When presented with a gradient of chemoattractant, many eukaryotic cells respond with polarized accumulation of the phospholipid Ptdlns(3,4,5)P 3 . This lipid asymmetry is one of the earliest readouts of polarity in neutrophils, Dictyostelium discoideum and fibroblasts. However, the mechanisms that regulate PtdlnsP 3 polarization are not well understood. Using a cationic lipid shuttling system, we have delivered exogenous PtdlnsP 3 to neutrophils. Exogenous PtdlnsP 3

Using bone marrow derived mast cells from SH2-containing inositol-5-phosphatase (SHIP) ؉/؉ and ؊/؊ mice, we found that the loss of SHIP leads to a dramatic increase in Steel Factor (SF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ), a substantial reduction in PI(3,4)P 2 , and no change in PI(4,5)P 2 levels. We also found that SF-induced activation of protein kinase B (PKB) is increased and prolonged in SHIP؊/؊ cells, due in large part to more PKB associating with the plasma membrane in these cells. Pretreatment of SHIP؊/؊ cells with 25 M LY294002 resulted in complete inhibition of SFinduced PI(3,4)P 2 , while still yielding PI(3,4,5)P 3 levels similar to those achieved in SHIP؉/؉ cells. This offered a unique opportunity to study the regulation of PKB by PI(3,4,5)P 3 , in the absence of PI(3,4)P 2 . Under these conditions, PKB activity was markedly reduced compared with that in SF-stimulated SHIP؉/؉ cells, even though more PKB localized to the plasma membrane. Although phosphoinositide-dependent kinase 1 mediated phosphorylation of PKB at Thr-308 was unaffected by LY294002, phosphorylation at Ser-473 was dramatically reduced. Moreover, intracellular delivery of PI(3,4)P 2 to LY294002-pretreated, SF-stimulated SHIP؊/؊ cells increased phosphorylation of PKB at Ser-473 and increased PKB activity. These results are consistent with a model in which SHIP serves as a regulator of both activity and subcellular localization of PKB.The src homology 2 (SH2 1 )-containing inositol phosphatase (SHIP) is a 145-kDa hemopoietic-specific signaling protein (1-3) that becomes both tyrosine-phosphorylated and associated with the adapter protein Shc in response to many cytokines and to B and T cell receptor engagement (4). SHIP has been shown to inhibit immune receptor activation in both mast cells and B cells by binding to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibition motif of the inhibitory co-receptor Fc␥RIIB and inhibiting Fc⑀R1-and B cell receptor-induced calcium influx, respectively (5, 6). In addition, SHIP has been shown, even in the absence of Fc␥RIIB coclustering, to play a "gatekeeper" role in IgE-mediated mast cell degranulation by setting the threshold for and limiting the degranulation process (7,8).In 1996 when we and others first reported the cloning of SHIP (1-3), we demonstrated its ability, in vitro, to hydrolyze the 5Ј-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) but not from PI(4,5)P 2 . More recently, however, by modifying the in vitro assay conditions, SHIP was found capable of readily hydrolyzing PI(4,5)P 2 to PI(4)P (9, 10). To resolve its phospholipid substrate specificity and to gain some insight into the normal role that SHIP plays in vivo, we generated a SHIP knockout mouse by homologous recombination in embryonic stem cells (11). Although these mice are viable and fertile, they suffer from progressive splenomegaly, massive myeloid infiltration of the lungs, wasting, and a shortened lifespan (11,12). Interestingly, granulocyte/macroph...

Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P3 production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P3 production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P3 production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.

Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a ''knockin'' allele of the G protein-coupled receptor (GPCR)-activated PI3K␥, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K-and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.chemotaxis ͉ signal transduction ͉ inflammation

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P 3 ) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P 3 intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P 3 caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phosphoAkt and GLUT4. Intracellular delivery of PI(3,4,5)P 3 using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P 3 was not reproduced by carrier-PI 4,5-bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P 3 elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P 3 did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of exogenously delivered PI(3,4,5)P 3 to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport.