Role of phosphatidylinositol 3 ' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in Dictyostelium

We show that cells lacking two Dictyostelium class I phosphatidylinositol ( PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated wit h the PI3K inhibitor LY294002 are unable to properly polarize, are very def ective in the temporal, spatial, and quantitative regulation of chemoattrac tant-mediated filamentous (F)-actin polymerization, and chemotax very slowl y. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate that in response to chemo attractants three PH domain-containing proteins do not localize to the lead ing edge in pi3k1/2-null cells, and the translocation is blocked in wild-ty pe cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerzation at the le ading edge and have chemotaxis pheno-types that are distinct from those des cribed previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor prot ein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge du ring pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.