| Protein kinases, control so many aspects of cell growth, differentiation, and metabolism that their catalytic activities must be under tight control. Various modes of phosphorylation and inter- or intramolecular interactions regulate kinases in response to appropriate cellular stimuli. Members of the family of p21-activated kinases (PAKs), which are stimulated by binding with the GTP-liganded form of Cdc42 or Rac, modulate assembly of the actin cytoskeleton and activate MAP kinase pathways. The crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1, shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. Central to this mechanism is an inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1. The IS domain positions a kinase inhibitor segment across the cleft of the kinase; GTPase binding refolds, part of the IS domain and unfolds the rest. To understand the conformational change during the inactive-active state transition, crystal structure of PAK1 kinase domain as well as a T423E mutant was determined. These structures reveal a two-phase activation process. During the first stage, association with GTP-bound Cdc42/Rac disrupts the dimeric PAK1, partly melts the IS domain and folds it into a new domain (PBD) such that the KI segment is destabilized and extracted from the cleft between two lobes of the kinase domain. Then in the second stage, with GTP-Cdc42/Rac still bound with the PBD of PAK1, the activation loop is released from the autoinhibited conformation and forms the “pseudoactive” state. Phosphorylation at T423 and further autophosphorylation at many other sites in PAK1 makes the activation process irreversible in the absence of protein phosphatases, even GTP-cdc42 dissociates from PAK1. In summary the autoinhibited and active conformation of PAK1 confirmed former biochemical data about autoinhibition and activation, and revealed a novel autoinhibition mechanism. |
