Identification and Characterization of Small GTPase-Associated Kinases

Rho-family signal-transduction cascades, which appear to be conserved in all eukaryotes, probably coordinate cytoskeletal reorganization and transcriptional activation via a variety of protein and lipid kinases, and other as-yet-undefined target proteins. The dissection of these cascades has begun with the identification of a growing number of Rho-p21 interacting proteins, though for the most part it remains to be established which of these proteins represents bona fide targets. Here we describe a method that was developed to analyze p21-interacting proteins in total protein extracts after their separation on sodium dodecyl sulfate (SDS)-polyacrylamide gels. In fact, both guanosine 5′ triphosphatase (GTPase)-activating proteins and GTPase-inhibitory proteins can be detected in situ , and the latter class have been successfully identified in expression screens. Novel p21-targets have now been characterized by this method, including the Cdc42-binding tyrosine kinase (ACK) (1 ); the p21-activated kinase (PAK) (2 ,3 ); the p160 RhoA-associated kinase (ROK) (4 ,5 ); and the neutrophil Wiscott-Aldrich Syndrome protein (WASP) (6 ). Deletion analysis of a number of cDNAs encoding these proteins have shown the p2l-binding domains encompass less than 60 amino acid residues (1 ,2 ).