A network involving TORC2/PKB and PLA2/PI3K pathways regulates chemotaxis in Dictostelium discoideum

Phosphotidylinositol 3,4,5-triphosphate (PIP3) which is made from phosphotidylinositol 4,5-phosphate (PIP2) by PI3 kinases and degraded by PI3 phosphatase PTEN plays an important role in directed cell migration. To identify the critical targets of PIP3 that link it to the cytoskeleton, we deleted secondary genes to reverse the deficiencies of pten- cells in Dictyostelium . The polarity defects in pten- cells correlate with elevated phosphorylations of PKB substrates. Deletion of AKT orthologue, PkbA, or a subunit of its activator TORC2, reduced the phosphorylations and suppressed the cytokinesis and chemotaxis defects in pten- cells. In these double mutants, the excessive PIP3 levels and, presumably, activation of other PIP3-binding proteins had little or no effect on the cytoskeleton. In bands with increased phosphorylation in pten- cells, we found PKB substrates, PI5K, GefS, GacG, and PakA. Disruption of PakA in pten- cells restored a large fraction of the cells to normal behavior. Consistently, expression of phosphomimetic PakA in pten- cells exacerbated the defects but non-phosphorylatable PakA had no effect. Thus, among many putative PIP 3-dependent events, phosphorylation of PKB substrates is the key downstream regulator of cell polarity.;Patatin-like phospholipase A2 (PLA2) acts in parallel to PIP3 to mediate chemotaxis. To understand how these different pathways act coordinately to control chemotaxis, especially the downstream F-actin polymerization event, we inhibited both PIP3 dependent and PIP3 independent pathways by treating plaA-piaA- cells with PI3K inhibitor LY. Chemotaxis response is almost completely abolished under this condition, and only a minimum amount of F-actin polymerization is detected. But Ras proteins that are upstream of PLA2/PI3K and TORC2/PKB are still activated. Hence, by combining genetic and pharmacological approaches, we generated a most severe defect in chemotaxis other than receptor/G proteins. This may have useful implications on positive feedback loop and cancer drug development.