Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (≥10(5)) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.     

Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase alpha (PI3Kalpha ). Functions of lipid kinase-deficient PI3Kalpha in signaling

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.     

ARAP3 is a PI3K- and rap-regulated GAP for RhoA

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.     

Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells

Recent studies have demonstrated that PH domains specific for PI(3,4,5)P3 accumulate at the leading edge of a number of migrating cells and that PI3Ks and PTEN associate with the membrane at the front and back, respectively, of chemotaxing Dictyostelium discoideum cells. However, the dependence of chemoattractant induced changes in PI(3,4,5)P3 on PI3K and PTEN activities have not been defined. We find that bulk PI(3,4,5)P3 levels increase transiently upon chemoattractant stimulation, and the changes are greater and more prolonged in pten- cells. PI3K activation increases within 5 s of chemoattractant addition and then declines to a low level of activity identically in wild-type and pten- cells. Reconstitution of the PI3K activation profile can be achieved by mixing membranes from stimulated pi3k1-/pi3k2- cells with cytosolic PI3Ks from unstimulated cells. These studies show that significant control of chemotaxis occurs upstream of the PI3Ks and that regulation of the PI3Ks and PTEN cooperate to shape the temporal and spatial localization of PI(3,4,5)P3.     

Phosphoinositide 3-kinase in disease: timing, location, and scaffolding

When PI3Ks are deregulated by aberrant surface receptors or modulators, accumulation of PtdIns(3,4,5)P3 leads to increased cell growth, proliferation and contact-independent survival. The PI3K/PKB/TOR axis controls protein synthesis and growth, while PtdIns(3,4,5)P3-mediated activation of Rho GTPases directs cell motility. PI3K activity has been linked to the formation of tumors, metastasis, chronic inflammation, allergy and cardiovascular disease. Although increased PtdIns(3,4,5)P3 is a well-established cause of disease, it is seldom known which PI3K isoform is implied. Recent work has demonstrated that PI3Kgamma contributes to the control of cAMP levels in the cardiac system, where the protein acts as a scaffold, but not as a lipid kinase.     

Emerging roles of phosphatidylinositol 3-monophosphate as a dynamic lipid second messenger

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration and survival. Disregulation of PI3K-activated pathways is implicated in different disease including diabetes and cancer. Among the different products of PI3Ks, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) has a well established role in signal transduction whereas the monophosphate phosphatidylinositol-3-phosphate (PtdIns-3-P) has been considered for a long time just a cellular component confined in endosomal structures. Only recently several evidence have indicated that PtdIns-3-P can also act as a dynamic intracellular second messenger. The role of PtdIns-3-P as mediator of crucial intracellular signals is therefore just beginning to be appreciated. Here we review some of the latest evidence showing that pools of PtdIns-3-P can be generated upon cellular stimulation in compartments different from the "classical" endosomal region. We describe several proteins that can be targets in mediating signals deriving from such stimulated PtdIns-3-P pools. In addition we describe the potential mechanism of switching on and off such signals. Taken together all this evidence suggest a novel, key role for PtdIns-3-P in signal transduction.     

Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes

Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of "comet tails" that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P(2) and PI(3,4,5)P(3) on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P(2) and PI(3,4,5)P(3) and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P(2) and PI(3,4,5)P(3) formation and signaling in endomembranes.     

Regulation of phosphoinositide 3-kinase signaling by oxidants: hydrogen peroxide selectively enhances immunoreceptor-induced recruitment of phosphatidylinositol (3,4) bisphosphate-binding PH domain proteins

Phosphoinositide 3-kinases (PI3Ks) generate several distinct lipid second messengers including phosphatidylinositol (3,4,5) trisphosphate (PIP3) and phosphatidylinositol (3,4) bisphosphate PI(3,4)P2. PI(3,4)P2 is produced with distinct kinetics and binds to distinct PH domain effector proteins; however, the regulation of this signaling pathway is poorly understood. Superoxides such as hydrogen peroxide are transiently produced after activation through various cell surface receptors and play important roles in immune and inflammatory responses. Here we use quantitative microscopy to examine the effect of peroxide on PI(3,4)P2-mediated mobilization of signaling proteins in B lymphocytes. Peroxide was found to induce dose-dependant membrane recruitment of the PI(3,4)P2-binding PH domain proteins Bam32, TAPP2 and Akt/PKB but not the PIP3-binding PH domain of Btk. Peroxide-induced membrane recruitment was found to be dependant on PI3K activity, with the p110delta isoform contributing much of the activity in the BJAB human B lymphoma model. Strikingly, peroxide co-stimulation enhanced antigen receptor-induced membrane recruitment of Bam32 and TAPP2, with combined stimulation exceeding the maximum achievable with either stimulus alone. Expression of the lipid phosphatase PTEN led to reduction of antigen receptor-induced membrane recruitment of TAPP2; however, peroxide costimulation could overcome the inhibitory effect of PTEN. Inhibition of the NADPH oxidase led to reduction of antigen receptor-induced membrane recruitment of TAPP2. Our results indicate that exogenous and endogenous superoxides can modulate the quality of the PI3K signal in lymphocytes by selectively increasing PI(3,4)P2-dependant signaling.     

PLA2 and PI3K/PTEN pathways act in parallel to mediate chemotaxis

Directed cell migration involves signaling events that lead to local accumulation of PI(3,4,5)P(3), but additional pathways act in parallel. A genetic screen in Dictyostelium discoideum to identify redundant pathways revealed a gene with homology to patatin-like phospholipase A(2). Loss of this gene did not alter PI(3,4,5)P(3) regulation, but chemotaxis became sensitive to reductions in PI3K activity. Likewise, cells deficient in PI3K activity were more sensitive to inhibition of PLA(2) activity. Deletion of the PLA(2) homolog and two PI3Ks caused a strong defect in chemotaxis and a reduction in receptor-mediated actin polymerization. In wild-type cells, chemoattractants stimulated a rapid burst in an arachidonic acid derivative. This response was absent in cells lacking the PLA(2) homolog, and exogenous arachidonic acid reduced their dependence on PI3K signaling. We propose that PLA(2) and PI3K signaling act in concert to mediate chemotaxis, and metabolites of PLA(2) may be important mediators of the response.     

Uncovering the PI3Ksome: Phosphoinositide 3-Kinases and Counteracting PTEN Form a Signaling Complex with Intrinsic Regulatory Properties

Production of the phosphoinositide lipid phosphatidylinositol (3,4,5)trisphosphate [PI(3,4,5)P₃, or PIP₃] by class I phosphoinositide 3-kinases (PI3Ks) is a major signaling mechanism whose deregulation contributes to serious diseases, including cancer. New findings suggest that tyrosine kinase receptor engagement results in the assembly of hetero-oligomeric PI3K complexes in which PI3Kα first activates PI3Kβ, and PI3K catalytic activity then promotes recruitment and activation of the PIP₃-removing tumor suppressor PTEN. Thus, PIP₃ production is fine-tuned through formation of an intrinsically regulated “PI3Ksome.”     

Class IA phosphatidylinositide 3-kinases, rather than p110 gamma, regulate formyl-methionyl-leucyl-phenylalanine-stimulated chemotaxis and superoxide production in differentiated neutrophil-like PLB-985 cells

Class I PI3Ks, through the formation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P(3)), are thought of as essential elements of the neutrophil response to chemotactic factors. Moreover, the recent development of PI3K-deficient mice and isoform-specific inhibitors enabled examinations of the contribution of the distinct PI3K isoforms in neutrophil activation. However, the results of these various studies are conflicting, and the exact role of the different PI3K isoforms is not yet clearly established, particularly in human cells. In the present study, we used a different approach to assess the role of the distinct PI3K isoforms in response to the chemotactic agent fMLP. We inhibited PI3K activities by the transient expression following nucleofection of dominant negative mutants of either p85alpha or p110gamma in the human myeloid cell line PLB-985, which can be induced to express a neutrophil-like phenotype. The data obtained with this approach showed that the production of PI(3,4,5)P(3) triggered by fMLP is biphasic, with a peak of production observed in a short time period that entirely depends on p110gamma activity, and a delayed phase that is mediated by class I(A) PI3K. We also provide evidence that the PI3K-dependent functional responses (i.e., superoxide production and chemotaxis) induced by the chemotactic factor mainly involve PI3K I(A) and, by implication, the delayed phase of PI(3,4,5)P(3) production, whereas p110gamma and the early peak of PI(3,4,5)P(3) do not play major roles in the initiation or the control of these responses.     

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.     

Gbetagammas and the Ras binding domain of p110gamma are both important regulators of PI(3)Kgamma signalling in neutrophils

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gbetagammas) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gbetagammas, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kgamma, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P(3), activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kgamma to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gbetagammas, p101 and the Ras-RBD interaction all have important roles in the regulation of PI(3)Kgamma in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kgamma effectors.     

SYK is upstream of phosphoinositide 3-kinase in B cell receptor signaling.

We have recently demonstrated that the D3-phosphoinositide phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) is critical for producing sustained calcium signals through its role in promoting the function of TEC family tyrosine kinases such as Bruton's tyrosine kinase. Although PtdIns-3,4,5-P(3) can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns-3,4,5-P(3) production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19. However, calcium signaling and other PI3K-dependent signals are relatively preserved in a LYN kinase-deficient B lymphocyte cell line, suggesting that an alternative pathway for PI3K activation exists. As SYK/ZAP70 kinases are upstream from many BCR-initiated signaling events, we directly analyzed SYK-dependent accumulation of both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) in B cell receptor signaling using both dominant negative and genetic knockout approaches. Both methods indicate that SYK is upstream of, and necessary for, a significant portion of BCR-induced PtdIns-3,4, 5-P(3) production. Whereas CD19 does not appear to be involved in this SYK-dependent pathway, the SYK substrate CBL is likely involved as the dominant negative SYK markedly attenuates CBL tyrosine phosphorylation and completely blocks the BCR-dependent association of CBL with p85 PI3K.     

Nuclear phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-kinase, Akt, and PTen: emerging key regulators of anti-apoptotic signaling and carcinogenesis

Inositol lipid-derived second messengers have long been known to have an important regulatory role in cell physiology. Phosphatidylinositol 3-kinase (PI3K) synthesizes the second messenger 3,4,5'-phosphatidylinositol trisphosphate (Ptdlns 3,4,5P3) which controls a multitude of cell functions. Down-stream of PI3K/PtdIns 3,4,5P3 is the serine/threonine protein kinase Akt (protein kinase B, PKB). Since the PI3K/ PtdIns 3,4,5P3 /Akt pathway stimulates cell proliferation and suppresses apoptosis, it has been implicated in carcinogenesis. The lipid phosphatase PTEN is a negative regulator of this signaling network. Until recently, it was thought that this signal transduction cascade would promote its anti-apoptotic effects when activated in the cytoplasm. Several lines of evidence gathered over the past 20 years, have highlighted the existence of an autonomous nuclear inositol lipid cycle, strongly suggesting that lipids are important components of signaling pathways operating at the nuclear level. PI3K, PtdIns(3,4,5)P3, Akt, and PTEN have been identified within the nucleus and recent findings suggest that they are involved in cell survival also by operating in this organelle, through a block of caspase-activated DNase and inhibition of chromatin condensation. Here, we shall summarize the most updated and intriguing findings about nuclear PI3K/ PtdIns(3,4,5)P3/Akt/PTEN in relationship with carcinogenesis and suppression of apoptosis.     

Phosphoinositides and phagocytosis

Phosphoinositide 3 kinases (PI3Ks)*Abbreviation used in this paper: PI3K, phosphoinositide 3 kinase. are known as regulators of phagocytosis. Recent results demonstrate that class I and III PI3Ks act consecutively in phagosome formation and maturation, and that their respective products, phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5]P(3)) and phosphatidylinositol 3-phosphate (PI[3]P), accumulate transiently at different stages. Phagosomes containing Mycobacterium tuberculosis do not acquire the PI(3)P-binding protein EEA1, which is required for phagosome maturation. This suggests a possible mechanism of how this microorganism evades degradation in phagolysosomes.     

Phosphoinositide 3-kinase-dependent activation of Rac

The monomeric GTPase Rac and the lipid kinase phosphoinositide 3-kinase (PI3K) are intracellular signalling enzymes that each regulate a huge range of cellular functions. Their signalling pathways overlap. Several pathways lead from PI3K activation via the production of the lipid second messenger phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P(3)) to the activation of guanine-nucleotide exchange factors (GEFs) that activate Rac. Vice versa, Rac can also stimulate the activation of PI3K, although the mechanism for this is unclear. We review here the evidence that links PI3K and Rac signalling pathways.     

Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells

3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelárová, H., and Meijer, A. J. (1997) Eur. J. Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3, 4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4, 5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC(50) 5-15 nM). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Galpha(i3) protein suggesting that both class III PI3K and trimeric G(i3) protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling.     

Intranuclear 3′-phosphoinositide metabolism and Akt signaling: New mechanisms for tumorigenesis and protection against apoptosis?

Lipid second messengers, particularly those derived from the polyphosphoinositide metabolism, play a pivotal role in multiple cell signaling networks. Phosphoinositide 3-kinase (PI3K) generate 3'-phosphorylated inositol lipids that are key players in a multitude of cell functions. One of the best characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt (protein kinase B, PKB). Recent findings have implicated the PI3K/Akt pathway in tumorigenesis because it stimulates cell proliferation and suppresses apoptosis. However, it was thought that this signal transduction network would exert its carcinogenetic effects mainly by operating in the cytoplasm. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid cycle, and strongly suggests that lipid molecules are important components of signaling pathways operating at the nuclear level. PI3K, its lipid product phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3), and Akt have been identified within the nucleus and recent data suggest that they counteract apoptosis also by operating in this cell compartment through a block of caspase-activated DNase and inhibition of chromatin condensation. In this review, we shall summarize the most updated and intriguing findings about nuclear PI3K/PtdIns(3,4,5)P3/Akt in relationship with tumorigenesis and suppression of apoptotic stimuli.     

PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.