The mechanism of PAK activation - Autophosphorylation events in both regulatory and kinase domains control activity

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc48-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PM 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 beta PAK, Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAH N-terminal regulatory domain play no direct role in activation, a phosphorylation of alpha PAK serine 144 or beta PAK 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 PM and Nck.