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7636
HTScan® PAK2 Kinase Assay Kit
Cellular Assay Kits
Assay Kit

HTScan® PAK2 Kinase Assay Kit #7636

Citations (0)
HTScan® PAK2 Kinase Assay Kit: Image 1

Figure 3. Dose dependence curve of PAK2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of substrate peptide (#1132) by PAK2 kinase. In a 50 µl reaction, increasing amounts of PAK2 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

HTScan® PAK2 Kinase Assay Kit: Image 2

Figure 5. Staurosporine inhibition of PAK2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of PAK2 substrate peptide (#1132) by PAK2 kinase. In a 50 µl reaction, 50 ng PAK1, 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.)

HTScan® PAK2 Kinase Assay Kit: Image 3

Figure 4. Peptide concentration dependence of PAK2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of substrate peptide (#1132) by PAK2 kinase. In a 50 µl reaction, 50 ng of PAK2 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

HTScan® PAK2 Kinase Assay Kit: Image 4

Figure 2. Time course of PAK2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of PAK2 substrate peptide (#1132) by PAK2 kinase. In a 50 µl reaction, 50 ng PAK2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)

HTScan® PAK2 Kinase Assay Kit: Image 5

Figure 1. PAK2 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 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: Tetra (LRRWSLG), 5 µg/50 µl, recombinant PAK1: 50 ng/50 µl.

To Purchase # 7636

Product Description

The kit provides a means of performing kinase activity assays with recombinant human PAK2 kinase. It includes active PAK2 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-peptide: 2,326 Daltons. GST-PAK2 Kinase: 88 kDa.

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 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, PAK5/7, 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). PAK family members are widely expressed, and often over expressed in human cancer (11, 12).

  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.
  11. Wen, Y.Y. et al. (2014) Expert Opin Ther Targets 18, 807-15.
  12. Molli, P.R. et al. (2009) Oncogene 28, 2545-55.

Pathways & Proteins

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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.