Phospho-PAK1 (Thr423)/PAK2 (Th

2601:

Western Blotting

Phospho-PAK1 (Thr423)/PAK2 (Thr402) Antibody detects endogenous PAK1, PAK2 and PAK3 only when phosphorylated at Thr423, Thr402 and Thr421, respectively. The antibody does not cross-react with phosphorylated PAK4, PAK5 or PAK6. The antibody does cross-react with phospho-Mst1 (Thr183) or phospho-Mst2 (Thr180).

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr423 of human PAK1. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western blot analysis of extracts from guinea pig neutrophils stimulated with 1 µM fMLP for indicated times, using Phospho-PAK1 (Thr423)/PAK2 (Thr402) Antibody (Provided by Drs. Qian Zhan and John Badwey, Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Massachusetts.)

Western Blotting

Western blot analysis of extracts from HeLa cells treated with 0.4 M sorbitol for indicted times, using Phospho-PAK1 (Thr423)/PAK2 (Thr402) Antibody (upper) or PAK2 antibody (lower).

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.

• Zhan, Q. et al. (2003) p21-Activated Kinase 2 in Neutrophils Can Be Regulated by Phosphorylation at Multiple Sites and by a Variety of Protein Phosphatases. The Journal of Immunology 171, 3785-3793. Applications: Western Blotting
• Rudrabhatla, R. S. et al. (2003) Modulation of Myosin Light-Chain Phosphorylation by p21-Activated Kinase 1 in Escherichia coli Invasion of Human Brain Microvascular Endothelial Cells. Infection and Immunity 71 (5), 2787-2797. Applications: Western Blotting
• Weber, D. S. et al. (2004) Phosphoinositide-Dependent Kinase 1 and p21-Activated Protein Kinase Mediate Reactive Oxygen Species-Dependent Regulation of Platelet-Derived Growth Factor-Induced Smooth Muscle Cell Migration. Circulation Research 94, 1219-1226. Applications: Western Blotting
• Harfouche, R. et al. (2005) FASEB J 19, 1728-30. Applications: Western Blotting
• Pandey, D. et al. (2009) Blood 114, 415-24. Applications: Western Blotting
• Siu, M.K. et al. (2010) Int J Cancer 127, 21-31. Applications: Western Blotting

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For Research Use Only. Not For Use In Diagnostic Procedures.