Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 an d Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link ext racellular stimuli to Pak1 activation. To separate activation of Pak1 by Cd c42 versus activation by Rae, we devised a genetic screen in yeast that ena bled us to create and identify Pak1 mutants that selectively couple to Cdc4 2 but not Rac1. We recovered several such Pak1 mutants and found that the r esidues most often affected lie within the p21 binding domain, a region pre viously known to mediate Pak1 binding to GTPases, but that several mutation s also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac 1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a G TPase other than Cdc42 to activate Pak1. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants t hat are unable to bind Rae are nonetheless well activated by calf serum, im plying that this stimulus may induce Pak activation independent of Rae.