| Abstract
| - Cdc42 and Rac are highly homologous members of the Rho family of small G proteins thatinteract with several downstream effector proteins thereby causing cytoskeletal rearrangements, cellproliferation, and differentiation. While some effectors, such as the tyrosine kinase, ACK, and the scaffoldprotein, WASP, are unique to Cdc42, others, such as the serine-threonine kinase, PAK, are shared withRac. Previous mutagenesis studies identified Val42 and Leu174 as residues that selectively affect bindingof Cdc42 to ACK and WASP but not to PAK. However, it is unclear whether these discriminatory residuesare sufficient determinants of specificity. In this study we sought to introduce “gain-of function” mutationsinto Rac to allow it to bind to ACK and WASP, thereby revealing all specificity determinants. Thirteenmutations were made changing Rac residues to those in Cdc42. Equilibrium binding constants of allmutant Rac proteins to ACK, WASP, and PAK were measured. A combination of seven mutations (S41A,A42V, N43T, D47G, N52T, W56F, and R174L) was determined to be necessary to change the bindingaffinity of Rac for ACK from negligible (Kd< 1 μM) to a comparable affinity to Cdc42 (Kd 25 nM).These mutations are not confined to interface residues. We interpret these data to indicate the importanceof the structure of regions of the protein distinct from the contact residues. None of these mutant Racproteins bound WASP with a similar affinity to Cdc42. Hence, residues as yet unidentified, outside theinterface, must be necessary for binding WASP.
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