alphaPix 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 alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 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 alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 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 alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
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Daniels
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Zenke
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Bokoch
D000818:Animals; D019556:COS Cells; D004789:Enzyme Activation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D020662:Guanine Nucleotide Exchange Factors; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D054462:p21-Activated Kinases
D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases