TI - The mechanism of PAK activation . Autophosphorylation events in both regulatory and kinase domains control activity . AB - The p21-activated kinases ( PAKs ) , in common with many kinases , undergo multiple autophosphorylation events upon interaction with appropriate activators . The Cdc42 -induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck . Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both alpha- and betaPAK . Both in vivo and in vitro analyses demonstrate that , although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation , a phosphorylation of alphaPAK serine 144 or betaPAK serine 139 , which lie in the kinase inhibitory domain , significantly contribute to activation . By contrast , sphingosine-mediated activation is independent of this residue , indicating a different mode of activation . Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck .