Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases.     

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.     

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.     

G Protein beta gamma subunits act on the catalytic domain to stimulate Bruton's agammaglobulinemia tyrosine kinase.

G proteins are critical cellular signal transducers for a variety of cell surface receptors. Both alpha and betagamma subunits of G proteins are able to transduce receptor signals. Several direct effect molecules for Gbetagamma subunits have been reported; yet the biochemical mechanism by which Gbetagamma executes its modulatory role is not well understood. We have shown that Gbetagamma could directly increase the kinase activity of Bruton's tyrosine kinase (Btk) whose defects are responsible for X chromosome-linked agammaglobulinemia in patients. The well characterized interaction of Gbetagamma with the PH (pleckstrin homology)/TH (Tec-homology) module of Btk was proposed to be the underlying activation mechanism. Here we show that Gbetagamma also interacts with the catalytic domain of Btk leading to increased kinase activity. Furthermore, we showed that the PH/TH module is required for Gbetagamma-induced membrane translocation of Btk. The membrane anchorage is also dependent on the interaction of Btk with phosphatidylinositol 3,4,5-trisphosphate, the product of phosphoinositide 3-kinase. These data support a dual role for Gbetagamma in the activation of Btk signaling function, namely membrane translocation and direct regulation of Btk catalytic activity.     

PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.     

Different subcellular localization and phosphoinositides binding of insulin receptor substrate protein pleckstrin homology domains

Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.     

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.     

PTEN: life as a tumor suppressor

PTEN, a tumor suppressor located at chromosome 10q23, is mutated in a variety of sporadic cancers and in two autosomal dominant hamartoma syndromes. PTEN is a phosphatase which dephosphorylates phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3), an important intracellular second messenger, lowering its level within the cell. By dephosphorylating PtdIns-3,4,5-P3, PTEN acts in opposition to phosphatidylinositol 3-kinase (PI3K), which has a pivotal role in the creation of PtdIns-3,4,5-P3. PtdIns-3,4,5-P3 is necessary for the activation of Akt, a serine/threonine kinase involved in cell growth and survival. By blocking the activation of Akt, PTEN regulates cellular processes such as cell cycling, translation, and apoptosis. In this review, we will discuss the identification of PTEN, its mutational status in cancer, its role as a regulator of PI3K, and its domain structure.     

Interaction of Btk and Akt in B cell signaling

Reactive oxygen species (ROS) or reactive oxygen intermediates (ROIs) mediate complex signaling involving multiple pathways. In this report, we demonstrate for the first time that endogenous Bruton's tyrosine kinase (Btk) and Akt can interact with each other in DT40 chicken B cells and human Nalm6 B cells and that this interaction is inducible following H2O2 stimulation. This interaction is supported by visualizing the co-localization of Btk and Akt in the perinuclear region and membrane ruffles in COS-7 cells. We have also shown the involvement of phosphatidylinositol 3-kinase (PI 3-K) and Btk in the phosphorylation of Akt following stimulation by hydrogen peroxide (H2O2). Interestingly, Akt phosphorylation was found in the presence of Btk even in the absence of oxidative stress. In addition, we have investigated the involvement of PI 3-K in the MAPKs and ERK and JNK phosphorylation, in the presence or absence of Btk. Phosphorylation of both ERK and JNK increased when the PI 3-K pathway was inhibited and both pathways were modulated positively by Btk. Taken together, based on the study of endogenous conditions, we show the novel interaction of Btk and Akt in H2O2 signaling in B cells.     

The intermolecular interaction between the PH domain and the C-terminal domain of Arabidopsis dynamin-like 6 determines lipid binding specificity

Dynamin and its related proteins are a group of mechanochemical proteins involved in the modulation of lipid membranes in various biological processes. Here we investigate the nature of membrane binding of the Arabidopsis dynamin-like 6 (ADL6) involved in vesicle trafficking from the trans-Golgi network to the central vacuole. Fractionation experiments by continuous sucrose gradients and gel filtration revealed that the majority of ADL6 is associated with membranes in vivo. Amino acid sequence analysis revealed that ADL6 has a putative pleckstrin homology (PH) domain. In vitro lipid binding assays demonstrated that ADL6 showed high affinity binding to phosphatidylinositol 3-phosphate (PtdIns-3-P) and that the PH domain was responsible for this interaction. However, the PH domain alone binds equally well to both PtdIns-3-P and phosphatidylinositol 4-phosphate (PtdIns-4-P). Interestingly, the high affinity binding of the PH domain to PtdIns-3-P was restored by a protein-protein interaction between the PH domain and the C-terminal region. In addition, deletion of the inserted regions within the PH domain results in high affinity binding of the PH domain to PtdIns-3-P. These results suggest that ADL6 binds specifically to PtdIns-3-P and that the lipid binding specificity is determined by the interaction between the PH domain and the C-terminal domain of ADL6.     

Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton's tyrosine kinase

Bruton's tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD19 mutant in which tyrosine residues previously shown to mediate CD19 interaction with PI3-K, Y484 and Y515, were changed to phenylalanine. Taken together, the data presented indicate that BCR aggregation-driven CD19 phosphorylation functions to promote Btk activation via recruitment and activation of PI3-K. Resultant phosphatidylinositol 3,4,5-trisphosphate probably functions to localize Btk for subsequent phosphorylation and activation by Src and Syk family kinases.     

Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells.     

PTEN expression causes feedback upregulation of insulin receptor substrate 2

PTEN is a tumor suppressor that antagonizes phosphatidylinositol-3 kinase (PI3K) by dephosphorylating the D3 position of phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3). Given the importance of PTEN in regulating PtdIns-3,4,5-P3 levels, we used Affymetrix GeneChip arrays to identify genes regulated by PTEN. PTEN expression rapidly reduced the activity of Akt, which was followed by a G(1) arrest and eventually apoptosis. The gene encoding insulin receptor substrate 2 (IRS-2), a mediator of insulin signaling, was found to be the most induced gene at all time points. A PI3K-specific inhibitor, LY294002, also upregulated IRS-2, providing evidence that it was the suppression of the PI3K pathway that was responsible for the message upregulation. In addition, PTEN, LY294002, and rapamycin, an inhibitor of mammalian target of rapamycin, caused a reduction in the molecular weight of IRS-2 and an increase in the association of IRS-2 with PI3K. Apparently, PTEN inhibits a negative regulator of IRS-2 to upregulate the IRS-2-PI3K interaction. These studies suggest that PtdIns-3,4,5-P3 levels regulate the specific activity and amount of IRS-2 available for insulin signaling.     

Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate

BACKGROUND: The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. RESULTS: We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P4). The inositol Ins (1,3,4,5)P4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-delta. A second Ins (1,3,4,5)P4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P4 to the wild type (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. CONCLUSIONS: Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.     

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec

IL-2 inducible T-cell kinase (Itk) is a Tec family non-receptor tyrosine kinase involved in signaling downstream of the T-cell receptor. Itk contains an amino-terminal Pleckstrin Homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate, recruiting Itk to the plasma membrane upon T-cell receptor activation. In addition to phosphoinositide binding, accumulating data suggest that the Itk PH domain likely mediates additional interactions outside of the phosphoinositide ligand binding pocket. The structural basis for additional PH domain functions remains elusive because of the poor recombinant expression and in vitro solution behavior of the Itk PH domain. Here, we determine that the lone α-helix in the Itk PH domain is responsible for the poor solution properties and that mutation of just two residues in the Itk α-helix to the corresponding amino acids in Btk or Tec dramatically improves the soluble recombinant expression and solution behavior of the Itk PH domain. We present this double mutant as a valuable tool to characterize the structure and function of the Itk PH domain. It is also interesting to note that the precise sites of mutation identified in this study appear as somatic mutations associated with cancerous tissue. Collectively, the findings suggest that the two helical residues in the Itk PH domain may serve an important and unique structural role in wild-type Itk that differentiates this tyrosine kinase from its related family members.     

Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4

In this study we have identified members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) as proteins to which the intracellular protein tyrosine kinase, Bruton's tyrosine kinase (Btk), binds. Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction. Furthermore, co-immunoprecipitation experiments revealed that Btk can also interact with key proteins involved in TLR4 signal transduction, namely, MyD88, Mal (MyD88 adapter-like protein), and interleukin-1 receptor-associated kinase-1, but not TRAF-6. The ability of Btk to interact with TLR4 and Mal suggests a role for Btk in lipopolysaccharide (LPS) signal transduction. Stimulation of the human monocytic cell line THP-1 with LPS resulted in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk was also stimulated after LPS stimulation. In addition, a dominant negative form of Btk inhibited TLR4-mediated activation of a nuclear factor kappaB (NFkappaB)-dependent reporter gene in HEK293 cells as well as LPS-induced activation of NFkappaB in the astrocytoma cell line U373 and the monocytic cell line RAW264.7. Further investigation revealed that the Btk-specific inhibitor, LFM-A13, inhibited the activation of NFkappaB by LPS in THP-1 cells. Our findings implicate Btk as a Toll/interleukin-1 receptor domain-binding protein that is important for NFkappaB activation by TLR4.     

Src family kinases mediate receptor-stimulated, phosphoinositide 3-kinase-dependent, tyrosine phosphorylation of dual adaptor for phosphotyrosine and 3-phosphoinositides-1 in endothelial and B cell lines

DAPP-1 (dual-adaptor for phosphotyrosine and 3-phosphoinositides-1) is a broadly distributed pleckstrin homology (PH) and Src homology 2 domain containing protein that can bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and can be phosphorylated on tyrosine 139 and internalised in response to activation of type I phosphoinositide 3-kinases (PI3K). Tyrosine phosphorylation of DAPP-1 appears important for appropriate intracellular targeting and creates a potential binding site for Src homology 2 domain-containing proteins. In endothelial cells overexpressing wild-type platelet-derived growth factor beta (PDGFbeta) receptors, which express Bmx and Src as their major Btk (Bruton's tyrosine kinase) family and Src family tyrosine kinases, respectively, PDGF can stimulate PI3K-dependent tyrosine phosphorylation of DAPP-1. Transient overexpression of Src most effectively, compared with Bmx and Syk, augments basal and PDGF-stimulated tyrosine phosphorylation of DAPP-1, whereas overexpression of dominant-negative Src, but not dominant-negative Bmx, inhibits PDGF-stimulated phosphorylation of DAPP-1. Cells expressing mutant PDGFbeta (Y579F/Y581F) receptors (which fail to bind and activate Src-type kinases) fail to tyrosine phosphorylate DAPP-1 in response to PDGF. We show that in DT40 chicken B cell lines, antibody stimulation leads to PI3K-dependent tyrosine phosphorylation of DAPP-1 that is lost in Lyn- or Syk-deficient cell lines but not Btk-deficient cell lines. PI3K-dependent activation of PKB is only lost in Syk-deficient lines. Finally, in vitro we find lipid-modified Src to be the most effective DAPP-1 tyrosine kinase (versus Syk, Lyn, Btk, and Bmx); phosphorylation of DAPP-1 but not Src autophosphorylation is stimulated approximately 10-fold by PtdIns(3,4,5)P(3) (IC(50) = 150 nm) and phosphatidylinositol 3,4-bisphosphate but not by their nonbiological diastereoisomers and depends on PH domain mediated binding of DAPP-1 to PtdIns(3,4,5)P(3)-containing membranes. We conclude that Src family kinases are responsible for tyrosine phosphorylation of DAPP-1 in vivo and that PI3K regulation is at the level of PH domain-mediated translocation of DAPP-1 to PI3K products in the membrane.