Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity

p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in viv o by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphin gosine, which stimulate autophosphorylation and phosphorylation of exogenou s substrates, The mechanism of Pak activation by these agents remains uncle ar. We investigated Pak kinase activation in more detail to gain insight in to the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation, We present biochemical evi dence that an autoinhibitory domain (ID) contained within amino acid residu es 67-150 of Pak1 interacts with the carboxyl-terminal kinase domain and th at this interaction is regulated in a GTPase-dependent fashion. Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited in trans by recombina nt ID peptide, indicating similarities in their mode of activation. However , Pak1, which was autophosphorylated in response to either GTPase or sphing osine, is highly active and is insensitive to inhibition by the ID peptide, We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and e nzymatic activity. Phosphorylation studies suggested that the stimulatory e ffect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation.