Joint work with Yoichiro Kamimura, Meghdad Rahdar, Jane Borleis, Yu Long, Sandra de Keijzer, Jonathan Franca-Koh, Kristen Franson, Michelle Tang, and Stacey S. Willard (Department of Cell Biology, Johns Hopkins University, Baltimore, MD, USA 21205). The mechanisms of sensing shallow gradients of extracellular signals is remarkably similar in Dictyostelium amoebae and mammalian leukocytes. An extensive series of studies have indicated that the upstream components and reactions in the signaling pathway are quite uniform while downstream responses such as PI (3,4,5)P3 accumulation and actin polymerization are sharply localized towards the high side of the gradient. Uniform stimuli transiently recruit and activate PI3Ks and cause PTEN to be released from the membrane while gradients of chemoattractant cause PI3Ks and PTEN to bind to the membrane at the front and the back of the cell, respectively. This reciprocal regulation provides robust control of PIP3 and leads to its sharp accumulation at the anterior. A similar PIP3-based "polarity circuit" plays a key role in cytokinesis where PI3Ks and PTEN move to and function at the poles and furrow, respectively, of the dividing cell. Disruption of PTEN broadens PI localization and actin polymerization in parallel, leading to vigorous extension of lateral pseudopodia; however, lowered levels of PIP3 do not greatly interfere with either chemotaxis or cytokinesis, suggesting that additional pathways act in parallel. A screen to identify redundant pathways revealed a gene with homology to patatin-like phospholipase A2. Loss of this gene did not alter PIP3 regulation, but chemotaxis became sensitive to reductions in PI3K activity. Likewise, cells deficient in PI3K activity were more sensitive to inhibition of PLA2 activity. Deletion of the PLA2 homologue and two PI3Ks caused a strong defect in chemotaxis and a reduction in receptor-mediated actin polymerization. We propose that PLA2 and PI3K signaling act in concert to mediate chemotaxis and arachidonic acid metabolites may be important mediators of the response. Evidence has suggested that PKB signaling plays a role in cell motility and that TorC2 can regulate the actin cytoskeleton. We have recently shown that activation of TorC2 and PKB occurs at the leading edge of chemotaxing cells and plays a critical role in directed cell migration. Within seconds of stimulation of chemotactically sensitive cells, two PKB homologs, PKBA and PKBR1, transiently phosphorylate at least seven proteins. The enzymes are activated by phosphorylation of their hydrophobic motifs (HMs) through TorC2 and subsequent phosphorylation of their activation loops (ALs). Activation of PKBR1, a myristoylated form persistently bound to the membrane, does not require PI(3,4,5)P3. Cells deficient in PKBR1 or TorC2, lack most of the phosphorylated substrates and are specifically impaired in directional sensing. Thus, temporal and spatial activation of PKB signaling by TorC2 is a critical event in directed cell migration that can act independently of localized PI(3,4,5)P3.