Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110. The expression of p110 siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110 siRNA induced CDK inhibitor p27^KIP1 levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110 siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110 and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27^KIP1 levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.     

Measles virus interacts with and alters signal transduction in T-cell lipid rafts

By a contact-dependent surface interaction, the measles virus (MV) glycoprotein complex induces a pronounced inhibition of T-cell proliferation. We now show that MV directly interacts with glycosphingolipid-enriched membrane microdomains on human primary T cells and alters recruitment and segregation of membrane proximal signaling components. Contact-dependent interference with T-cell receptor-stimulated tyrosine phosphorylation and Ca mobilization is a late event seen 24 h after MV treatment. In contrast, stimulated recruitment of pleckstrin homology domain-containing proteins such as Akt and Vav is inhibited early after MV contact, as is segregation of the activated Akt kinase from rafts. Tyrosine phosphorylation of the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), p85, is apparently normal then, yet this protein fails to partition to the lipid raft fraction, and this is associated with stable expression of its negative regulator Cbl-b. Thus, by interaction with lipid rafts, MV contact initially targets recruitment of PI3K by preventing stimulated Cbl-b degradation and activation of PI3K-dependent signaling components.     

Expression of the RAE-1 family of stimulatory NK-cell ligands requires activation of the PI3K pathway during viral infection and transformation

Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.     

Class IA phosphoinositide 3-kinase modulates basal lymphocyte motility in the lymph node

Recruitment of PI3K to the cell membrane is an indispensable step in normal lymphocyte proliferation and activation. In this study we identify PI3K as an important signaling molecule for maintaining basal T and B lymphocyte motility and homing in the intact lymph node. Pharmacological inhibition of PI3K catalytic isoforms exerted broad effects on basal lymphocyte motility, including changes in homing kinetics, localization of B cells within the lymph node, and reduced cell velocities. Lymphocytes deficient in either or both of the class IA PI3K regulatory subunits p85alpha and p85beta also exhibited reduced velocities, with the magnitude of reduction depending upon both cell type and isoform specificity. B cells deficient in p85alpha exhibited gross morphological abnormalities that were not evident in cells treated with a PI3K inhibitor. Our results show, for the first time, that class IA PI3Ks play an important role in regulating basal lymphocyte motility and that p85alpha regulatory subunit expression is required to maintain B cell morphology in a manner independent of PI3K catalytic function. Moreover, we demonstrate distinct roles for catalytic domain function and class IA PI3K regulatory domain activity in lymphocyte motility, homing, and homeostatic localization of mature resting B cells.     

Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P₃] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P₃ levels.     

Cross-talk between phosphatidylinositol 3-kinase and sphingomyelinase pathways as a mechanism for cell survival/death decisions

Peptide hormones act to regulate apoptosis through activation of multiple pro- and anti-apoptotic signaling cascades of which lipid signaling events represent an important facet of the cellular rheostat that determines survival and death decisions. Activation of sphingomyelinase, which generates ceramide, is an intermediate in cellular stress responses and induction of apoptosis in many systems. Conversely, phosphatidylinositol 3-kinase (PI3K) is a critical signaling molecule involved in regulating cell survival and proliferation pathways. In the present study, we investigate cross-talk between the PI3K and sphingomyelinase pathways as a mechanism for regulation of cell survival/death decisions. We show that phorbol ester, insulin-like growth factor 1, and a constitutively active PI3K suppress both tumor necrosis factor-induced apoptosis and ceramide generation. Conversely, inhibition of the PI3K pathway with expression of a kinase-dead PI3K both prevented survival signaling and enhanced tumor necrosis factor-induced ceramide generation. The ability of exogenous sphingomyelinase to induce ceramide generation was partially suppressed by expression of constitutively active PI3K and enhanced by inhibition of PI3K suggesting that cross-talk between PI3K and ceramide generation within cells is regulated subsequent to activation of sphingomyelinase.     

SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.     

Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT1/2 inhibit RELMbeta induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELMbeta, a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELMbeta expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxo1 nuclear localization, and robust induction of RELMbeta expression in wild-type, but not IL-4Ralpha knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4Ralpha.     

Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.     

Phosphatidylinositol-3 phosphatase myotubularin-related protein 6 negatively regulates CD4 T cells

Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell.     

Serum withdrawal-induced accumulation of phosphoinositide 3-kinase lipids in differentiating 3T3-L6 myoblasts: distinct roles for Ship2 and PTEN

Phosphoinositide 3-kinase (PI3K) activation and synthesis of phosphatidylinositol-3,4-bisphosphate (PI-3,4-P₂) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P₃) lipids mediate growth factor signaling that leads to cell proliferation, migration, and survival. PI3K-dependent activation of Akt is critical for myoblast differentiation induced by serum withdrawal, suggesting that in these cells PI3K signaling is activated in an unconventional manner. Here we investigate the mechanisms by which PI3K signaling and Akt are regulated during myogenesis. We report that PI-3,4-P₂ and PI-3,4,5-P₃ accumulated in the plasma membranes of serum-starved 3T3-L6 myoblasts due to de novo synthesis and increased lipid stability. Surprisingly, only newly synthesized lipids were capable of activating Akt. Knockdown of the lipid phosphatase PTEN moderately increased PI3K lipids but significantly increased Akt phosphorylation and promoted myoblast differentiation. Knockdown of the lipid phosphatase Ship2, on the other hand, dramatically increased the steady-state levels of PI-3,4,5-P₃ but did not affect Akt phosphorylation and increased apoptotic cell death. Together, these results reveal the existence of two distinct pools of PI3K lipids in differentiating 3T3-L6 myoblasts: a pool of nascent lipids that is mainly dephosphorylated by PTEN and is capable of activating Akt and promoting myoblast differentiation and a stable pool that is dephosphorylated by Ship2 and is unable to activate Akt.     

Physical and functional interactions between protein tyrosine phosphatase alpha, PI 3-kinase, and PKCdelta.

The somatostatin analogue, TT-232 inhibits cell proliferation and induces apoptosis in a variety of tumor cells both in vivo and in vitro. While the early transient activation of Erk/MAPK was found to be important for the induction of cell cycle arrest, the signaling pathway leading to the activation of Erk/MAPK had not been fully established. Here we present evidence that activation of the Erk/MAPK pathway by TT-232 involves PI 3-kinase, PKCdelta and the protein tyrosine phosphatase alpha (PTPalpha). We show a physical interaction of PI 3-kinase and PKCdelta with PTPalpha and show that the tyrosine phosphatase plays a role in the activation of MAPK. In this process, PTPalpha Ser-180 and Ser-204 phosphorylation is critical for the induction of phosphatase activity, which is required for dephosphorylation of pp60(c-src). Taken together, we demonstrate the physical and functional association between PI 3-kinase, PKCdelta and PTPalpha in a signaling complex that mediates the antitumor activity of the somatostatin analogue TT-232.     

IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway

IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN-gamma secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4(+) T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4(+) T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN-gamma induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is critical for mediating IL-12-induced CD4(+) T cell responses such as T cell proliferation and survival.     

ICOS ligation recruits the p50alpha PI3K regulatory subunit to the immunological synapse

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85alpha subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50alpha, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50alpha accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50alpha plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50alpha at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50alpha is known to carry a stronger lipid kinase activity compared with p85alpha. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50alpha is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.     

PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy

Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85alpha regulatory and a p110alpha catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In this study, we used the potent PI3K inhibitor wortmannin and small interfering RNA (siRNA) targeting the p85alpha and p110alpha subunits to determine whether total or selective PI3K inhibition would abrogate the proliferative response of the liver after partial hepatectomy in mice. Hepatic resection is associated with an induction in PI3K activity; total PI3K blockade with wortmannin and selective inhibition of p85alpha or p110alpha with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest time points. Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85alpha or p110alpha, as reflected by a paucity of F4/80-positive cells. Additionally, PI3K inhibition led to an aberrant architecture in the regenerating hepatocytes characterized by vacuolization, lipid deposition, and glycogen accumulation; these changes were not noted in the sham livers. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, most likely by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.     

The p110delta isoform of phosphoinositide 3-kinase controls clonal expansion and differentiation of Th cells

The role of PI3K in T cell activation and costimulation has been controversial. We previously reported that a kinase-inactivating mutation (D910A) in the p110delta isoform of PI3K results in normal T cell development, but impaired TCR-stimulated cell proliferation in vitro. This proliferative defect can be overcome by providing CD28 costimulation, which raises the question as to whether p110delta activity plays a role in T cell activation in vivo, which occurs primarily in the context of costimulation. In this study, we show that the PI3K signaling pathway in CD28-costimulated p110delta D910A/D910A T cells is impaired, but that ERK phosphorylation and NF-kappaB nuclear translocation are unaffected. Under in vitro conditions of physiological Ag presentation and costimulation, p110delta D910A/D910A T cells showed normal survival, but underwent fewer divisions. Differentiation along the Th1 and Th2 lineages was impaired in p110delta D910A/D910A T cells and could not be rescued by exogenous cytokines in vitro. Adoptive transfer and immunization experiments in mice revealed that clonal expansion and differentiation in response to Ag and physiological costimulation were also compromised. Thus, p110delta contributes significantly to Th cell expansion and differentiation in vitro and in vivo, also in the context of CD28 costimulation.     

PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% (P < 0.001; chi(2) analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.     

A Role for phosphoinositide 3-kinase in the control of cell division and survival during retinal development

Neurogenesis in the retina requires the concerted action of three different cellular processes: proliferation, differentiation, and apoptosis. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. p110alpha has been shown to regulate cell division and survival. Little is known of its function in development, however, as p110alpha knockout mice exhibit CNS defects, but death at early embryonic stages impairs further study. Here, we examine the role of PI3K in mouse retina development by expressing an activating form of PI3K regulatory subunit, p65(PI3K), as a transgene in the retina. Mice expressing p65(PI3K) showed severely disrupted retina morphogenesis, with ectopic cell masses in the neuroepithelium that evolved into infoldings of adult retinal cell layers. These changes correlated with an altered cell proliferation/cell death balance at early developmental stages. Nonetheless, the most affected cell layer in adult retina was that of photoreceptors, which correlated with selectively increased survival of these cells at developmental stages at which cell division has ceased. These results demonstrate the relevance of accurate PI3K regulation for normal retinal development, supporting class IA PI3K involvement in induction of cell division at early stages of neurogenesis. These data also show that, even after cell division decline, PI3K activation mediates survival of differentiated neurons in vivo.     

PI3K in lymphocyte development,differentiation and activation

Phosphoinositide 3-kinases (PI3Ks) regulate numerous biological processes, including cell growth, differentiation, survival, proliferation, migration and metabolism. In the immune system, impaired PI3K signalling leads to immunodeficiency, whereas unrestrained PI3K signalling contributes to autoimmunity and leukaemia. New insights into the role of PI3Ks in lymphocyte biology have been derived from gene-targeting studies, which have identified the PI3K subunits that are involved in B-cell and T-cell signalling. In particular, the catalytic subunit p110delta seems to be adapted to transmit antigen-receptor signalling in B and T cells. Additional recent work has provided new insights into the molecular interactions that lead to PI3K activation and the signalling pathways that are regulated by PI3K.