p21-activated protein kinase γ-PAK is translocated and activated in response to hyperosmolarity -: Implication of Cdc42 and phosphoinositide 3-kinase in a two-step mechanism for γ-PAK activation

A member of the family of p21-activated protein kinases, gamma-PAK, has cytostatic properties and is activated during apoptosis and in response to DNA damage. To determine whether gamma-PAK is activated by other types of cell stress and to assess its mechanism of activation, the response of gamma-PAK to hyperosmotic stress was examined. In 3T3-L1 mouse fibroblasts, there are two pools of gamma-PAK: the majority of the protein kinase is soluble and has low specific activity, whereas gamma-PAK associated with the particulate fraction has significantly higher specific activity. Hyperosmolarity promotes translocation of gamma-PAK from the soluble to the particulate fraction; this parallels activation of the protein kinase. Activation but not translocation of gamma-PAK is wortmannin-sensitive, suggesting the involvement of a phosphoinositide 3-kinase-related activity. gamma-PAK translocation in response to hyperosmolarity parallels Cdc42 translocation to the particulate fraction in vivo and can be induced in vitro by guanosine 5'-3-O-(thio)triphosphate. Cotransfection of gamma-PAK with constitutively active Cdc42 induces gamma-PAK activation and translocation, whereas inactive Cdc42 inhibits both processes in response to hyperosmotic stress, suggesting that Cdc42 has a role in the translocation and activation of gamma-PAK. alpha-PAK is not activated in response to hyperosmolarity in 3T3-L1 cells. A two-step model of gamma-PAK activation is presented.