Revision 1

#7636Store at -80C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#Q13177

Entrez-Gene Id:

5062

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO. Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 5 mM DTT, 15 mM reduced glutathione and 20% glycerol.
Store at –80°C.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human PAK2 (Met1-Arg534) (GenBank Accession No. NM_002577) with an amino-terminal GST tag. The protein was purified by one-step affinity purification using glutathione-agarose.

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 overexpressed in human cancer (11,12).

  1. Knaus, U.G. and Bokoch, G.M. (1998) Int. J. Biochem. Cell Biol. 30, 857-62.
  2. Daniels, R.H. et al. (1998) EMBO J. 17, 754-64.
  3. King, C.C. et al. (2000) J. Biol. Chem. 275, 41201-9.
  4. Manser, E. et al. (1997) Mol. Cell. Biol. 17, 1129-43.
  5. Gatti, A. et al. (1999) J. Biol. Chem. 274, 8022-8.
  6. Lei, M. et al. (2000) Cell 102, 387-97.
  7. Chong, C. et al. (2001) J. Biol. Chem. 276, 17347-53.
  8. Zhao, Z. et al. (2000) Mol. Cell. Biol. 20, 3906-17.
  9. Abo, A. et al. (1998) EMBO J. 17, 6527-40.
  10. Qu, J. et al. (2001) Mol. Cell. Biol. 21, 3523-33.
  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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。
Revision 1
#7636

HTScan® PAK2 Kinase Assay Kit

HTScan® PAK2 Kinase Assay Kit: Image 1 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用数量递增的 PAK2 和 1.5 µM 底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® PAK2 Kinase Assay Kit: Image 2 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng PAK1、1.5 µM 底物肽、20 µM ATP 和数量递增的 staurosporine 反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® PAK2 Kinase Assay Kit: Image 3 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔在室温下用 50 ng PAK2 和浓度递增的底物肽反应 30 分钟。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® PAK2 Kinase Assay Kit: Image 4 Expand Image
图 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. 在 50 µl 反应体积中，每个反应孔用 50 ng PAK2 和 1.5 µM 底物肽。（DELFIA® 是 PerkinElmer, Inc. 的注册商标）
HTScan® PAK2 Kinase Assay Kit: Image 5 Expand Image
图 1. 使用以下反应条件在放射分析中测量 PAK2 激酶活性：60 mM HEPES-NaOH、pH 7.5、3 mM MgCl2、3 mM MnCl2、3 µM 原钒酸钠、1.2 mM DTT、ATP（变量）、2.5 µg/50 µl PEG20.000，底物：Tetra (LRRWSLG)、5 µg/50 µl，重组 PAK1：50 ng/50 µl。
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Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
仅供研究使用。不得用于诊断流程。