Cell Signaling Technology

Product Pathways - Cytoskeletal Signaling

PAK2 Kinase #7635

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.

Description

Purified recombinant full length human PAK2 kinase, supplied as a GST fusion protein.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct containing 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 chromatography using glutathione-agarose.

Gel Staining

Gel Staining

Figure 1. The purity of the GST-PAK2 fusion protein was analyzed using SDS/PAGE followed by anti-PAK2 Western blot (A) or Silver stain (B).

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 2. 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 and 50 ng/50 µl Recombinant PAK2.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

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.)


Quality Control

The theoretical molecular weight of the GST-PAK2 fusion protein is 88 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. PAK2 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure PAK2 activity using HTScan™ PAK2 Kinase Assay Kit #7636 [Fig.3].

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).

  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.

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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.

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