USE OF A FLUORESCENCE SPECTROSCOPIC READOUT TO CHARACTERIZE THE INTERACTIONS OF CDC42HS WITH ITS TARGET EFFECTOR, MPAK-3/

The family of p21-activated kinases (PAKs) has been shown to contain a domain that can independently bind to the Ras-like proteins Cdc42Hs a nd Pac. We have expressed a 72 amino acid recombinant form of this p21 -binding domain (PBD) from mPAK-3 in Eschreichia Coli for use in struc ture-function studies. The protein can be purified on a nickel affinit y resin due to a hexa-His tag that is incorporated onto the amino term inus of the domain. PBD binds to Cdc42Hs in a guanine nucleoti-dedepen dent manner as demonstrated by a novel fluorescence assay that takes a dvantage of the spectroscopic properties of N-methylanthraniloyl (Mant )-guanine nucleotides. Ionic strength has little effect on the affinit y of PBD for Cdc42Hs, but alkaline pH values tend to weaken the intera ction. We have shown that the inhibition of the GTPase activity of Cdc 42Hs, as well as a previously undescribed inhibition of guanine nucleo tide dissociation, is mediated by the PBD portion of the mPAK-3 molecu le. These findings suggest that PBD binding alters the geometry of the guanine nucleotide binding site on Cdc42Hs, perhaps as an outcome of the target/effector molecule binding in close proximity to the nucleot ide domain. We therefore tested if mutations in the effector region of Cdc42Hs (32-40), which in Ras are very close to the guanine nucleotid e binding site, had any effect on PBD binding. Changing tyrosine 32 to lysine (Y32K) resulted in a small (5-fold) inhibition of PBD binding, but the very conservative mutation D38E yielded at least a 50-fold de crease in affinity. Finally, the catalytic domain of the GTPase activa ting protein, Cdc42-GAP, was shown to inhibit PBD binding in a competi tive manner, indicating that this target molecule and the negative reg ulator (GAP) bind to overlapping sites on the Cdc42Hs molecule.