αPix stimulates p21-activated kinase activity through exchange factor-dependent and -independent mechanisms

Activation of pal-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 alpha Pix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alpha Pix(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 alpha Pix. The activation of wild-type Pak1 by alpha Pix(155-545) also requires that alpha Pix(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 alpha Pix(155-545) and by a mutant of alpha Pix(155-545) that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTP gamma S-loaded Cdc42 was also enhanced by recombinant alpha Pix(155-545) in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alpha Pix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.