Characterization of Cdc42 and its mutant Cdc(T35A)

by Hargrave, Robynetta R.; Adams, Paul D.

Discovering the role of oncogenic ras proteins can give understanding to the pathogenesis of human cancer. Ras proteins control cycling between guanosine triphosphate (GTP) and guanosine diphosphate (GDP), the states in which the protein is in its active and inactive states, resp. The protein cell division cycle protein Cdc42, a member of the Rho subfamily of Ras proteins, is involved in cell morphogenesis through a Ras GTPase cycle to activate basic cellular functions. The protein has been shown to stimulate DNA synthesis and initiate a protein cascade that begins with the activation of the p21-activated serine/theorine kinases (PAK). Cdc42Hs has been shown to interact with a minimal GTPase-binding domain peptide of p21-activated kinase, PBD46. This goal of this work was to express, purify, and label a mutant protein, Cdc(T35A), with a site-specific fluorophore, sNDB, in order to examine the mutant protein's ability to bind PBD46 relative to wild type.