Cdc42 Mutants Exhibit Differential Binding to PBD46 of mPAK Using Flourescence and Isothermal Titration Calorimetry

by Durchman, Jeremy E.; Coleman, Heath B.; Adams, Paul D.

Cdc42 is a member of the Ras superfamily of proteins, and one of its functions is to regulate signal transduction processes through interactions with a variety of downstream effectors. One such effector is p21-activated kinase (PAK), which interacts with flexible effector-binding regions of Cdc42 to regulate GTP hydrolysis. The mol. details of a complex of Cdc42 with a minimal GTPase-binding domain peptide deriv. of mPAK contg. 46 amino acids (PBD46) have been well characterized. We seek to investigate if there are differences in the binding of PBD46 to various Cdc42 mutant constructs relative to wild type. The mutations in these Cdc42 constructs are in regions of the protein thought to be important for normal signaling function. In-vitro binding assays were performed to qual. show if there are differences in binding of PBD46 to the Cdc42 mutants relative to wild type. Isothermal titrn. calorimetry and fluorescence spectroscopy were used to characterize the thermodn. of PBD46 binding to the mutant Cdc42 constructs for comparison to wild type. The data suggests that the mutant Cdc42 constructs display different binding affinities with PBD46 relative to wild type, which leads to the possibility that the mutations facilitate different interactions with effector mols. that may alter normal signal transduction processes regulated by this Ras protein.