRIB (or PBD) domain near the amino terminus of PAK causes autophosphorylation and conformational changes in PAK (1). Phosphorylation of PAK1 at Thr423 by PDK induces activation of PAK1 (3). Several autophosphorylation sites have been identified, including serines 199 and 204 of PAK1 and serines 192 and 197 of PAK2 (4,5). Because the autophosphorylation sites are located in the amino-terminal inhibitory domain, it has been hypothesized that modification in this region prevents the kinase from reverting to an inactive conformation (6). Research indicates that phosphorylation of Ser144 of PAK1 or Ser139 of PAK3 (located in the kinase inhibitory domain) affects kinase activity (7). Phosphorylation of Ser21 of PAK1 or Ser20 of PAK2 regulates binding with the adaptor protein Nck (8). More recently identified family members including PAK4, PAK5 and PAK6 have lower sequence similarity with PAK1-3 in the amino-terminal regulatory region (9). Phosphorylation of Ser474 of PAK4, a site analogous to Thr423 of PAK1, may play a pivotal role in regulating the activity and function of PAK4 (10).
- Knaus, U.G. and Bokoch, G.M. (1998) Int. J. Biochem. Cell Biol. 30, 857-862.
- Daniels, R.H. et al. (1998) EMBO J. 17, 754-764.
- King, C.C. et al. (2000) J. Biol. Chem. 275, 41201-41209.
- Manser, E. et al. (1997) Mol. Cell. Biol. 17, 1129-1143.
- Gatti, A. et al. (1999) J. Biol. Chem. 274, 8022-8028.
- Lei, M. et al. (2000) Cell 102, 387-397.
- Chong, C. et al. (2001) J. Biol. Chem. 276, 17347-17353.
- Zhao, Z. et al. (2000) Mol. Cell. Biol. 20, 3906-3917.
- Abo, A. et al. (1998) EMBO J. 17, 6527-6540.
- Qu, J. et al. (2001) Mol. Cell. Biol. 21, 3523-3533.
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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.
