Product Pathways - Cytoskeletal Signaling
PAK1 Kinase #7632
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 PAK1 kinase, supplied as a GST fusion protein.
Source / Purification
The GST-PAK1 fusion protein was produced using a baculovirus expression system with a construct expressing full length human PAK1 (Met1-His545) (GenBank Accession No. NM_002576) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-PAK1 fusion protein was analyzed using SDS/PAGE followed by anti-PAK1 Western blot (A) or Coomassie stain (B)..
Kinase Assay - Radiometric
Figure 2. PAK1 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 25 ng/50 µl Recombinant PAK1.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of PAK1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 to detect phosphorylation of substrate peptide (#1132) by PAK1 kinase. In a 50 µl reaction, increasing amounts of PAK1 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-PAK1 fusion protein is 90 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain and Western blot [Fig.1]. PAK1 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure PAK1 activity using HTScan™ PAK1 Kinase Assay Kit #7633 [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).
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Application References
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