PAK-PBD protein agarose beads (PAK02)

Material
The Rac/Cdc42 (p21) binding domain (PBD) of the human p21 activated kinase 1 protein (PAK) protein has been expressed as a GST-fusion protein in E. coli. This protein binds binds specifically to GTP-bound, and not GDP-bound, Rac and Cdc42 proteins. The domain can therefore be used to specifically precipitate active, GTP-bound Rac and Cdc42 as well as to specifically block the activity of Rac and Cdc42 in vitro and in vivo.

The GST-PAK-PBD contains residues 67-150 of PAK. This region includes the highly conserved CRIB motif plus sequences required for the high affinity interaction with GTP-Rac and GTP-Cdc42.

The protein is supplied in a glutathione agarose bound format and is shipped lyophilized. The beads are colored for ease of use. This product is used in our Cdc42 and Rac activation assay Biochem Kits™ (Cat. # BK034 and BK035, repectively). The GST-PAK-PBD is also available as a free protein (Cat. # PAK01).

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-PAK-PBD protein is >80% pure (see Figure 2).

Biological Activity
PAK-PBD protein specifically recognizes and binds the active, GTP-bound, forms of the Rac and Cdc42 proteins. It has a much lower affinity for the inactive, GDP-bound, forms of Rac and Cdc42. When coupled to a colored glutathione sepharose matrix, the PAK-PBD protein beads become a convienent tool for assaying the activity of the Rac and Cdc42 proteins. The quality control biological assay for PAK-PBD protein beads consists of a Rac protein pulldown from human platelet extracts loaded with either GTPγS (Cat. # BS01) or GDP.

Examples of publications where this product was used:
Autieri, M. V., Kelemen, S. E. and Wendt, K. W. (2003). AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration. Circ. Res. 92, 1107-1114.

Bi, Y. and Williams, J. A. (2005). A role for Rho and Rac in secretagogue induced amylase release by pancreatic acini. Am. J. Physiol. 289, C22-33.

Borm, B., Requardt, R. P., Herzog, V. and Kirfel, G. (2005). Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization. Exp. Cell Res. 302, 83-95.

Chiang, S. H., Hwang, J., Legendre, M., Zhang, M., Kimura, A. and Saltiel, A. R. (2003). TCGAP, a multidomain Rho GTPase-activating protein involved in insulin-stimulated glucose transport. EMBO J. 22, 2679-2691.

Saito, S., Liu, X. F., Kamijo, K., Raziuddin, R., Tatsumoto, T., Okamoto, I., Chen, X., Lee, C. C., Lorenzi, M. V., Ohara, N. et al. (2004). Deregulation and mislocalization of the cytokinesis regulator ECT2 activate the Rho signaling pathways leading to malignant transformation. J. Biol. Chem. 279, 7169-7179.

Wang, S. E., Wu, F. Y., Shin, I., Qu, S. and Arteaga, C. L. (2005). Transforming growth factor β (TGF-β)-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-β function. Mol. Cell. Biol. 25, 4703-4715.