Fluorescence assays of Cdc42 interactions with target/effector proteins

The goal of these studies was to examine the interactions between the GTP-binding protein Cdc42 and its target/effectors by fluorescence spectroscopy. We have inserted fluorescent reporter groups at two distinct sites on Cdc42: N-methylanthraniloyl- (Mant-) derivatized nucleotides were complexed to the nucleotide-binding site of Cdc42, while a fluorescent succinimidyl ester was covalently attached to lysine 150. These two sites are separated by about 30 A on the Cdc42 molecule. Thus, the attachment of reporter groups to these sites enables the effects of target/effector binding to be viewed over a significant portion of the GTP-binding protein surface. We have taken advantage of fluorescence changes occurring at both sites to compare the interactions of activated Cdc42 with the limit binding domains from the following target/effectors: the serine/threonine kinase PAK, the tyrosine kinase ACK-2, and the RasGAP-related protein IQGAP. In addition, a unique lysine residue on the Cdc42-binding domain of ACK-2 (GBD-ACK) was covalently modified with a fluorescent succinimidyl ester. The distances separating this reactive lysine from the nucleotide binding site and lysine 150 of Cdc42 were determined by fluorescence resonance energy transfer and yielded a picture for Cdc42/GBD-ACK interactions that is consistent with recent NMR structural determinations for Cdc42/effector complexes. The changes in reporter group fluorescence at the reactive lysine of GBD-ACK, which were induced by the binding of activated Cdc42, were also examined. Overall, the results of these studies suggest not only that Cdc42 can induce conformational changes within an effector but also that in a reciprocal fashion the target/effectors induce conformational changes that span a significant distance on the GTP-binding protein.