STRUCTURAL REQUIREMENTS FOR PAK ACTIVATION BY RAC GTPASES

The Rho family GTPases, Rad and Rac2, regulate a variety of cellular f unctions including cytoskeletal reorganization, the generation of reac tive oxygen species, G(1) cell cycle progression and, in concert with Ras, oncogenic transformation, Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase pal-activated k inase (PAK) is a prime candidate for mediating some of Rac's cellular effects, This report shows that Rad binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structura l elements on Rac and PAK that are important for optimal binding and a ctivation. The most notable difference between the highly homologous R ac isomers is the composition of their C-terminal polybasic domains, M utation of these six basic residues in Rac1 to neutral amino acids dra matically decreased the ability of Rad to bind PAK1 and almost complet ely abolished its ability to stimulate PAK activity. Moreover, replaci ng the highly charged polybasic domain of Rad with the less charged do main of Rac2 (and vice versa) completely reversed the PAK binding/acti vation properties of the two Rac isomers, Thus, polybasic domain diffe rences account for the disparate abilities of Rac1 and Rac2 to activat e PAK, PAH proteins also contain a basic region, consisting of three c ontiguous lysine residues (Lys(66)-Lys(67)-Lys(68)), which lies outsid e of the previously identified Cdc42/Rac-binding domain. Mutation of t hese Lys residues to neutral residues decreased PAK binding to activat ed Rad and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rad, Rac2, and Cdc42 proteins in vivo, In contrast, mutation of lysin es 66-68 to basic Arg residues did not decrease (and in some cases enh anced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effec tor domain that may allow the two Rac isomers to activate different ef fector proteins. In addition, our results indicate that a basic region in PAK is required for PAR activation and that binding of Rac/Cdc42 t o PAK is not sufficient for kinase activation.