Production and use of a cell permeable inhibitor of group A Paks (TAT-PID) to analyze signal transduction.
The Rho-family GTPases Cdc42 and Rac regulate a large number of important cellular processes, including motility, adhesion, proliferation, and survival. Among the key effectors for these GTPases are the p21-activated kinases. Although no specific chemical inhibitor has been developed against these enzymes, an inhibitory peptide derived from the N-terminus of these kinases is able to act in trans to suppress the activity of the full-length kinase. Here, we describe a method to deliver the inhibitory fragment into cells, using the recently described TAT system for protein transduction. This method is easy to use and is effective for transducing many different cell types, including those refractory to standard plasmid transfection. Use of the TAT-based inhibitor provides a specific means to suppress a single group of Cdc42 and Rac effectors, which is useful in analyzing their function.
Beeser
Alexander
A
Tumor Cell Biology Program, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, USA.
Chernoff
Jonathan
J
D000818:Animals; D019556:COS Cells; D002463:Cell Membrane Permeability / Q000235:genetics; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D015696:Gene Products, tat / Q000235:genetics*; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D014161:Transduction, Genetic; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D015696:Gene Products, tat; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C515903:PAK1 protein, human; C515907:PAK2 protein, human; C515911:PAK3 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein