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7651
HTScan® PAK4 Kinase Assay Kit
Cellular Assay Kits

HTScan® PAK4 Kinase Assay Kit #7651

This product is discontinued

Kinase Assay - Radiometric Image 1

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.

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Kinase Assay - DELFIA Image 2

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.)

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Kinase Assay - DELFIA Image 3

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.)

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Kinase Assay - DELFIA Image 4

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.)

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Kinase Assay - DELFIA Image 5

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.)

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Product Includes Quantity (with Count) Solution Color
Phospho-PLK (Ser137) Antibody 1 x 30 µl
Kinase Buffer (10X) 9802 1 x 15 ml
ATP (10 mM) 9804 1 x 1 ml

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 Formula:

Peptide substrate, biotin-PLK (Ser137): 1945 Daltons, GST-PAK4: 94 kDa.

Peptide Core Sequence:

RRS*LL

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 Ser199 and Ser204 of PAK1 and Ser192 and Ser197 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 at Ser144 of PAK1 or Ser139 of PAK3 (located in the kinase inhibitory domain) affects kinase activity (7). Phosphorylation at Ser21 of PAK1 or Ser20 of PAK2 regulates binding with the adaptor protein Nck (8). PAK4, PAK5, and PAK6 have lower sequence similarity with PAK1-3 in the amino-terminal regulatory region (9). Phosphorylation at Ser474 of PAK4, a site analogous to Thr423 of PAK1, may play a pivotal role in regulating the activity and function of PAK4 (10).

  1. Knaus, U.G. and Bokoch, G.M. (1998) Int. J. Biochem. Cell Biol. 30, 857-862.
  2. Daniels, R.H. et al. (1998) EMBO J. 17, 754-764.
  3. King, C.C. et al. (2000) J. Biol. Chem. 275, 41201-41209.
  4. Manser, E. et al. (1997) Mol. Cell. Biol. 17, 1129-1143.
  5. Gatti, A. et al. (1999) J. Biol. Chem. 274, 8022-8028.
  6. Lei, M. et al. (2000) Cell 102, 387-397.
  7. Chong, C. et al. (2001) J. Biol. Chem. 276, 17347-17353.
  8. Zhao, Z. et al. (2000) Mol. Cell. Biol. 20, 3906-3917.
  9. Abo, A. et al. (1998) EMBO J. 17, 6527-6540.
  10. Qu, J. et al. (2001) Mol. Cell. Biol. 21, 3523-3533.
Entrez-Gene Id
10298
Swiss-Prot Acc.
O96013
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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