αPix Stimulates p21-activated Kinase Activity through Exchange Factor-dependent and -independent Mechanisms

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of αPix (amino acids 155–545), we observe stimulation of Pak1 kinase activity when αPix 155–545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind αPix. The activation of wild-type Pak1 by αPix 155–545 also requires that αPix 155–545 retain functional exchange factor activity. However, the Pak1 H83,86L mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by αPix 155–545 and by a mutant of αPix 155–545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPγS-loaded Cdc42 was also enhanced by recombinant αPix 155–545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with αPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.