RAC INSERT REGION IS A NOVEL EFFECTOR REGION THAT IS IMPLICATED IN THE ACTIVATION OF NADPH OXIDASE, BUT NOT PAK65

The small GTPase Rac assembles with the cytosolic p47(phox) and p67(ph ox) and the membrane-associated flavocytochrome b(558) to form the mul ticomponent respiratory burst oxidase. Mutation of amino acids in a re gion of Rac (residues 26-45), homologous to an effector region in Pas, was previously shown to interfere with Rac binding to the oxidase. He rein wc have elucidated an additional region in Rac involved in regula ting oxidase activity. Rho family small GTPases contain a 12-amino aci d ''insert'' region (residues 124-135) that is not present in Pas, Poi nt mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. App arent binding constants (based on EC(50) values) of the mutant Rac pro teins for the oxidase are at least 13-25-fold higher than for wild-typ e Rac, Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different role s for these two protein regions: mutations in the insert region but no t the 26-45 effector region resulted in an increase in the EC(50) for p67(phox). Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidas e, insert region mutations did not affect activation of PAK. We propos e that the combinatorial use of the 26-45 effector region and the inse rt region provides the Rho family GTPases with versatility in their sp ecificity for several downstream targets.