Secondary literature sources for the PI3K_p85B domain

For each of the hand selected primary papers associated with the PI3K_p85B domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

Zhou K, Takegawa K, Emr SD, Firtel RA
Mol Cell Biol. 1995; 15: 5645-56

Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5 encodes a functional homolog of yeast Vps34p. These observations indicate that in D. discoideum, different PI kinases regulate distinct cellular processes, including cell growth, development, and protein trafficking.

Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85.

Zheng Y, Bagrodia S, Cerione RA
J Biol Chem. 1994; 269: 18727-30

The Ras-like GTPase Cdc42 is essential for cell polarity and bud site assembly in Saccharomyces cerevisiae by regulating cell cycle-dependent reorganization of cortical cytoskeletal elements. However, its role in mammalian cells is unknown. To identify potential effectors of Cdc42Hs, we incubated lysates from NIH 3T3 fibroblasts or PC12 cells with immobilized glutathione S-transferase (GST)-Cdc42Hs fusion proteins bound to different guanine nucleotides and observed a specific association between the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase) and GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-bound GST-Cdc42Hs. Recombinant p85 formed a complex with GTP gamma S-bound GST-Cdc42Hs and with a GTPase-defective GTP-bound GST-Cdc42Hs-Q61L mutant, but not with a GTP gamma S-bound, effector domain GST-Cdc42HsT35A mutant. Both the Rho-GAP homology domain of p85 and the Cdc42Hs-GAP competitively inhibited the binding of recombinant p85 to Cdc42Hs. In addition, PI 3-kinase activity immunoprecipitated from cell lysates with anti-p85 antibody was stimulated 2-4-fold by GST-Cdc42-GTP gamma S. Similar interactions were observed between p85 and GST-Rac1-GTP gamma S but not between p85 and GST-RhoA-GTP gamma S. These findings suggest that PI 3-kinase, through the Rho-GAP homology domain of p85, can couple to the effector domain of Cdc42Hs and that p85 may be a target for the GTP-bound forms of Cdc42Hs and Rac1.

Controlled overexpression of selected domains of the P85 subunit of phosphatidylinositol 3-kinase reverts v-Ha-Ras transformation.

Zhang QX, Davis ID, Baldwin GS
Biochim Biophys Acta. 1996; 1312: 207-14

Selected domains of the regulatory p85 subunit of phosphatidylinositol 3-kinase have been expressed under the control of the tetracycline transactivator in NIH 3T3 fibroblasts transformed by the v-Ha-Ras oncogene. The domains expressed were the SH3 domain, the BCR homology domain, the region between the two SH2 domains which contains the p110 binding site (the inter SH2 (IS) domain), and the C-terminal (CT) domain (containing both SH2 domains and the IS domain). The levels of IS or SH3 domain expressed in the presence of tetracycline were sufficient to reverse the transforming effects of v-Ha-Ras, and no further inhibition of proliferation was observed when expression was increased 7-fold by removal of tetracycline. In contrast inhibition of proliferation by the CT domain was observed only when the level of expression was increased 5-fold by removal of tetracycline. Overexpression of the BCR domain of p85 had no effect on v-Ha-Ras transformation. Expression of the IS domain disrupted the interaction of the p85 regulatory subunit with the p110 catalytic subunit. These results indicate that the association of the p85 subunit of PI 3-kinase with the p110 subunit is necessary for v-Ha-Ras-induced transformation in NIH 3T3 cells.

CD28-mediated up-regulation of beta 1-integrin adhesion involves phosphatidylinositol 3-kinase.

Zell T, Hunt SW 3rd, Mobley JL, Finkelstein LD, Shimizu Y
J Immunol. 1996; 156: 883-6

Cross-linking of the CD28 Ag on T cells results in increased beta 1-integrin-mediated adhesion to fibronectin. Chimeric contructs containing the CD28 cytoplasmic domain fused to the extracellular and transmembrane regions of CD2 were expressed in HL60 cells to investigate CD28-mediated regulation of adhesion. Ab cross-linking of the CD2/28 chimera resulted in increased beta 1-dependent adhesion of HL60 transfectants to fibronectin. Induced binding was completely inhibited by the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Cross-linking of the CD2/28 chimera also induced association of the p85 subunit of PI 3-K with the CD2/28 cytoplasmic domain. In contrast, cross-linking of a CD2/28 chimera containing a tyrosine to phenylalanine substitution in the YMNM motif did not result in increased adhesion to fibronectin and did not lead to association of the chimera with PI 3-K. These results directly implicate the YMNM motif and PI 3-K in the regulation of beta 1-integrin activity by the CD28 Ag.

Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit.

Yu J, Zhang Y, McIlroy J, Rordorf-Nikolic T, Orr GA, Backer JM
Mol Cell Biol. 1998; 18: 1379-87

We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.

Inhibition of phosphatidylinositol 3-kinase by c-Abl in the genotoxic stress response.

Yuan ZM, Utsugisawa T, Huang Y, Ishiko T, Nakada S, Kharbanda S, Weichselbaum R, Kufe D
J Biol Chem. 1997; 272: 23485-8

Activation of phosphatidylinositol (PI) 3-kinase by growth factors results in phosphorylation of phosphatidylinositol lipids at the D3 position. Although PI 3-kinase is essential to cell survival, little is known about mechanisms that negatively regulate this activity. Here we show that the c-Abl tyrosine kinase interacts directly with the p85 subunit of PI 3-kinase. Activation of c-Abl by ionizing radiation exposure is associated with c-Abl-dependent phosphorylation of PI 3-kinase. We also show that phosphorylation of p85 by c-Abl inhibits PI 3-kinase activity in vitro and in irradiated cells. These findings indicate that c-Abl negatively regulates PI 3-kinase in the stress response to DNA damage.

The lipid kinase activity of the phosphatidylinositol 3-kinase is affected by its intrinsic protein kinase activity.

Woscholski R, Kodaki T, Parker PJ
Biochem Soc Trans. 1995; 23: 14-14

Modulation of the substrate specificity of the mammalian phosphatidylinositol 3-kinase by cholesterol sulfate and sulfatide.

Woscholski R, Kodaki T, Palmer RH, Waterfield MD, Parker PJ
Biochemistry. 1995; 34: 11489-93

The substrate specificity of the purified, mammalian phosphatidylinositol 3-kinase is subject to modulation by detergents, which are able to switch substrate specificity in vitro in favor of PtdInsP2. This effect of the detergents is due to an activation of the phosphatidylinositol biphosphate 3-kinase activity, while the phosphatidylinositol 3-kinase activity is inhibited. The selective inhibition of the phosphatidylinositol 3-kinase activity (p110 alpha/p85 alpha) is shown here also to be observed by employing cholesterol sulfate or sulfatide at low micromolar concentrations, whereas cholesterol and androsterone sulfate fail to inhibit. These naturally occurring sulfated lipids have at these concentrations no effect on the phosphatidylinositol bisphosphate 3-kinase activity but inhibit the manganese-dependent intrinsic protein kinase activity, thus switching substrate specificity toward the more highly phosphorylated inositol lipids. Cholesterol sulfate and sulfatide inhibit the free catalytic subunit p110 alpha but fail to inhibit the homologous phosphatidylinositol 3-kinase from Saccharomyces cerevisiae (Vps34p), suggesting that these sulfated lipids act specifically on the mammalian phosphatidylinositol 3-kinase. Consistent with this specificity, the regulatory subunit (p85), which is not conserved in the yeast enzyme, is found to play an important role for the affinity of these inhibitors. The implications for the phosphatidylinositol 3-kinase activity in vivo are discussed.

A comparison of demethoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase.

Woscholski R, Kodaki T, McKinnon M, Waterfield MD, Parker PJ
FEBS Lett. 1994; 342: 109-14

The mammalian Ptdlns 3-kinase is shown to be inhibited by low nanomolar concentrations of demethoxyviridin, an antifungal agent structurally related to wortmannin. The inhibitory potency of both compounds could be observed in purified Ptdlns 3-kinase whether or not the regulatory subunit (p85 alpha) was present, suggesting that the inhibitors bind to the catalytic subunit (p110) of the Ptdlns 3-kinase. These inhibitors also show similar potency against the intrinsic p85-phosphorylating activity of the p110-kinase. However, the structurally related Ptdlns 3-kinase from Saccharomyces cerevisiae (Vps34p) is not inhibited by either compound. Both inhibitors target the mammalian Ptdlns 3-kinase in vitro and in vivo, implying that these compounds should be useful in suppressing Ptdlns 3-kinase in mammalian systems. The inhibitors did not affect the mammalian Ptdlns 4-kinase, but they are able to inhibit a membrane-associated Ptdlns 4-kinase from Schizosaccharomyces pombe.

Biochemical characterization of the free catalytic p110 alpha and the complexed heterodimeric p110 alpha.p85 alpha forms of the mammalian phosphatidylinositol 3-kinase.

Woscholski R, Dhand R, Fry MJ, Waterfield MD, Parker PJ
J Biol Chem. 1994; 269: 25067-72

The regulatory (p85 alpha) and catalytic (p110 alpha) subunits of the mammalian phosphatidylinositol 3-kinase have been expressed in insect cells using the baculovirus sytem. The free catalytic subunit p110 alpha and the coexpressed heterodimeric complex of p85 alpha and p110 alpha were purified and their enzymological properties compared. While many kinetic parameters were similar, the coexpressed complex was found to have a 20-fold higher Km for ATP in comparison with the free catalytic subunit p110 alpha using phosphatidylinositol 4,5-bisphosphate as a substrate; no significant difference was detectable when phosphatidylinositol was used. Reconstitution of the p110 alpha.p85 alpha complex in vitro showed that it had the properties of the free p110 alpha and not the p110 alpha.p85 alpha in vivo complex. Therefore, a post-translational modification dependent upon the presence of the regulatory subunit p85 alpha rather than the physical subunit interaction itself is responsible for the observed properties of the lipid kinase activity of the p110 alpha.p85 alpha complex. Phosphatase treatment of the purified lipid kinase complex reduced the high Km for ATP, suggesting that a phosphorylation of the heterodimeric complex (p85 alpha.p110 alpha) caused this effect. This mode of regulation is discussed in the context of lipid kinase activation in vivo.

AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid binding domain.

Welters P, Takegawa K, Emr SD, Chrispeels MJ
Proc Natl Acad Sci U S A. 1994; 91: 11398-402

The cDNA encoding phosphatidylinositol (PI) 3-kinase was cloned from Arabidopsis thaliana, and the derived amino acid sequence (AtVPS34) has a significantly higher homology to yeast PI 3-kinase (VPS34) than to the mammalian (p110). The protein has two conserved domains: a catalytic site with the ATP-binding site near the C terminus and a calcium-dependent lipid-binding domain near the N terminus. The plant cDNA does not rescue a yeast vps34 deletion mutant, but a chimeric gene in which the coding sequence for the C-terminal third of VPS34 is replaced by the corresponding sequence from the plant gene does rescue the yeast mutant. PI 3-kinase activity is detectable in extracts from plants that overexpress the plant PI 3-kinase. Expression of antisense constructs gives rise to second-generation transformed plants severely inhibited in growth and development.

Direct association of Grb2 with the p85 subunit of phosphatidylinositol 3-kinase.

Wang J, Auger KR, Jarvis L, Shi Y, Roberts TM
J Biol Chem. 1995; 270: 12774-80

Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown to play a key role in growth factor signaling pathways, although its signaling mechanism has not been fully elucidated. Using the yeast interaction trap system, we have identified Grb2 as a PI 3-kinase interacting protein. Our experiments demonstrate that p85, the regulatory subunit of PI 3-kinase, interacts with Grb2 in vivo, and this interaction is independent of growth factor stimulation. The direct association between Grb2 and p85 was reconstituted in vitro with glutathione S-transferase fusion proteins. Domain analyses and peptide competition indicate that the association is mediated by the SH3 domains of Grb2 and the proline-rich motifs of p85 and that only one SH3 domain is required for minimal binding. The interaction does not displace the catalytic subunit of PI 3-kinase but is exclusive of Sos. Signaling through PI 3-kinase, therefore, may involve the ubiquitous adapter Grb2, which serves as a convergence point for multiple pathways.

Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110 alpha (PIK3CA) gene.

Volinia S, Hiles I, Ormondroyd E, Nizetic D, Antonacci R, Rocchi M, Waterfield MD
Genomics. 1994; 24: 472-7

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110 alpha subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3.

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Volinia S, Dhand R, Vanhaesebroeck B, MacDougall LK, Stein R, Zvelebil MJ, Domin J, Panaretou C, Waterfield MD
EMBO J. 1995; 14: 3339-48

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Interleukin-7 induces the association of phosphatidylinositol 3-kinase with the alpha chain of the interleukin-7 receptor.

Venkitaraman AR, Cowling RJ
Eur J Immunol. 1994; 24: 2168-74

The recently characterized receptor for interleukin (IL)-7 (IL-7R) includes a unique alpha chain as well as a common gamma chain shared with the receptors for IL-2 and IL-4. Engagement of the IL-7R activates the intracellular enzyme phosphatidylinositol (PtdIns) 3-kinase but the mechanism of PtdIns 3-kinase activation and the molecular basis of its interaction with IL-7R are not known. Here we show that IL-7 causes the 85-kDa regulatory subunit of PtdIns 3-kinase (p85), and PtdIns 3-kinase activity, to associate with the IL-7R. This interaction can be ascribed to ligand-induced phosphorylation of a single Tyr residue in the receptor's unique alpha chain. Herbimycin A, a specific protein tyrosine kinase inhibitor, suppresses not only tyrosine phosphorylation of the IL-7R but also its association with p85. A phosphopeptide corresponding to the sequence surrounding Tyr449 in the cytoplasmic tail of the IL-7R alpha chain, but not its non-phosphorylated analogue or phosphopeptides coincident with the sequences surrounding other alpha chain Tyr residues, efficiently competes out p85 binding. Replacement of Tyr449 with Phe results in a loss of p85 binding. Finally, soluble forms of the src homology 2 domains of p85, which bind directly to phosphotyrosyl peptides, specifically inhibit the association of p85 with the IL-7R. Thus, PtdIns 3-kinase recruitment occurs through a single, phosphotyrosine dependent recognition motif surrounding Tyr449 in the IL-7R alpha chain. This motif corresponds to a canonical sequence for p85 binding, Tyr(P)-X-X-Met. Since the closely related IL-2R and IL-4R also activate PtdIns 3-kinase but are devoid of such canonical motifs, our results suggest that the mechanism by which IL-7R recruits and activates PtdIns 3-kinase differs fundamentally from that used by the other receptors. PtdIns 3-kinase may, therefore, play a unique and important role in the biological response to IL-7.

Cross-talk between the insulin and angiotensin signaling systems.

Velloso LA, Folli F, Sun XJ, White MF, Saad MJ, Kahn CR
Proc Natl Acad Sci U S A. 1996; 93: 12490-5

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.

Role of PI 3-kinase in mitogenesis.

Varticovski L, Harrison-Findik D, Keeler ML, Susa M
Biochim Biophys Acta. 1994; 1226: 1-11

Expression of dominant negative mutant SHPTP2 attenuates phosphatidylinositol 3'-kinase activity via modulation of phosphorylation of insulin receptor substrate-1.

Ugi S, Maegawa H, Kashiwagi A, Adachi M, Olefsky JM, Kikkawa R
J Biol Chem. 1996; 271: 12595-602

To clarify the role of protein-tyrosine phosphatase (PTPase) containing Src homology 2 regions (SHPTP2) in insulin signaling, either wild-type or mutant SHPTP2 (delta PTP; lacking full PTPase domain) was expressed in Rat 1 fibroblasts overexpressing human insulin receptors. In response to insulin, phosphorylation of insulin receptor substrate 1 (IRS-1), IRS-1-associated PTPase activities and phosphatidylinositol (PI) 3'-kinase activities were slightly enhanced in wild-type cells when compared with those in the parent cells transfected with hygromycin-resistant gene alone. In contrast, introduction of delta PTP inhibited insulin-induced association of IRS-1 with endogenous SHPTP2 and impaired both insulin-stimulated phosphorylation of IRS-1 and activation of PI 3'-kinase. Furthermore, decreased content of p85 subunit of PI 3'-kinase was also found in mutant cells. Consistently, the insulin-stimulated mitogen-activated protein kinase activities and DNA synthesis were also enhanced in wild-type cells, but impaired in mutant cells. Thus, the interaction of SHPTP2 with IRS-1 may be associated with modulation of phosphorylation levels of IRS-1, resulting in the changes of PI 3'-kinase and mitogen-activated protein kinase activity. Furthermore, an impaired insulin signaling in mutant cells may be partly reflected in a decreased content of p85 protein of PI 3'-kinase.

Dominant negative effect of the truncated p110 subunit of phosphatidylinositol-3 kinase.

Takayanagi J, Kimura K, Nishioka N, Akimoto K, Moriya S, Ohno S, Fukui Y
Biochem Mol Biol Int. 1996; 39: 721-8

Effects of p110, the catalytic subunit of PI-3 kinase, on induction of TPA response element-driven promoter by EGF was examined. The induction was enhanced by co-expression of the wild type of p110. The truncated p110 mutants containing the binding site for p85 but missing the catalytic activity repressed the induction. A mutant with no binding activity to p85 did not show this effect. These results suggest that PI-3 kinase is involved in signal transduction of EGF and that the truncated p110s capable of binding to p85 serves as a dominant negative reagent for PI-3 kinase.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nurnberg B, et al.
Science. 1995; 269: 690-3

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

p120cbl is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermal growth factor in PC12 and other cells.

Soltoff SP, Cantley LC
J Biol Chem. 1996; 271: 563-7

Although epidermal growth factor (EGF) activates phosphoinositide (PI) 3-kinase activity in a number of types of cells or cell lines, in most cases that we have investigated the p85 regulatory subunit of PI 3-kinase does not appear to bind directly to the EGF receptor. Previously we demonstrated that EGF-dependent activation of PI 3-kinase activity in A431 cells is accompanied by the binding of p85 to ErbB3, an EGF receptor homologue. However, this mechanism did not explain the large activation of PI 3-kinase activity that was found in PC12 and A549 cells, which possess little or no ErbB3. Here we provide evidence that the p120cbl proto-oncoprotein is an intracellular adapter protein that associates with PI 3-kinase and thus is involved in the EGF-dependent activation of this enzyme in these two cell lines. Using an anti-p120cbl antibody, we immunoprecipitated the EGF receptor from PC12 cells and PI 3-kinase activity from PC12 and A549 cells in an EGF-dependent fashion. Treatment of PC12 cells with nerve growth factor or insulin stimulated large increases in PI 3-kinase activity that was immunoprecipitated using anti-Tyr(P) antibody but not using anti-p120cbl antibody. In EGF-treated PC12 cells, the tyrosine phosphorylation of p120cbl displayed similar kinetics to the activation of PI 3-kinase as measured by both in vivo lipid production and lipid kinase assays conducted using anti-p120cbl and anti-Tyr(P) immunoprecipitates. The use of glutathione S-transferase fusion proteins of various domains of p85 demonstrated that p120cbl associated with both the SH2 and SH3 domains of p85. p120cbl was also present in A431 cells and offers an additional pathway by which EGF can activate PI 3-kinase in these cells.

Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity.

Singh SS, Chauhan A, Murakami N, Chauhan VP
Biochemistry. 1996; 35: 16544-9

Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI 4,5-P2) and regulate the concentration of monomeric actin. We report here that profilin and gelsolin stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a concentration-dependent manner. This effect is specific to profilin and gelsolin because other cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity. In addition to lipid kinase activity, PI 3-kinase also has protein kinase activity: it phosphorylates proteins (p85 subunit of PI 3-kinase). However, the protein kinase activity of PI 3-kinase was not affected in the presence of profilin. Kinetic analysis, as a function of varying concentrations of ATP and PI 4,5-P2, showed that profilin affects the Vmax of PI 3-kinase without affecting k(m). Profilin may also affect PI 3-kinase activity by its direct association to the enzyme because dot-blot analysis using antibody to glutathione S-transferase (GST) suggested that GST-85 kDa, a fusion protein of PI 3-kinase, binds to profilin. However, PI 3-kinase did not affect the actin-sequestering ability of profilin (determined by pyrene-labeled actin), which indicates that actin and p85 do not share a common binding site on profilin. These studies suggest that profilin and gelsolin may control the generation of 3-OH phosphorylated phosphoinositides, which in turn may regulate the actin polymerization.

[Structure-functional characteristics of heterodimeric phosphatidylinositol-3-kinase and molecular mechanisms of its conjugation with other components of the signaling system]

Shpakov AO
Tsitologiia. 1999; 41: 975-91

This review presents literary data and results of the author own studies on structural and functional characteristics of regulatory (p55/p85) and catalytic (p110) subunits of heterodimeric phosphatidylinositol-3-kinases (PI-3-kinases), and on molecular mechanisms of their functional conjugation with other signaling system components, regulated by insulin and growth factors. Various models simulating the interaction of regulatory subunits of PI-3-kinase and of their substrates (insulin receptor sustrate proteins phosphorylated on tyrosin residues) with molecules of receptors-tyrosinekinases have been considered. Mechanisms of the functional conjugation between regulatory and catalytic enzyme subunits are discussed, with special reference to a possible role of the coiled-coil interactions in this process.

CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.

Schneider H, Prasad KV, Shoelson SE, Rudd CE
J Exp Med. 1995; 181: 351-5

CTLA-4 is a T cell antigen that is structurally related to CD28 and serves as a high affinity ligand for the B cell antigen B7-1/2. Unlike CD28, the function of CTLA-4 is unclear, although reports have implicated the antigen in the costimulation of T cells. Recently, phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the costimulatory function of CD28 by virtue of its ability to bind to a pYMNM motif within the cytoplasmic tail of the antigen. In this study, we show that CTLA-4 can also associate with PI 3-kinase as detected by lipid kinase analysis and immunoblotting with anti-p85 antiserum. High pressure liquid chromatographic separation of deacylated lipids showed the presence of a peak corresponding to PI-3-P. Anti-CTLA-4 ligation of the receptor induced a significant increase in the levels of precipitable PI 3-kinase activity. Peptide binding studies revealed that the NH2- and COOH-terminal SH2 domains of p85 bind the CTLA-4 cytoplasmic pYVKM motif with an affinity (ID50: 0.6 and 0.04 microM), that is similar to CD28. CTLA-4 binding to PI 3-kinase provides further evidence that CTLA-4 is not an inert counterreceptor, but rather is coupled to an intracellular signaling molecule with the capacity to regulate cell growth.

Monocyte colony-stimulating factor stimulates binding of phosphatidylinositol 3-kinase to Grb2.Sos complexes in human monocytes.

Saleem A, Kharbanda S, Yuan ZM, Kufe D
J Biol Chem. 1995; 270: 10380-3

Monocyte colony-stimulating factor (M-CSF) is required for the proliferation of mononuclear phagocytes. The activated M-CSF receptor associates with phosphatidylinositol 3-kinase (PI 3-kinase). In the present studies, we demonstrate that M-CSF also induces direct interaction of PI 3-kinase (p85 alpha subunit) with the SH2/SH3 adaptor protein Grb2. Tyrosine-phosphorylated PI 3-kinase interacts with the SH2 domain of Grb2. A pYRNE (pY408) site in PI 3-kinase is potentially involved in this interaction. The results also demonstrate that the PI 3-kinase.Grb2 complex associates with the guanine nucleotide exchange protein Sos. Since Sos binds to the SH3 domains of Grb2 and thereby associates with Ras at the cell membrane, formation of the PI 3-kinase.Grb2.Sos complex provides a potential mechanism for growth factor-induced interactions of PI 3-kinase and Ras.

Real-time assay of the interaction of a GST fusion protein with a protein ligate using resonant mirror technique.

Rubio I, Buckle P, Trutnau H, Wetzker R
Biotechniques. 1997; 22: 269-71

Regulation of phosphatidylinositol 3'-kinase by tyrosyl phosphoproteins. Full activation requires occupancy of both SH2 domains in the 85-kDa regulatory subunit.

Rordorf-Nikolic T, Van Horn DJ, Chen D, White MF, Backer JM
J Biol Chem. 1995; 270: 3662-6

Phosphatidylinositol 3'-kinase (PI 3'-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3'-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3'-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3'-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3'-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3'-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3'-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3'-kinase by tyrosylphosphorylated proteins requires occupancy of both SH2 domains in p85.

Phosphatidylinositol 3' kinase: one of the effectors of Ras.

Rodriguez-Viciana P, Marte BM, Warne PH, Downward J
Philos Trans R Soc Lond B Biol Sci. 1996; 351: 225-31

Ras proteins are proto-oncogene products that are critical components of signalling pathways leading from cell surface receptors to control of cellular proliferation, morphology and differentiation. the ability of Ras to activate the MAP kinase pathway through interaction with the serine/threonine kinase Raf is now well established. However, recent work has shown that Ras can also interact directly with the catalytic subunit of phosphatidylinositol 3' kinase and is involved in control of the lipid kinase in intact cells. A model is presented in which both tyrosine phosphoprotein interaction with the regulatory p85 subunit and Ras. GTP interaction with the catalytic p110 subunit is required to achieve optimal activation of phosphatidylinositol 3'kinase in response to extracellular stimuli. The ability of Ras to regulate phosphatidylinositol 3' kinase may be important both in Ras control of cellular morphology through the actin cytoskeleton and also in Ras control of DNA synthesis.

Phosphoinositide 3-kinase activation and platelet function.

Rittenhouse SE
Blood. 1996; 88: 4401-14

Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways.

Reif K, Nobes CD, Thomas G, Hall A, Cantrell DA
Curr Biol. 1996; 6: 1445-55

BACKGROUND: Phosphatidylinositol 3'-hydroxyl kinase (PI 3-kinase) is activated by many growth factor receptors and is thought to exert its cellular functions through the elevation of phosphatidylinositol (3,4,5)-triphosphate levels in the cell. PI 3-kinase is required for growth-factor induced changes of the actin cytoskeleton which are mediated by the GTPases Rac and Rho. Recently, a role for Rac and Rho in regulating gene transcription has become evident. RESULTS: Here, we show that membrane targeting of the p110 catalytic subunit, but not the p85 regulatory subunit, of PI 3-kinase generates a constitutively active enzyme that allows us to assess the relative contribution of PI 3-kinase activation to a particular cellular response. Expression of this active PI 3-kinase induced actin reorganization in the form of Rac-mediated lamellipodia and focal complexes, and Rho-mediated stress fibres and focal adhesions. However, expression of active PI 3-kinase did not induce the Ras/Rac/Rho signalling pathways that regulate gene transcription controlled by the c-fos promoter, the c-fos serum response element or the transcription factors Elk-1 and AP-1. CONCLUSIONS: Our results demonstrate that PI 3-kinase induces a selective subset of cellular responses, but is not sufficient to stimulate the full repertoire of Rac- or Rho-mediated responses.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Reedijk M, Liu X, van der Geer P, Letwin K, Waterfield MD, Hunter T, Pawson T
EMBO J. 1992; 11: 1365-72

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.

Structure-activity studies of phosphorylated peptide inhibitors of the association of phosphatidylinositol 3-kinase with PDGF-beta receptor.

Ramalingam K, Eaton SR, Cody WL, Lu GH, Panek RL, Waite LA, Decker SJ, Keiser JA, Doherty AM
Bioorg Med Chem. 1995; 3: 1263-72

Phosphorylated pentapeptides derived from Tyr751 of the PDGF-beta receptor (pTyr751-Val-Pro-Met-Leu, pTyr = phosphotyrosine) were prepared to examine their ability to inhibit the association of the C-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) with the PDGF-beta receptor. Peptidic analogs were prepared to examine the importance of the amine and carboxy terminus and specific amino acids via alanine/D-amino acid scans and site specific modifications. Several of these peptides had submicromolar activity. In particular, it was shown that neutralization of the amine and carboxy terminus led to analogs with enhanced activity. In addition, it was determined that only minimal modifications were allowed for pTyr and Met, while the other positions were quite tolerant of modification.

T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif.

Prasad KV, Cai YC, Raab M, Duckworth B, Cantley L, Shoelson SE, Rudd CE
Proc Natl Acad Sci U S A. 1994; 91: 2834-8

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)-MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.

Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase.

Patki V, Virbasius J, Lane WS, Toh BH, Shpetner HS, Corvera S
Proc Natl Acad Sci U S A. 1997; 94: 7326-30

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3'-phosphoinositides on membrane trafficking in yeast and mammalian cells.

Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositol 3-kinase activity.

Pandey A, Lazar DF, Saltiel AR, Dixit VM
J Biol Chem. 1994; 269: 30154-7

The Eph/Eck subfamily of receptor protein tyrosine kinases is currently the largest subfamily of receptor protein tyrosine kinases with a dozen members (Van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10, 251-337). Using the cytoplasmic domain of Eck as bait in a yeast two-hybrid screen of mouse embryonic and T-cell cDNA libraries, it was discovered that the p85 subunit of phosphatidylinositol 3-kinase bound Eck. Further, using glutathione S-transferase fusion proteins, it was found that the C-terminal src homology 2 domain of the p85 subunit specifically interacted with Eck. Additionally, Eck coimmunoprecipitated with p85 in ligand activated cells confirming their interaction in vivo. In keeping with the above observations, activation of Eck by its ligand, B61, increased phosphatidylinositol 3-kinase activity. This is the first description of a signal transduction pathway initiated by any member of the Eph/Eck family.

p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase.

Panchamoorthy G, Fukazawa T, Miyake S, Soltoff S, Reedquist K, Druker B, Shoelson S, Cantley L, Band H
J Biol Chem. 1996; 271: 3187-94

We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3-kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120cbl in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors.

In vitro association of the phosphatidylinositol 3-kinase regulatory subunit (p85) with the human insulin receptor.

Ottinger EA, Hui TY, Man Z, Barany G, Bernlohr DA
Int J Pept Protein Res. 1995; 46: 346-53

The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its beta-subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S-transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated beta-subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C-gamma 1 (PLC gamma 1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15-residue tyrosine-phosphorylated peptide corresponding to the carboxyl-terminal region of the insulin receptor, but not by phosphopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N + C SH2 domain-insulin receptor complex with an IC0.5 of 2.3 +/- 0.35 microM, whereas a 10-residue phosphopeptide derived from the insulin receptor substrate 1 (IRS-1) competed with an IC0.5 of 0.54 +/- 0.10 microM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl-terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS-1.

Phosphatidylinositol 3-kinase is involved in TRE-dependent gene expression in response to arginine vasopressin.

Nishioka N, Akimoto K, Moriya S, Takayanagi J, Fukui Y, Hirai S, Mizuno K, Kosaka K, Ohno S
Biochem Biophys Res Commun. 1995; 215: 1037-42

We identified arginine vasopressin (AVP) as a potent activator of TPA-response element (TRE)-dependent gene expression in rat 3Y1 fibroblasts. In order to examine the involvement of phosphatidylinositol 3-kinase (PI3-kinase) in TRE-mediated gene expression, we examined the effect of the overexpression of PI3-kinase mutant. The overexpression of p110 alpha, the catalytic subunit of PI3-kinase, enhances TRE-reporter expression in response to AVP. On the other hand, the overexpression of the p110-EcoS mutant, which contains the binding site for the regulatory p85 subunit but lacks the catalytic domain, results in decreased TRE reporter expression in response to AVP. These results suggest that PI3-kinase is involved in TRE-dependent gene expression in response to AVP.

Novel functions of phosphatidylinositol 3-kinase in terminally differentiated cells.

Nakanishi S, Yano H, Matsuda Y
Cell Signal. 1995; 7: 545-57

Importance of phosphatidylinositol 3-kinase (PI 3-kinase) in signalling pathways leading to growth stimulation has already been reviewed in this journal and others. Evidence has now been accumulating that PI 3-kinase is involved in transmission of activation signals in terminally differentiated cells, especially signals starting from receptors which have no intrinsic tyrosine kinase domain. The pioneer works showed the presence of PI 3-kinase activity and the accumulation of the reaction products of PI 3-kinase correlated with the cell responses. However, these studies were done in only limited cell responses such as respiratory burst in neutrophils and degranulation in platelets. Recent finding of a potent and selective inhibitor of PI 3-kinase, wortmannin, reported from three independent groups including us, gave a new and powerful tool not only to confirm the suggested functions but also to reveal new functions of PI 3-kinase such as histamine release from antigen-stimulated mast cells/basophils and glucose uptake in insulin-stimulated adipocytes. Nearly one hundred papers which describe the action of wortmannin on various cells have been reported during one year after the publication of the discovery of wortmannin as PI 3-kinase inhibitor, suggesting possible involvement of the enzyme in the diverse cell responses besides cell proliferation.

Erythropoietin-dependent association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated erythropoietin receptor.

Miura O, Nakamura N, Ihle JN, Aoki N
J Biol Chem. 1994; 269: 614-20

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The Epo receptor (EpoR) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, Epo induces tyrosine phosphorylation of cellular substrates including the EpoR. To explore the functional significance of receptor tyrosine phosphorylation, we examined the possible interaction of the receptor with the 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase. After Epo stimulation, p85 was found to associate with the tyrosine-phosphorylated 72-kDa form of EpoR as well as a 92-kDa phosphotyrosyl protein, and PI 3-kinase activity was detectable in anti-EpoR immunoprecipitates. Anti-EpoR blotting of anti-p85 immunoprecipitates revealed that p85 binds specifically to the 72-kDa form of the EpoR and not to unphosphorylated 66- and 64-kDa forms. Association of p85 with the EpoR was Epo dose- and time-dependent and correlated with tyrosine phosphorylation of the receptor. Consistent with a role for tyrosine phosphorylation of the EpoR, PI 3-kinase did not associate with a mitogenically active receptor mutant that lacked tyrosine-phosphorylation sites in the carboxyl-terminal region. A recombinant fusion protein containing the carboxyl-terminal SH-2 domain of p85 was shown to bind to tyrosine-phosphorylated EpoR in vitro. Taken together, these results indicate that, following Epo stimulation, the EpoR recruits PI 3-kinase to the cell membrane by binding between the carboxyl-terminal SH-2 domain of p85 and the tyrosine-phosphorylated carboxyl-terminal region of the receptor. The association with PI 3-kinase is, however, not required for the growth signal transduction from the EpoR.

Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.

McIlroy J, Chen D, Wjasow C, Michaeli T, Backer JM
Mol Cell Biol. 1997; 17: 248-55

We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway.

Association of phosphatidylinositol 3-kinase with the proto-oncogene product Cbl upon CD38 ligation by a specific monoclonal antibody in THP-1 cells.

Matsuo T, Hazeki K, Tsujimoto N, Inoue S, Kurosu H, Kontani K, Hazeki O, Ui M, Katada T
FEBS Lett. 1996; 397: 113-6

We reported that ecto-NAD+ glycohydrolase activity induced upon differentiation of HL-60 cells with retinoic acid is localized on the extracellular domain of CD38 and that CD38 ligation by a specific monoclonal antibody, HB-7, is followed by rapid tyrosine phosphorylation of cellular proteins including a proto-oncogene product, Cbl. In the present study, we investigated intracellular signaling linked to the HB-7-induced Cbl phosphorylation in dibutyryl cAMP-treated THP-1 cells. The 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase was immunoprecipitated with anti-Cbl antibody in a manner dependent on the tyrosine phosphorylation of Cbl. PI 3-kinase activity was also observed in the immunoprecipitated fractions containing tyrosine-phosphorylated Cbl. The phosphorylated form of Cbl, which had been separated from the CD38-stimulated cells, was capable of directly binding to a recombinant p85 fused to glutathione S-transferase. Thus, the direct association of tyrosine-phosphorylated Cbl with PI 3-kinase, possibly leading to the kinase activation, appeared to be involved in intracellular signaling caused by the CD38 ligation.

Specific association of phosphatidylinositol 3-kinase with the protooncogene product Cbl in Fc gamma receptor signaling.

Matsuo T, Hazeki K, Hazeki O, Katada T, Ui M
FEBS Lett. 1996; 382: 11-4

A tyrosine-phosphorylated protein with a molecular mass of 115 kDa was reported to be tightly associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, when the enzyme was essentially activated upon ligand engagement of Fc gamma receptors (Fc gamma R) leading to engulfment of IgG-coated erythrocytes by phagocytes [Ninomiya et al. (1994) J. Biol. Chem. 269, 22732-22737]. Here, the 115-kDa protein is identified as the product of human c-cbl protooncogene. Cross-linking of Fc gamma RII on the surface of THP-1 cells triggered (a) prominent tyrosine phosphorylation of Cbl, (b) activation of PI 3-kinase that was immunoprecipitated with the anti-Cbl or the anti-phosphotyrosine antibody, and (c) specific association of Cbl with p85. Thus, Cbl functions in phagocytes as a result of its association with PI 3-kinase in response to Fc gamma R ligation.

Comparison of calcium-dependent conformational changes in the N-terminal SH2 domains of p85 and GAP defines distinct properties for SH2 domains.

Mahadevan D, Thanki N, McPhie P, Beeler JF, Yu JC, Wlodawer A, Heidaran MA
Biochemistry. 1994; 33: 746-54

Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)

A divalent metal ion binding site in the kinase insert domain of the alpha-platelet-derived growth factor receptor regulates its association with SH2 domains.

Mahadevan D, Thanki N, Aroca P, McPhie P, Yu JC, Beeler J, Santos E, Wlodawer A, Heidaran MA
Biochemistry. 1995; 34: 2095-106

To investigate the effects of metal ion binding to the alpha-PDGFR kinase insert domain, a PCR product representing amino acid residues 691-795 (104 amino acids) was bacterially expressed and purified. Secondary structure prediction and circular dichroism spectroscopy indicated this domain to be a mixed alpha + beta protein with a large coil/turn contribution. This 16 kDa, soluble, nonphosphorylated domain bound to 45Ca2+ and 65Zn2+ through a common shared site. Of the unlabeled divalent and trivalent metal ions tested, Ho3+ = Zn2+ > Ni2+ > Ca2+ = Mn2+ > Mg2+, Ba2+ in competing for 45Ca2+ binding to this domain. In the presence of Ca2+ ions, the conformation of the KI domain changed significantly, and this changed conformation was resistant to subtilisin proteolysis. However, in the presence of Zn2+ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn2+ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca2+ ions. In vitro binding studies using purified baculovirus-expressed alpha-PDGFR showed a marked increase in binding the p85 N-SH2 domain in the presence of Ca2+ or Zn2+ ions (KD = 0.5 microM), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the alpha-PDGFR with the p85 N-SH2 domain was tested in the presence of the KI domain. The nonphosphorylated KI domain was effective in competing with the alpha-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca2+ ions. Microinjection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI domain has a correctly folded three-dimensional structure compatible with biological activity. Together these findings indicate that the recombinant alpha-PDGFR KI domain binds the p85 N-SH2 domain and this binding is modulated by the presence of a novel divalent metal ion binding site within its structure.

A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction.

MacDougall LK, Domin J, Waterfield MD
Curr Biol. 1995; 5: 1404-15

BACKGROUND: Mammalian phosphoinositide 3-kinases (PI 3-kinases) are involved in receptor-mediated signal transduction and have been implicated in processes such as transformation and mitogenesis through their role in elevating cellular phosphatidylinositol (3,4,5)-trisphosphate. Additionally, a PI 3-kinase activity which generates phosphatidylinositol 3-phosphate has been shown to be required for protein trafficking in yeast. RESULTS: We have identified a family of three distinct PI 3-kinases in Drosophila, using an approach based on the polymerase chain reaction to amplify a region corresponding to the conserved catalytic domain of PI 3-kinases. One of these family members, PI3K_92D, is closely related to the prototypical PI 3-kinase, p110 alpha; PI3K_59F is homologous to Vps34p, whereas the third, PI3K_68D, is a novel PI 3-kinase which is widely expressed throughout the Drosophila life cycle. The PI3K_68D cDNA encodes a protein of 210 kDa, which lacks sequences implicated in linking p110 PI 3-kinases to p85 adaptor proteins, but contains an amino-terminal proline-rich sequence, which could bind to SH3 domains, and a carboxy-terminal C2 domain. Biochemical analyses demonstrate that PI3K_68D has a novel substrate specificity in vitro, restricted to phosphatidylinositol and phosphatidylinositol 4-phosphate, and is unable to phosphorylate phosphatidylinositol (4,5)-bisphosphate, the implied in vivo substrate for p110. CONCLUSIONS: A family of PI 3-kinases in Drosophila, including a novel class represented by PI3K_68D, is described. PI3K_68D has the potential to bind to signalling molecules containing SH3 domains, lacks p85-adaptor-binding sequences, has a Ca(2+)-independent phospholipid-binding domain and displays a restricted in vitro substrate specificity, so it could define a novel signal transduction pathway.

Functional association of CD7 with phosphatidylinositol 3-kinase: interaction via a YEDM motif.

Lee DM, Patel DD, Pendergast AM, Haynes BF
Int Immunol. 1996; 8: 1195-203

Human CD7 is a 40 kDa protein expressed on thymocytes, early T, B, NK and myeloid lineage cells in bone marrow, and on mature T and NK cells. Previous studies suggested human CD7 may be involved in T and NK cell activation and/or adhesion, and that CD7-mediated cell activation may be transduced via the lipid kinase phosphatidylinositol 3-kinase (Pi3-kinase), a heterodimeric cytosolic protein consisting of an 85 kDa adaptor subunit that is coupled to a 110 kDa catalytic subunit. It has recently been shown that a sequence motif present in the cytoplasmic tall of both human and mouse CD7 bound with high affinity to recombinant SH2 domains present in the p85 subunit of Pi3-kinase. In this work, we used co-precipitation with anti-CD7 mAb 3A1 and recombinant p85 SH2-GST fusion proteins and peptide competition analysis to demonstrate that the cytoplasmic tail of CD7 interacts with a functional Pi3-kinase via the pTyr-X-X-Met motif. Furthermore, we show that cross-linking of CD7 markedly increased the amount of Pi3-kinase activity associated with CD7. The interaction of CD7 with the Pi3-kinase signal transduction pathway provides a mechanism for the previously observed functional responses attributed to CD7-mediated T and NK cell activation.

Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system.

Lamothe B, Bucchini D, Jami J, Joshi RL
FEBS Lett. 1995; 373: 51-5

Interaction of the p85 subunit of PI 3-kinase with the insulin receptor (IR) and the IGF-1 receptor (IGF-1R) was investigated using the two-hybrid system by assessing for his3 and lacZ activation in S. cerevisiae. The experiments were performed with the cytoplasmic beta domain (wild type or mutated) of IR and IGF-1R and p85 or its subdomains (N + C-SH2, N-SH2, C-SH2, SH3 + N-SH2). The results of his3 activation indicated that p85, N + C-SH2 and C-SH2 interact with both IR beta and IGF-1R beta, whereas N-SH2 and SH3 + N-SH2 interact only with IR beta. Interaction of p85 and N+C-SH2 with IR beta (delta C-43) or IGF-1R beta(delta C-43) in which the C-terminal 43 amino acids (including the YXXM motif) were deleted, persisted. The internal binding site thus revealed was not altered by further mutating Y960/F for IR or Y950/F for IGF-1R. Activation of lacZ upon interaction of p85 with IR beta(delta C-43) was 4-fold less as compared to IR beta. This activation with p85 and IGF-1R beta was 4-fold less as compared to IR beta and was somewhat increased (2-fold) for IGF-1R beta (delta C-43). Thus, the C-terminal domain in IGF-1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3-kinase pathway.

Differential activities of protein tyrosine phosphatases in intact cells.

Lammers R, Bossenmaier B, Cool DE, Tonks NK, Schlessinger J, Fischer EH, Ullrich A
J Biol Chem. 1993; 268: 22456-62

We have employed transient co-overexpression of protein tyrosine phosphatases (PTPs) with a panel of receptor tyrosine kinases (RTKs) to investigate molecular parameters that regulate dephosphorylation activity and specificity in intact cells. Our results demonstrate clear differences in susceptibility of various forms of different RTKs to the action of PTP 1B, T-cell phosphatase (TC-PTP), and CD45, which suggests cellular compartmentalization as a major factor defining activity and overall function. TC-M PTP, a nonlocalized cytosolic mutant, is deregulated and is therefore able to efficiently suppress v-erbB- and v-fms-induced cell transformation, which is not observed with the intact TC-PTP or PTP 1B. The transmembrane PTP CD45 displays more selectivity but appears to be already active during transport to the cell surface. Dephosphorylation activity is also dependent on relative RTK/PTP expression levels and can be modulated by the SH2 domain-containing noncatalytic subunit of phosphatidylinositol 3'-kinase, p85. Overexpression of high affinity binding proteins could therefore contribute to RTK-induced cell transformation and cancer.

Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110beta is synergistically activated by the betagamma subunits of G proteins and phosphotyrosyl peptide.

Kurosu H, Maehama T, Okada T, Yamamoto T, Hoshino S, Fukui Y, Ui M, Hazeki O, Katada T
J Biol Chem. 1997; 272: 24252-6

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the betagamma subunits of G protein (Gbetagamma) from rat liver. The Gbetagamma-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110beta and p85 (or their related subunits). The stimulation by Gbetagamma was inhibited by the GDP-bound alpha subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of Gbetagamma was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110beta/p85alpha expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110alpha and p85 in the same rat liver, together with a recombinant p110alpha/p85alpha, was not activated by Gbetagamma, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110beta/p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.

Association of Phosphatidylinositol 3-Kinase Composed of p110beta-Catalytic and p85-Regulatory Subunits with the Small GTPase Rab5.

Kurosu H, Katada T
J Biochem (Tokyo). 2001; 130: 73-8

A family of phosphatidylinositol 3-kinases (PI 3-kinase), comprising three major classes (I-III) in terms of substrate specificity and regulation, play important roles in a variety of cell functions. We previously reported that the class-I heterodimeric PI 3-kinase consisting of p110beta-catalytic and p85-regulatory subunits is synergistically activated by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating trimeric G proteins. Here we report an additional unique feature of the p110beta/p85 PI 3-kinase. The small GTPase Rab5 was identified as a binding protein for the p110beta-catalytic subunit in a yeast two-hybrid screening system. The interaction appears to require at least two separated amino-acid sequences present specifically in the beta isoform of p110 and the GTP-bound form of Rab5. The expressions of constitutively active and dominant negative mutants of Rab5 in THP-1 cells induce the stimulation and inhibition, respectively, of protein kinase B activity, which is dependent on the PI 3-kinase product phosphatidylinositol 3,4,5-triphosphate. These results suggest that there is a specific interaction between GTP-bound Rab5 and the p110beta/p85 PI 3-kinase, leading to efficient coupling of the lipid kinase product to its downstream target, protein kinase B.

Radiolabeling of catalytic subunits of PI 3-kinases with 17 beta-hydroxy-16 alpha(-)[125I]iodowortmannin: identification of the G beta gamma-sensitive isoform as a complex composed of 46-kDa and 100-kDa subunits.

Kurosu H, Hazeki O, Kukimoto I, Honzawa S, Shibasaki M, Nakada M, Ui M, Katada T
Biochem Biophys Res Commun. 1995; 216: 655-61

A fungal metabolite, wortmannin, is a potent inhibitor of phosphatidylinositol (PI) 3-kinases. In the present study, we prepared a radiolabeled derivative of wortmannin, 17 beta-hydroxy-16 alpha(-)[125I]iodowortmannin. The compound bound tightly to a 110-kDa subunit in the previously identified isoform of PI 3-kinase (p85/p110), and also to a 100-kDa peptide in a partially purified preparation of another isoform of PI 3-kinase whose activity was markedly stimulated by the beta gamma subunits of GTP-binding proteins (G beta gamma). The binding to both peptides was inhibited by non-radiolabeled wortmannin and also by LY294002, another inhibitor of PI 3-kinases. An antibody against p85 recognized a 46-kDa peptide in the G beta gamma-sensitive isozyme and precipitated the 100-kDa peptide specifically labeled with 17 beta-hydroxy-16 alpha(-)[125I]iodowortmannin. These results suggested that the newly found isozyme was a complex composed of 46-kDa and 100-kDa peptides.

Phosphoinositide 3-kinase as an upstream regulator of the small GTP-binding protein Rac in the insulin signaling of membrane ruffling.

Kotani K, Hara K, Kotani K, Yonezawa K, Kasuga M
Biochem Biophys Res Commun. 1995; 208: 985-90

Membrane ruffling and the closely linked response of fluid-phase pinocytosis were investigated in Chinese hamster ovary cells that stably overexpress the human insulin receptor and a mutant 85-kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) that lacks a binding site for the catalytic 110-kDa subunit of this enzyme. Both membrane ruffling and pinocytosis induced by insulin were markedly impaired in these cells. Microinjection of Rac, a Ras-related small GTP-binding protein, induced membrane ruffling in human epidermoid carcinoma KB cells, and this effect of Rac was not blocked by coinjection of delta p85 or by exposure of cells to wortmannin, a specific PI 3-kinase inhibitor. These results suggest that PI 3-kinase is essential not only for insulin-stimulated membrane ruffling but also for pinocytosis, and that PI 3-kinase possibly functions upstream of Rac in the signal transduction pathway.

Expression of a constitutively active phosphatidylinositol 3-kinase induces process formation in rat PC12 cells. Use of Cre/loxP recombination system.

Kobayashi M, Nagata S, Kita Y, Nakatsu N, Ihara S, Kaibuchi K, Kuroda S, Ui M, Iba H, Konishi H, Kikkawa U, Saitoh I, Fukui Y
J Biol Chem. 1997; 272: 16089-92

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.

Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways.

Klippel A, Reinhard C, Kavanaugh WM, Apell G, Escobedo MA, Williams LT
Mol Cell Biol. 1996; 16: 4117-27

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Finally, targeting of pl10* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.

A region of the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo.

Klippel A, Escobedo JA, Hu Q, Williams LT
Mol Cell Biol. 1993; 13: 5560-6

Phosphatidylinositol (PI) 3-kinase is a heterodimer consisting of an 85-kDa subunit (p85) and 110-kDa subunit (p110). The 85-kDa noncatalytic subunit, which contains two Src homology 2 (SH2) domains, one SH3 domain, and a domain homologous to the carboxy terminus of the breakpoint cluster region gene product, is known to mediate the association of the PI 3-kinase complex with activated growth factor receptors. We previously demonstrated that the C-terminal SH2 domain of p85 is responsible for the interaction of PI 3-kinase with phosphorylated platelet-derived growth factor receptor. To define the region in p85 that directs the complex formation with the PI 3-kinase catalytic subunit, a series of truncated p85 mutants was analyzed for association with p110 in vivo. We found that a fragment of p85 containing the region between the two SH2 domains was sufficient to promote the interaction with p110 in vivo. The complex between the fragment of p85 and p110 had PI 3-kinase activity that was comparable in magnitude to the activity of p110 associated with full-length p85. The binding with p110 was abolished when this domain in p85 was disrupted. These results identify a novel structural and functional element that is responsible for localizing the catalytic subunit of PI 3-kinase.

Phosphatidylinositol-3 kinase in fission yeast: a possible role in stress responses.

Kimura K, Miyake S, Makuuchi M, Morita R, Usui T, Yoshida M, Horinouchi S, Fukui Y
Biosci Biotechnol Biochem. 1995; 59: 678-82

A DNA fragment coding for a part of a putative phosphatidylinositol 3 kinase was cloned from Schizosaccharomyces pombe by cross-hybridization with Saccharomyces cerevisiae VPS34 gene, a yeast homologue of mammalian PI-3 kinase. The clone contained an open reading frame of 797 amino acids but lacked the initiation codon, ATG. The predicted amino acid sequence was homologous to those of S. cerevisiae VPS34 and mammalian PI-3 kinase genes. Disruption of the gene resulted in extremely low levels of PI-3-P and higher levels of PI-4-P, supporting the idea that the gene codes for the PI-3 kinase of S. pombe. The disruptants harbored large vacuoles and were sensitive to stresses such as high temperature or high concentration of monovalent and divalent cations.

Ionizing radiation stimulates a Grb2-mediated association of the stress-activated protein kinase with phosphatidylinositol 3-kinase.

Kharbanda S, Saleem A, Shafman T, Emoto Y, Taneja N, Rubin E, Weichselbaum R, Woodgett J, Avruch J, Kyriakis J, et al.
J Biol Chem. 1995; 270: 18871-4

The stress-activated protein (SAP) kinases are induced by tumor necrosis factor, oncoproteins, and UV light. The present studies demonstrate that ionizing radiation (IR) activates p54 SAP kinase. IR-induced activation of SAP kinase is associated with binding to the SH2/SH3-containing adaptor protein Grb2. This interaction is mediated by the SH3 domains of Grb2 and the proline-rich sequence PPPKIP in the carboxy-terminal region of SAP kinase. We also demonstrated that SAP kinase and the p85 alpha-subunit of phosphatidylinositol (PI) 3-kinase form a complex in irradiated cells. The results indicate that this complex involves binding of the p85 alpha subunit of PI 3-kinase to the SH2 domain of Grb2. The functional role of linking SAP kinase to PI 3-kinase is further supported by the finding that wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. These results suggest that the cellular response to IR may include regulation of SAP kinase by a PI 3-kinase-dependent signaling pathway.

Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.

Katagiri H, Asano T, Ishihara H, Inukai K, Shibasaki Y, Kikuchi M, Yazaki Y, Oka Y
J Biol Chem. 1996; 271: 16987-90

To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody. p110alpha overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Katada T, Kurosu H, Okada T, Suzuki T, Tsujimoto N, Takasuga S, Kontani K, Hazeki O, Ui M
Chem Phys Lipids. 1999; 98: 79-86

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.

Role of PI 3-kinase and SH-PTP2 in insulin action.

Kasuga M
Diabet Med. 1996; 13: 879-879

Insulin bonding to the alpha subunit of its receptor activates the tyrosine kinase of the beta subunit. This induces the tyrosine-phosphorylation of insulin receptor substrates-1 (IRS-1) and a complex formation between IRS-1 and other proteins including GRB2, SH-PTP2, Nck, and phosphoinositide (PI) 3-kinase consisting of a regulatory 85-kDa subunit and a catalytic 110-kDa subunit. We found that insulin-induced binding of the PI 3-kinase activity to IRS-1 generates the signal to activate glucose transport and membrane ruffling. In contrast, insulin-induced binding of SH-PTP2 to IRS-1 generates the signal to activate the Ras protein.

The Src-family kinase, Fyn, regulates the activation of phosphatidylinositol 3-kinase in an interleukin 2-responsive T cell line.

Karnitz LM, Sutor SL, Abraham RT
J Exp Med. 1994; 179: 1799-808

The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein tyrosine kinase (PTK) activity. One IL-2-regulated PTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol 3-kinase (PI3-kinase) as well as the IL-2-dependent activation of PI3-kinase in T cells; however, the role of Fyn in IL-2-induced PI3-kinase activation remains unclear. In this report, we demonstrate that IL-2 stimulation triggers tyrosine phosphorylation of the p85 subunit of PI3-kinase in the murine T cell line, CTLL-2. Lysates prepared from growth factor-deprived and IL-2-stimulated T cells reconstituted both the binding of CTLL-2 cell-derived Fyn to and the IL-2-inducible tyrosine phosphorylation of exogenously added recombinant p85. Furthermore, overexpression of wild-type Fyn in these cells enhanced both the basal and IL-2-mediated activation of PI3-kinase. Additional studies of the Fyn-PI3-kinase interaction demonstrated that the Src homology 3 (SH3) domain of Fyn constitutes a direct binding site for the p85 subunit of PI3-kinase. These results support the notion that Fyn may be directly involved in the activation of the downstream signaling enzyme, PI3-kinase, in IL-2-stimulated T cells.

Phosphoinositide 3-kinase binds constitutively to alpha/beta-tubulin and binds to gamma-tubulin in response to insulin.

Kapeller R, Toker A, Cantley LC, Carpenter CL
J Biol Chem. 1995; 270: 25985-91

Recently we reported the localization of phosphoinositide 3-kinase (PI 3-kinase) by immunofluorescence to microtubule bundles and the centrosome (Kapeller, R., Chakrabarti, R., Cantley, L., Fay, F., and Corvera, S. (1993) Mol. Cell. Biol. 13, 6052-6063). In complementary experiments we used the recombinant p85 subunit of PI 3-kinase to identify proteins that associate with phosphoinositide 3-kinase and found that phosphoinositide 3-kinase associates with alpha/beta-tubulin. The association occurs in vivo but was not significantly affected by growth factor stimulation. We localized the region of p85 that interacts with alpha/beta-tubulin to the inter-SH2 domain. These results support the immunofluorescence data and show that p85 directly associates with alpha/beta-tubulin. We then determined whether phosphoinositide 3-kinase associates with gamma-tubulin. We found a dramatic growth factor-dependent association of phosphoinositide 3-kinase with gamma-tubulin. Phosphoinositide 3-kinase associates with gamma-tubulin in response to insulin and, to a lesser extent, in response to platelet-derived growth factor. Neither epidermal growth factor nor nerve growth factor treatment of cells results in association of phosphoinositide 3-kinase and gamma-tubulin. Phosphoinositide 3-kinase is also immunoprecipitated with antibodies to pericentrin in response to insulin, indicating that phosphoinositide 3-kinase is recruited to the centrosome. Neither phosphoinositide 3-kinase activity, nor intact microtubules are necessary for the association. Treatment of cells with 0.5 M NaCl dissociates gamma-tubulin from the centrosome and disrupts the association of phosphoinositide 3-kinase with pericentrin, but not gamma-tubulin. Recombinant p85 binds to gamma-tubulin from both insulin stimulated and quiescent cells. These results suggest that the association of phosphoinositide 3-kinase with gamma-tubulin is direct. These data suggest that phosphoinositide 3-kinase may be involved in regulating microtubule responses to insulin and platelet-derived growth factor.

Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton.

Kapeller R, Chakrabarti R, Cantley L, Fay F, Corvera S
Mol Cell Biol. 1993; 13: 6052-63

Phosphatidylinositol (PI)-3' kinase catalyzes the formation of PI 3,4-diphosphate and PI 3,4,5-triphosphate in response to stimulation of cells by platelet-derived growth factor (PDGF). Here we report that tyrosine-phosphorylated PDGF receptors, the p85 subunit of PI-3' kinase (p85), and activated PI-3' kinase are found in isolated clathrin-coated vesicles within 2 min of exposure of cells to PDGF, indicating that both receptor and activated PI-3' kinase enter the endocytic pathway. Immunofluorescence analysis of p85 in serum-starved cells revealed a punctate/reticular staining pattern, concentrated in the perinuclear region and displaying high focal concentration at the centrosome. In addition, partial coalignment of p85 with microtubules was observed after optical sectioning microscopy and image reconstruction. The association of p85 with the microtubule network was further evidenced by the microtubule-depolymerizing drug nocodazole, which caused a redistribution of p85 from the perinuclear region to the cell periphery. Interestingly, the most significant effect of PDGF on the distribution of p85 was an increase in the staining intensity of this protein in the perinuclear region, and this effect was eliminated by prior treatment of cells with nocodazole. These results suggest that PDGF receptor-p85 complexes internalize and transit in association with the microtubule cytoskeleton. In addition, the high concentration of p85 in intracellular structures in the absence of PDGF stimulation suggests additional roles for this protein independent of its association with receptor tyrosine kinases.

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

Jucker M, Feldman RA
J Biol Chem. 1995; 270: 27817-22

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl.

Jain SK, Susa M, Keeler ML, Carlesso N, Druker B, Varticovski L
Blood. 1996; 88: 1542-50

BCR/abl is a chimeric oncogene implicated in the pathogenesis of human chronic myelogenous leukemia. Expression of the BCR/abl gene induces hematologic malignancies in transgenic mice and transformation of interleukin-3-dependent hematopoietic cells. The mechanism of BCR/abl-mediated transformation of hematopoietic cells is poorly understood and involves activation of at least two signaling pathways, p21ras and PI 3-kinase. Here we report that PI 3,4-P2 and PI 3,4,5-P3, the enzymatic products of PI 3-kinase, accumulate in metabolically labeled transformed hematopoietic cells, in contrast to our previous report on the lack of accumulation of PI 3-kinase products in nontransformed NIH 3T3 fibroblasts that express p210 BCR/abl. Transformed cells also have increased PI 3-kinase activity in total cell extracts and membrane fractions. Activation of PI 3-kinase occurs by occupancy of SH2 domains of PI 3-kinase regulatory subunit, p85, by phosphorylated YXXM motifs. Therefore, we investigated whether BCR/abl binds to p85 and whether this binding is mediated by interaction of p85 SH2 domains with YXXM motif of BCR/abl. Association of p210 BCR/abl with p85 in immune complexes and with p85 SH2 domains was evident in hematopoietic cells that express the wt p210 BCR/abl. However, the binding of BCR/abl to p85 SH2 domains was abolished in cells expressing mutant, temperature-sensitive (ts) p210 BCR/abl in which the tyrosine in the YXXM motif of p210 BCR/abl was replaced by histidine. Despite lack of direct interaction with p85 SH2 domains, expression of ts p210 BCR/abl resulted in rapid, time-dependent activation of total and membrane-associated PI 3-kinase and increased PI 3-kinase activity in anti-P-tyr and anti-abl immunoprecipitates. These data suggest that BCR/abl-induced activation of PI 3-kinase in hematopoietic cells does not require binding of p85 SH2 domains to BCR/abl gene product and involves interaction with other tyrosine phosphorylated intermediate proteins.

p85alpha gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-Kinase), p50alpha, p55alpha, and p85alpha, with different PI 3-kinase activity elevating responses to insulin.

Inukai K, Funaki M, Ogihara T, Katagiri H, Kanda A, Anai M, Fukushima Y, Hosaka T, Suzuki M, Shin BC, Takata K, Yazaki Y, Kikuchi M, Oka Y, Asano T
J Biol Chem. 1997; 272: 7873-82

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85alpha, but the SH3 and bcr homology domains of p85alpha were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85alpha gene product. We suggest that this protein be called p50alpha. Northern blotting using a specific DNA probe corresponding to p50alpha revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50alpha protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50alpha. Taken together, these observations indicate that the p85alpha gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85alpha, p55alpha, and p50alpha), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50alpha forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85alpha and p55alpha associate only with p110 that can be wortmannin-labeled. Furthermore, p50alpha exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50alpha expression in the liver and its marked responsiveness to insulin, p50alpha appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three alpha isoforms has a different function and may have specific roles in various tissues.

Heterodimeric deoxynucleoside kinases of Lactobacillus acidophilus R-26: functional assignment of subunits using limited proteolysis controlled by end-product inhibitors.

Ikeda S, Ma GT, Ives DH
Biochemistry. 1994; 33: 5328-34

Heterodimeric quaternary structures for two enzyme complexes from Lactobacillus acidophilus R-26 exhibiting deoxycytidine kinase/deoxyadenosine kinase (I) and deoxyguanosine kinase/deoxyadenosine kinase(II) activities have been proven by the following steps: (1) separation of each complex into two components on SDS-PAGE at pH 6.6; (2) N-terminal amino acid sequencing of each component; (3) functional assignment of each component by differential limited proteolysis. The third step was facilitated by the finding that the binding of a specific end-product inhibitor dNTP, to each kinase active site makes the corresponding kinase subunit resistant to trypsin, while leaving the heterologous kinase subunit susceptible to proteolysis. Analysis on SDS-PAGE has revealed only two fragments (15.8 and 11.0 kDa) following proteolysis of dCyd kinase/dAdo kinase (I) with trypsin in the presence of dATP. This may indicate that the kinase polypeptide chain (27.2 kDa) not protected by dNTP is cut by trypsin at a single specific site, with concomitant loss of activity. Thus, this work presents a unique approach to the clarification of structure and function of enzymes composed of heterologous subunits.

Fine mapping of human PI 3-kinase associated p85 alpha transcripts in the YAC contig surrounding the spinal muscular atrophy gene.

Huschenbett J, Gasch A, Katzer A, Speer A
Hum Genet. 1994; 94: 427-31

During a search for transcribed sequences within the gene region for autosomal recessive spinal muscular atrophy (SMA), two cDNA clones were isolated from a human fetal brain and an adult spinal cord cDNA library, respectively, by use of the cosmid LA96B (LAS96). The clones sized 950 bp and 1733 bp detect a 7.7-kb transcript in all tested human tissues. An additional transcript of 6.6 kb is detectable in brain and kidney, and faintly in skeletal muscle and liver. Using comparative human Northern blot analysis, the isolated LA96B cDNA clones could be identified as parts of the 3' untranslated region from the phosphatidylinositol 3 (PI3)-kinase associated p85 alpha transcripts; these were unknown up to now. The 5' end of the gene was mapped to YAC-EFTA:A, whereas the 3' end was localized within the distal overlapping YAC 85 flanking the SMA candidate gene region.

Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase.

Hu Q, Klippel A, Muslin AJ, Fantl WJ, Williams LT
Science. 1995; 268: 100-2

Phosphatidylinositol (Pl)-3 kinase is one of many enzymes stimulated by growth factors. A constitutively activated mutant, p110, that functions independently of growth factor stimulation was constructed to determine the specific responses regulated by Pl-3 kinase. The p110 protein exhibited high specific activity as a Pl-3 kinase and as a protein kinase. Expression of p110 in NIH 3T3 cells induced transcription from the fos promoter. Co-expression of dominant negative Ras blocked this response. When expressed in Xenopus laevis oocytes, p110 increased the amount of guanosine 5'-triphosphate-bound Ras, caused activation of the Ras effector Raf-1, and induced Ras-dependent oocyte maturation. These findings show that Pl-3 kinase can stimulate diverse Ras-dependent cellular processes, including oocyte maturation and fos transcription.

Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

Hu P, Mondino A, Skolnik EY, Schlessinger J
Mol Cell Biol. 1993; 13: 7677-88

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.

Phosphorylation of cbl after stimulation of Nb2 cells with prolactin and its association with phosphatidylinositol 3-kinase.

Hunter S, Koch BL, Anderson SM
Mol Endocrinol. 1997; 11: 1213-22

Stimulation of Nb2 cells with PRL results in the rapid phosphorylation of a 120-kDa protein identified as the adapter protein cbl on tyrosine residues. Maximal phosphorylation of cbl occurs at 20 min after PRL stimulation and declines thereafter. Stimulation with as little as 5 nM PRL resulted in the phosphorylation of cbl; increasing the concentration of PRL to 100 nM had only a minimal effect upon the phosphorylation of cbl. The cbl protein appears to be constitutively associated with grb2 and the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). The constitutive association of cbl with the p85 subunit of PI 3-kinase was observed in Nb2 cells as well as in 32Dcl3 cells transfected with either the rat Nb2 (intermediate) form of the PRL receptor or the long form of the human PRL receptor. A glutathione S-transferase fusion protein encoding the SH3 domain of the p85 subunit of PI 3-kinase bound to cbl in lysates of both unstimulated and PRL-stimulated Nb2 cells; however, neither of the SH2 domains of p85 bound to cbl under the same conditions. PRL stimulation increased the cbl-associated PI kinase activity. The majority of PI kinase activity appeared to be cbl-associated after PRL stimulation. These results suggest that cbl may function as an adapter protein in PRL-mediated signaling events and regulate activation of PI 3-kinase. Our model suggests that the p85 subunit of PI 3-kinase is constitutively associated with cbl through binding of the p85 SH3 domain to a proline-rich sequence in cbl. After the tyrosine phosphorylation of cbl, an SH2 domain(s) of p85 binds to a specific phosphorylation site(s) in cbl, leading to the activation of PI 3-kinase.

The gene that encodes the phosphatidylinositol-3 kinase regulatory subunit (p85 alpha) maps to chromosome 13 in the mouse.

Hoyle J, Yulug IG, Egan SE, Fisher EM
Genomics. 1994; 24: 400-2

Directed mutagenesis of deoxyguanosine site at arginine 79 up-regulates turnover on deoxyadenosine kinase subunit of heterodimeric enzyme from Lactobacillus acidophilus R26.

Hong YS, Ma GT, Ives DH
J Biol Chem. 1995; 270: 6602-6

Examination of conserved motifs on the cloned subunits of the deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidophilus R-26 has begun with the Asp-Arg-Ser (DRS) motif. Replacement of Asp-78 of both subunits with Glu, Ala, or Asn reduced dGK and dAK activities to less than 0.2%, whereas replacement of Arg-79 with Lys, either on both subunits in tandem (R79K), or on the dGK subunit only (R79K:dGK), yielded active but kinetically modified enzymes. These were partially purified, and their kinetic and regulatory properties were analyzed. For dAK activity, the Vmax of the R79K:dGK enzyme was increased 28-fold, with no change in the limiting Km for dAdo, but with a slightly reduced Km for MgATP. The V/K efficiency ratio of dAK was also increased 29-fold, but that of dGK was decreased to 5-10% due to a 10-fold increase in Km for dGuo and a reduced Vmax. Therefore, the R79K substitution seems to have a greater effect on dGuo binding than on that of dAdo, but dGK modification appears to produce a stimulatory conformational effect on the opposite subunit, resembling the known unidirectional activation of dAK by either dGuo or dGTP.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

Holt KH, Olson L, Moye-Rowley WS, Pessin JE
Mol Cell Biol. 1994; 14: 42-9

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

N-terminal domains of the class ia phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signaling.

Hill KM, Huang Y, Yip SC, Yu J, Segall JE, Backer JM
J Biol Chem. 2001; 276: 16374-8

Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading edge of the cell. We have now examined the role of the p85alpha regulatory subunit in greater detail. Microinjection of recombinant p85alpha into MTLn3 cells blocked both EGF-stimulated mitogenic signaling and lamellipod extension. In contrast, a truncated p85(1-333), which lacks the SH2 and iSH2 domains and does not bind p110, had no effect on EGF-stimulated mitogenesis but still blocked EGF-stimulated lamellipod extension. Additional deletional analysis showed that the SH3 domain was not required for inhibition of lamellipod extension, as a construct containing only the proline-rich and breakpoint cluster region (BCR) homology domains was sufficient for inhibition. Although the BCR domain of p85 binds Rac, the effects of the p85 constructs were not because of a general inhibition of Rac signaling, because sorbitol-induced JNK activation in MTLn3 cells was not inhibited. These data show that the proline-rich and BCR homology domains of p85 are involved in the coupling of p85/p110 PI 3-kinases to regulation of the actin cytoskeleton. These data provide evidence of a distinct cellular function for the N-terminal domains of p85.

Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.

Hiles ID, Otsu M, Volinia S, Fry MJ, Gout I, Dhand R, Panayotou G, Ruiz-Larrea F, Thompson A, Totty NF, et al.
Cell. 1992; 70: 419-29

Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.

Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.

He TC, Zhuang H, Jiang N, Waterfield MD, Wojchowski DM
Blood. 1993; 82: 3530-8

Using an active, HAI epitope-tagged form of the murine erythropoietin (EPO) receptor and via direct coimmunoprecipitation, the p85 regulatory subunit of phosphatidyl inositol-3 kinase (p85/PI3-K) is shown to associate with the EPO receptor in transfected FDC-P1 cell lines. Coimmunoprecipitation of p85 with epitope-tagged EPO receptors was observed initially in FDC-HER cells labeled metabolically with [32P]orthophosphate, and association of these factors was confirmed by Western analyses of receptor immunoprecipitates using p85 antiserum. Interestingly, this association occurred in the absence of ligand, and exposure of FDC-HER cells to EPO did not detectably affect levels of receptor-associated p85 or overall levels of p85 phosphorylation. However, EPO was observed to stimulated the rapid formation of phosphatidylinositol 32P-phosphate in FDC-HER and FDC-ER cells. Through baculovirus-mediated expression of epitope-tagged EPO receptor forms in SF9 cells, domains for p85 association were mapped. Analyses of receptor forms with cytosolic truncations and deletions delineated a candidate subdomain for p85 binding to an essential extended box-2 region (P329-E374; including a putative motif for SH2 binding, Y343LVL). These findings extend a mechanistic alignment between the EPO receptor and protein tyrosine kinase-encoding receptors that likewise activate PI3-K, and expand the importance of further defining pathways to PI3-K activation.

Inhibitory effect of wortmannin on phosphatidylinositol 3-kinase-mediated cellular events.

Hazeki O, Hazeki K, Katada T, Ui M
J Lipid Mediat Cell Signal. 1996; 14: 259-61

Insulin-induced activation of phosphoinositide 3-kinase in Fao cells.

Hayashi T, Okamoto M, Yoshimasa Y, Inoue G, Yamada K, Kono S, Shigemoto M, Suga J, Kuzuya H, Nakao K
Diabetologia. 1996; 39: 515-22

Phosphoinositide 3-kinase (PI3-kinase) plays a crucial role in insulin signal transduction. We studied the molecular mechanism of the insulin-induced activation of PI3-kinase in rat hepatoma Fao cells using an antibody against the 110-kDa catalytic subunit (p110) and two against the 85-kDa regulatory subunit (p85 alpha). PI3-kinase activity increased 1.6-fold in anti-p85 immunoprecipitates after insulin stimulation, whereas it did not increase when cell lysates were first immunoprecipitated with anti-phosphotyrosine or anti-insulin receptor substrate-1 (IRS-1), then with anti-p85, suggesting that the PI3-kinase which associates with tyrosyl phosphoproteins including IRS-1 is responsible for the increase in kinase activity. The activated PI3-kinase molecules constituted 4-6% of the total PI3-kinase, and their specific activity was 11-14 times higher than that of the basal state. Anti-p110 recognized the catalytically active form of p110, and immunoprecipitated p110 only after exposure to insulin. Hence, the epitope of anti-p110, P200-C215, seems to be included in the portion of p110, the conformation of which is changed by insulin stimulation. We conclude that, in response to insulin stimulation, only a small fraction of p85 in the PI3-kinase pool associates with tyrosyl phosphoproteins including IRS-1, and that the specific activity of p110 is increased presumably through a conformational change including the P200-C215 region.

The alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase is phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor.

Hayashi H, Nishioka Y, Kamohara S, Kanai F, Ishii K, Fukui Y, Shibasaki F, Takenawa T, Kido H, Katsunuma N, et al.
J Biol Chem. 1993; 268: 7107-17

We have shown previously that insulin stimulated the tyrosine phosphorylation of the alpha-type 85-kDa subunit (p85) of phosphatidylinositol (PI) 3-kinase in vitro and in vivo. In the present work, we identified the major tyrosine phosphorylation sites of the alpha-type p85 by the insulin receptor. [32P]Phosphopeptides obtained from lysylendopeptidase digestion of phosphorylated alpha-type p85 in intact cells after insulin treatment were analyzed using reverse-phase high performance liquid chromatography and thin layer electrophoresis. The tyrosine phosphorylation sites of alpha-type p85 in vivo were assigned to three major phosphopeptides, designated p1, p2, and p3. Highly purified insulin receptor also phosphorylated the purified p85 of PI 3-kinase from the bovine thymus at p1. The purified glutathione S-transferase (GST)-p85 (alpha-type) fusion protein and its truncated proteins from Escherichia coli were also phosphorylated by the purified insulin receptor at p1, p2, and p3 in vitro. Analysis of [32P]phosphopeptide of the truncated GST-p85 (alpha-type) fusion proteins and radiosequence analysis revealed that the p1, p2, and p3 phosphopeptides were phosphorylated at tyrosines 607, 580, and 368, respectively. In addition, phenylalanine substitutions at tyrosine 607 and 580 reduced the p1 and p2 phosphopeptides in vivo, respectively. We conclude that the alpha-type p85 of PI 3-kinase was phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor in vivo.

Specific association of the beta isoform of the p85 subunit of phosphatidylinositol-3 kinase with the proto-oncogene c-cbl.

Hartley D, Meisner H, Corvera S
J Biol Chem. 1995; 270: 18260-3

Phosphatidylinositol-3 kinase (PI-3 kinase) has been implicated in cellular events such as mitogenic signaling, actin organization, and receptor sorting. The p85 subunit of PI-3 kinase contains multiple domains capable of protein-protein interactions that may contribute to mediate the multiple physiological functions of this enzyme. Here, we demonstrate that antibodies raised against the p85 subunit of PI-3 kinase immunoprecipitate a single tyrosine-phosphorylated protein of 120 kDa (pp120) from lysates of activated Jurkat T cells and A20 B cells. This protein is the only significant phosphotyrosine-containing protein in p85 immunoprecipitates from these cells, and it cannot be detected in immunoprecipitates of other signaling proteins such as PLC gamma. Furthermore, antibodies specific for the beta isoform of p85 but not antibodies specific for the alpha isoform immunoprecipitate this tyrosine-phosphorylated protein. pp120 completely comigrates with the proto-oncogene c-cbl, which is a 120 kDa protein product abundant in lymphoid cells. Furthermore, immunoblots of p85 immunoprecipitates using antibodies raised against c-cbl detect a band at exactly the position of pp120. In addition, p85 can be detected in immunoblots of c-cbl immunoprecipitates. Thus, pp120 appears to correspond to c-cbl. A direct association between c-cbl and p85 can be observed in vitro using a fusion protein comprising the Src homology 2 (SH2) domains of p85, and this binding is abolished by phenyl phosphate, suggesting that the interaction is mediated through phosphotyrosine-SH2 domain interactions. Thus, these results show important functional differences between the alpha and beta isoforms of p85 in vivo and point to c-cbl as a potentially important mediator of some of the functions of PI-3 kinase in intact cells.

Association of phosphatidylinositol 3-kinase with SHC in chronic myelogeneous leukemia cells.

Harrison-Findik D, Susa M, Varticovski L
Oncogene. 1995; 10: 1385-91

Expression of p210 BCR/abl oncoprotein transforms hematopoietic cells. P210 BCR/abl tyrosine kinase induces tyrosine phosphorylation of Shc, and activation of p21ras and PI 3-Kinase. Here we show that PI 3-Kinase associates with Shc in cells transformed by BCR/abl oncoprotein. Immunoprecipitation of Shc from cells expressing p210 BCR/abl had 7.5-fold increase in PI 3-Kinase activity compared to parental cells. Tyrosine phosphorylated Shc specifically bound to the C-SH2 domain of the p85 subunit of PI 3-Kinase. The p85 SH3 domain also interacted with Shc in cell lysates from parental and transformed cells. The binding of p85 SH3 domain to Shc was substantially higher in BCR/abl transformed than in parental cells. Phenylphosphate blocked p85 SH2 mediated association with Shc but enhanced the binding of the p85 SH3 domain to Shc. The N-terminal proline-rich region of Shc between A263 and N273 specifically blocked the interaction of p85 SH3 domain with Shc. Our results indicate that PI 3-Kinase interacts with Shc directly in hematopoietic cells which express p210 BCR/abl oncoprotein.

Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments.

Guo X, Ghalayini AJ, Chen H, Anderson RE
Invest Ophthalmol Vis Sci. 1997; 38: 1873-82

PURPOSE: Phosphatidylinositol 3-kinase (PI 3-kinase) plays important roles in mitogenesis, vesicular trafficking, actin rearrangement, and prevention of apoptotic cell death in nonocular tissues. This investigation looked for whether PI 3-kinase is present in bovine rod photoreceptors and if light has any effect on its activity. METHODS: Bleached (BROS) and dark-adapted (DROS) rod outer segments were prepared from frozen bovine retinas and immunoblotted or immunoprecipitated with antibodies against the regulatory (p85) or catalytic (p110) subunits of PI 3-kinase. Kinase activity was measured in the immunoprecipitates and the reaction products were identified by high-performance liquid chromatography (HPLC). The amount of PI 3-kinase in membrane and cytosol fractions was determined by densitometry of immunoblots. RESULTS: Immunoblot analysis showed the presence of 85 and 110 kDa proteins in ROS. PI 3-kinase immunoprecipitated by anti-p85 antibody converted PI to PI-3-P and PI-4-P to PI-3,4-P2, as determined by HPLC analysis of the deacylated products. The PI 3-kinase activity in these ROS preparations was sensitive to wortmannin, a potent inhibitor of PI 3-kinase, at low concentrations (IC50 3 nM). Immunoprecipitates (IPs) showed activity twice as high in BROS as in DROS. The IPs of ROS membranes but not cytosol maintained the light-dark difference. However, measurement of anti-p85 and anti-p110 immunoreactivities on western blots of ROS, ROS membranes, and ROS cytosol did not show any light-dark differences. CONCLUSIONS: PI 3-kinase is present in bovine rod outer segments and its activity appears to be greater in light-adapted retinas.

Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein.

Gout I, Middleton G, Adu J, Ninkina NN, Drobot LB, Filonenko V, Matsuka G, Davies AM, Waterfield M, Buchman VL
EMBO J. 2000; 19: 4015-25

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38.

Fukazawa T, Reedquist KA, Panchamoorthy G, Soltoff S, Trub T, Druker B, Cantley L, Shoelson SE, Band H
J Biol Chem. 1995; 270: 20177-82

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma 1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.

Structural organization and alternative splicing of the murine phosphoinositide 3-kinase p85 alpha gene.

Fruman DA, Cantley LC, Carpenter CL
Genomics. 1996; 37: 113-21

Phosphoinositide 3-kinase is a lipid and protein kinase composed of a 110-kDa catalytic subunit and an 85-kDa (p85) or 55-kDa (p55) regulatory subunit. In mammals, at least two genes encode catalytic subunits, and at least three genes encode regulatory subunits. Here we report the cloning and structural analysis of the mouse p85 alpha gene. The translated portion of mouse p85 alpha is encoded by 15 exons that span at least 40 kb. We have cloned an alternatively spliced form of p85 alpha from both mouse and rat cDNA libraries. This splice variant encodes a unique 5'-untranslated region, start codon, and 6-amino-acid aminoterminus followed by the carboxyterminal 418 amino acids of p85 alpha. A corresponding exon is present within the p85 alpha genomic locus. In vitro transcription and translation of the splice variant cDNA generate a protein of approximately 45 kDa that is reactive with an anti-p85 alpha antiserum. Northern blot analysis of mouse tissues reveals differential expression of full-length and alternatively spliced p85 alpha, with the splice variant most abundant in the liver.

Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase.

Dombrosky-Ferlan PM, Corey SJ
Oncogene. 1997; 14: 2019-24

Ligand binding of multi-chain antigen receptors and hematopoietin/cytokine receptors results in rapid activation of protein tyrosine kinase (PTK)-dependent signalling molecules such as phosphatidylinositol 3-kinase (PI 3-kinase). Co-precipitation studies have shown that Src-related PTK, such as Lyn, associates with the p85 regulatory subunit of PI 3-kinase via SH2 and SH3 domain binding with their cognate ligands. More recent studies have shown that the proto-oncogene product Cbl co-precipitates with p85 following engagement of cytokine and antigen receptors. As opposed to in vitro co-precipitation studies, the yeast two-hybrid screen reveals in vivo protein-protein interactions. Using the yeast two-hybrid screen, we demonstrate an in vivo association of Lyn's SH3 and SH2 domains with the proline-rich domain of Cbl. Lyn's SH3 and SH2 domains do not interact with p85 in the yeast two-hybrid screen, as would be predicted from glutathione-S-transferase (GST) fusion protein pull-down or co-immunoprecipitation studies from whole cell lysates. However, the SH3 domain of p85 interacts with the proline-rich domain of Cbl. When yeast were transformed with catalytic Lyn, an interaction between p85's SH2 domain and Cbl occurred. From the data, we propose the following three step process of PI 3-kinase activation: (1) complexes of Lyn-Cbl and Cbl-p85 exist without ligand stimulation, (2) upon ligand binding, Lyn becomes active and phosphorylates Cbl, and (3) Cbl's tyrosine phosphorylated residue serves as a docking site for the SH2 domains of p85 - thereby stabilizing the complex and activating PI 3-kinase. The yeast two-hybrid system can be used to dissect the precise mechanisms of in vivo protein-protein interactions, including those between phosphotyrosine and SH2-containing proteins.

Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase.

Didichenko SA, Tilton B, Hemmings BA, Ballmer-Hofer K, Thelen M
Curr Biol. 1996; 6: 1271-8

BACKGROUND: Phosphoinositide 3-kinase (PI 3-kinase) activity is required for mitogenic signaling and for secretory responses. Cell activation is presumed to cause the translocation of PI 3-kinase from the cytosol to the plasma membrane where the kinase interacts with its substrate phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted and therefore constitutively active kinase could help elucidate the role of PI 3-kinase in intracellular signaling. RESULTS: The membrane-targeting sequence of Ha-Ras, containing the consensus sequence for palmitoylation and farnesylation, was fused to the carboxyl terminus of p110 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor directed PI 3-kinase to the membrane and led to constitutively elevated phosphatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Expression of membrane-targeted PI 3-kinase resulted in the continuous activation of downstream effectors, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synthase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was marginal in transfectants expressing non-membrane-targeted PI 3-kinase. Multiple phosphorylation of the cytosolic factor p47phox is required for the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the expression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47phox. CONCLUSIONS: Targeting of PI 3-kinase to the site of its preferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways.

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Dhand R, Hiles I, Panayotou G, Roche S, Fry MJ, Gout I, Totty NF, Truong O, Vicendo P, Yonezawa K, et al.
EMBO J. 1994; 13: 522-33

Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.

Functional interactions of phosphatidylinositol 3-kinase with GTPase-activating protein in 3T3-L1 adipocytes.

DePaolo D, Reusch JE, Carel K, Bhuripanyo P, Leitner JW, Draznin B
Mol Cell Biol. 1996; 16: 1450-7

The role of phosphatidylinositol (PI) 3-kinase in specific aspects of insulin signaling was explored in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity by LY294002 or wortmannin significantly enhanced basal and insulin-stimulated GTPase-activating protein (GAP) activity in 3T3-L1 adipocytes. Furthermore, removal of the inhibitory influence of PI 3-kinase on GAP resulted in dose-dependent decreases in the ability of insulin to stimulate p21ras. This effect was specific to adipocytes, as inhibition of PI 3-kinase did not influence GAP in either 3T3-L1 fibroblasts, Rat-1 fibroblasts, or CHO cells. Immunodepletion of either of the two subunits of the PI 3-kinase (p85 or p110) yielded similar activation of GAP, suggesting that catalytic activity of p110 plays an important role in controlling GAP activity in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity in 3T3-L1 adipocytes resulted in abrogation of insulin-stimulated glucose uptake and thymidine incorporation. In contrast, effects of insulin on glycogen synthase and mitogen-activated protein kinase activity were inhibited only at higher concentrations of LY294002. It appears that in adipocytes, P1 3-kinase prevents activation of GAP. Inhibition of PI 3-kinase activity or immunodepletion of either one of its subunits results in activation of GAP and decreases in GTP loading of p21ras.

Thymocyte activation induces the association of phosphatidylinositol 3-kinase and pp120 with CD5.

Dennehy KM, Broszeit R, Garnett D, Durrheim GA, Spruyt LL, Beyers AD
Eur J Immunol. 1997; 27: 679-86

CD5 is a glycoprotein expressed on thymocytes, T cells, and a subset of B cells. Antibody-mediated cross-linking studies or studies on CD5 knockout mice implicate CD5 as a co-stimulatory or negative regulatory molecule. CD5 is rapidly phosphorylated on tyrosine (Y) residues following Tcell activation. Y429 and Y441 occur in an imperfect immunoreceptor tyrosine-based activation motif (ITAM)-like sequence. We investigated whether phosphatidylinositol (PI) 3-kinase, which binds to tyrosine-phosphorylated ITAM, interacts with CD5 following T cell activation. PI 3-kinase activity and the regulatory p85 subunit of PI 3-kinase associated with CD5 in pervanadate-stimulated, but not in unstimulated thymocytes. Cellular p85 as well as the recombinant Src homology 2 (SH2) domains of p85 bound a tyrosine-phosphorylated peptide encompassing Y463 with approximately threefold greater affinity than a doubly tyrosine-phosphorylated Y429-Y441 peptide. Binding of the C-SH2 domain to the Y463 phosphopeptide, together with preferential binding of the N-SH2 domain to the Y429-Y441 phosphopeptide, suggests a bivalent interaction. A 120-kDa phosphoprotein (pp120) associated with CD5 and specifically with the Y429-Y441 phosphopeptide in stimulated thymocytes. We conclude that stimulation of thymocytes with pervanadate induces the recruitment of PI 3-kinase and pp120 to CD5.

Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor.

Damen JE, Mui AL, Puil L, Pawson T, Krystal G
Blood. 1993; 81: 3204-10

The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as we and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. We now present data showing that, in response to Ep, the 85-Kd regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) becomes immunoprecipitable with antiphosphotyrosine antibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine-phosphorylated EpR, either directly or through a 93- or 70-Kd tyrosine-phosphorylated intermediate. The activity of this EpR associated PI 3-kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3-kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylated motifs on the EpR.

Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.

Damen JE, Cutler RL, Jiao H, Yi T, Krystal G
J Biol Chem. 1995; 270: 23402-8

We recently reported that phosphatidylinositol (PI) 3-kinase becomes associated with the activated erythropoietin receptor (EpR), most likely through the Src homology 2 (SH2) domains within the p85 subunit of PI-3 kinase and one or more phosphorylated tyrosines within the EpR. We have now investigated this interaction in more detail and have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these fusion proteins to SDS-denatured EpRs, that this binding is direct. Moreover, both in vitro competition studies, involving phosphorylated peptides corresponding to the amino acid sequences flanking the eight tyrosines within the intracellular domain of the EpR, and in vivo studies with mutant EpRs bearing tyrosine to phenylalanine substitutions, indicate that phosphorylation of Tyr503 within the EpR is essential for the binding of PI 3-kinase. The presence of PI 3-kinase activity in EpR immunoprecipitates from DA-3 cells infected with wild-type but not Y503F EpRs confirms this finding. Our results demonstrate that the SH2 domains of p85 can bind, in addition to their well established Tyr-Met/Val-X-Met consensus binding sequence, a Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of erythropoietin-induced tyrosine phosphorylations and proliferation of wild-type and Y503F EpR-infected DA-3 cells revealed no differences. However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the erythropoietin-induced proliferation of both cell types, suggesting that PI 3-kinase is activated in Y503F EpR expressing cells. This was confirmed by carrying out PI 3-kinase assays with anti-phosphotyrosine immunoprecipitates from erythropoietin-stimulated Y503F EpR-infected DA-3 cells and suggested that PI 3-kinase has a role in regulating erythropoietin-induced proliferation, but at a site distinct from the EpR.

Production of monoclonal antibodies specific to the carboxyl terminal region of the 85 kDa subunit of phosphatidylinositol 3-kinase: use of the antibodies in recognition of mutant p85.

Daduang S, Nagata S, Matsuda M, Yamori T, Onodera K, Fukui Y
Immunol Cell Biol. 1995; 73: 134-9

We have established two hybridomas producing mAb to the carboxyl terminal region of phosphatidylinositol-3 kinase 85 kDa subunit type alpha (p85 alpha). Analysis using deletion mutants of p85 revealed that epitopes for the two mAb were located on the border of the src homology 2 (SH2) sequence located at the carboxyl end of p85. They immunoprecipitated free p85 efficiently, but reactivity to p85 bound to p110 was very weak. Together with the mAb which we have reported previously, a panel of mAb that covered the various parts of p85 alpha was obtained. Using this panel, we characterized two mutants of p85 (70 and 50 kDa) expressed in the human colon carcinoma cell line, HCC2998. No wild-type p85 was detected in these cells. A mAb specific to the carboxyl terminal region detected p70 but not p50, suggesting that this region is missing in p50. The panel of mAb is a useful tool to use to analyse mutant forms of p85.

Interaction of the Flt-1 tyrosine kinase receptor with the p85 subunit of phosphatidylinositol 3-kinase. Mapping of a novel site involved in binding.

Cunningham SA, Waxham MN, Arrate PM, Brock TA
J Biol Chem. 1995; 270: 20254-7

We have examined the interactions of the p85 regulatory subunit of phosphatidylinositol 3-kinase with the endothelium-specific Flt-1 receptor tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tail of the Flt-1 receptor in order to identify the site(s) that is responsible for the p85 interactions. A single tyrosine to phenylalanine change at position 1213 inhibits the binding of both p85 SH2 domains. Phosphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophosphorylation. The amino acids following this tyrosine are VNA and thus define a novel binding site for p85.

CD28-mediated costimulation in the absence of phosphatidylinositol 3-kinase association and activation.

Crooks ME, Littman DR, Carter RH, Fearon DT, Weiss A, Stein PH
Mol Cell Biol. 1995; 15: 6820-8

T-cell activation involves two distinct signal transduction pathways. Antigen-specific signaling events are initiated by T-cell receptor recognition of cognate peptide presented by major histocompatibility complex molecules. Costimulatory signals, which are required for optimal T-cell activation and for overcoming the induction of anergy, can be provided by the homodimeric T-cell glycoprotein CD28 through its interaction with the counterreceptors B7-1 and B7-2 on antigen-presenting cells. Ligation of CD28 results in its phosphorylation on tyrosines and the subsequent recruitment and activation of phosphatidylinositol 3-kinase (PI 3-kinase). It has been suggested that the induced association of CD28 and PI 3-kinase is required for costimulation. We report here that ligation of CD19, a heterologous B-cell receptor that also associates with and activates PI 3-kinase upon ligation, failed to costimulate interleukin-2 production. Moreover, pharmacological inhibition of PI 3-kinase activity failed to block costimulation mediated by CD28. By mutational analysis, we demonstrate that disruption of PI 3-kinase association with CD28 also did not abrogate costimulation. These results argue that PI 3-kinase association with CD28 is neither necessary nor sufficient for costimulation of interleukin-2 production. Finally, we identify specific amino acid residues required for CD28-mediated costimulatory activity.

The stress-activated phosphatidylinositol 3-phosphate 5-kinase Fab1p is essential for vacuole function in S. cerevisiae.

Cooke FT, Dove SK, McEwen RK, Painter G, Holmes AB, Hall MN, Michell RH, Parker PJ
Curr Biol. 1998; 8: 1219-22

Polyphosphoinositides have many roles in cell signalling and vesicle trafficking [1-3]. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a recently discovered PIP2 isomer, is ubiquitous in eukaryotic cells and rapidly accumulates in hyperosmotically stressed yeast. PI(3,5)P2 is synthesised from PI(3)P in both yeast and mammalian cells [4,5]. A search of the Saccharomyces cerevisiae genome database identified FAB1, a gene encoding a PIP kinase homologue and potential PI(3)P 5-kinase. Fab1p shows PI(3)P 5-kinase activity both in vivo and in vitro. A yeast strain in which FAB1 had been deleted was unable to synthesise PI(3,5)P2, either in the presence or absence of osmotic shock. A loss of PI(3,5)P2 was observed also in a temperature-sensitive FAB1 strain at the non-permissive temperature. A recombinant glutathione-S-transferase (GST)-Fab1p fusion protein was shown to have selective PI(3)P 5-kinase activity in vitro. Thus, we have demonstrated that Fab1p is a PI(3)P-specific 5-kinase and represents a third class of PIP kinase activity, which we have termed type III. Deletion of the FAB1 gene produces a loss of vacuolar morphology [6]; it is therefore concluded that PI(3,5)P2, the lipid product of Fab1p, is required for normal vacuolar function.

RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex.

Chiu MI, Katz H, Berlin V
Proc Natl Acad Sci U S A. 1994; 91: 12574-8

Rapamycin is a potent immunosuppressant that blocks the G1/S transition in antigen-activated T cells and in yeast. The similar effects of rapamycin in animal cells and yeast suggest that the biochemical steps affected by rapamycin are conserved. Using a two-hybrid system we isolated mammalian clones that interact with the human FK506/rapamycin-binding protein (FKBP12) in the presence of rapamycin. Specific interactors, designated RAPT1, encode overlapping sequences homologous to yeast Tor, a putative novel phosphatidylinositol 3-kinase. A region of 133 amino acids of RAPT1 is sufficient for binding to the FKBP12/rapamycin complex. The corresponding region in yeast Tor contains the serine residue that when mutated to arginine confers resistance to rapamycin. Introduction of this mutation into RAPT1 abolishes its interaction with the FKBP12/rapamycin complex.

Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase.

Chen HC, Guan JL
Proc Natl Acad Sci U S A. 1994; 91: 10148-52

The focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To gain insight into FAK function, we examined the potential interaction of FAK with intracellular signaling molecules containing the Src homology 2 domains. We report here the stable association of FAK with phosphatidylinositol 3-kinase (PI3-kinase; EC 2.7.1.137) in NIH 3T3 mouse fibroblasts. This interaction was stimulated by cell adhesion concomitant with FAK activation. We also found that recombinant FAK bound to the p85 subunit of PI 3-kinase directly in vitro and that autophosphorylation of recombinant FAK in vitro increased its binding to PI 3-kinase. We detected increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase during cell adhesion and observed direct phosphorylation of p85 by FAK in vitro. Together, these results suggest that PI 3-kinase may be a FAK substrate in vivo and serve as an effector of FAK.

Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase.

Chen HC, Appeddu PA, Isoda H, Guan JL
J Biol Chem. 1996; 271: 26329-34

We have shown previously that cell adhesion or platelet-derived growth factor (PDGF) promotes the in vivo association of focal adhesion kinase (FAK) with phosphatidylinositol (PI) 3-kinase. In vitro experiments indicated that this interaction was mediated by the p85 subunit of PI 3-kinase and dependent on the tyrosine phosphorylation of FAK. Here we report data suggesting that the major autophosphorylation site of FAK (Tyr-397) is the binding site for the SH2 domains of p85 in vitro and is also required for the association of FAK with PI 3-kinase in vivo. We also show that Tyr-397 is responsible for the increased FAK:PI 3-kinase association upon PDGF stimulation, implying that no additional site of FAK was involved in its binding to PI 3-kinase after PDGF stimulation. Finally, we present evidence that the interaction of PI 3-kinase with Tyr-397 of FAK stimulates its activity. Together, these results suggest that FAK activation and autophosphorylation at Tyr-397 may lead to its association with PI 3-kinase through the SH2 domains of p85, which can subsequently activate PI 3-kinase during cell adhesion.

p110delta, a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes.

Chantry D, Vojtek A, Kashishian A, Holtzman DA, Wood C, Gray PW, Cooper JA, Hoekstra MF
J Biol Chem. 1997; 272: 19236-41

We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110delta. In addition, we have independently isolated p110delta from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110delta phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110delta is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system.

Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains.

Chalupny NJ, Aruffo A, Esselstyn JM, Chan PY, Bajorath J, Blake J, Gilliland LK, Ledbetter JA, Tepper MA
Eur J Immunol. 1995; 25: 2978-84

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.

Phosphoinositide 3-kinase and the regulation of cell growth.

Carpenter CL, Cantley LC
Biochim Biophys Acta. 1996; 1288: 116-116

Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit.

Carpenter CL, Auger KR, Chanudhuri M, Yoakim M, Schaffhausen B, Shoelson S, Cantley LC
J Biol Chem. 1993; 268: 9478-83

Tyrosine-phosphorylated peptides based on the regions of polyoma virus middle t antigen and the platelet-derived growth factor receptor that bind phosphoinositide 3-kinase are shown to activate this enzyme 2-3-fold in vitro. The concentrations of the peptides required to activate the enzyme are at least 10-1000-fold higher than the dissociation constants of these peptides for the individual SH2 domains of the 85-kDa subunit (KD < 100 nM). Doubly phosphorylated peptides are more effective than singly phosphorylated peptides. The results suggest that a fraction of the cellular phosphoinositide 3-kinase has SH2 domains with relatively low affinity for phosphopeptides and that binding of phosphopeptides to these enzymes causes activation. Thus, SH2 domains may be involved not only in recruiting the enzyme but also in regulating activity.

Cyclic peptide inhibitors of phosphatidylinositol 3-kinase p85 SH2 domain binding.

Burke TR Jr, Nomizu M, Otaka A, Smyth MS, Roller PP, Case RD, Wolf G, Shoelson SE
Biochem Biophys Res Commun. 1994; 201: 1148-53

Cyclic hexameric peptides based on the amino acid sequence "Gly-Xxx-Val-Pro-Met-Leu", where Xxx is either phosphotyrosyl (pTyr) residue or a hydrolytically stable pTyr mimetic, were examined for their ability to bind to the C-terminal SH2 domain of the p85 phosphoinositol 3-kinase (PI 3-kinase). The cyclic peptides retained significant binding affinity relative to their linear counterparts. Potency varied depending on Xxx in the order: phosphonomethyl phenylalanine (Pmp, ID50 = 5.2 microM) < phosphonodifluoromethyl phenylalanine (F2Pmp, ID50 = 2.2 microM) < pTyr (ID50 = 1.0 microM), with Xxx = Tyr being inactive (ID50 > 500 M). Greatly reduced potency was observed when Xxx was of the unnatural D-configuration. The cyclic peptides represent conformationally constrained ligands which should be useful in the development of p85 SH2 domain-directed inhibitors.

Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

Bonnefoy-Berard N, Liu YC, von Willebrand M, Sung A, Elly C, Mustelin T, Yoshida H, Ishizaka K, Altman A
Proc Natl Acad Sci U S A. 1995; 92: 10142-6

Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting and enzymatic assays as one of the 14-3-3-binding proteins in resting or activated cells. Moreover, endogenous 14-3-3 and PI3-K were coimmunoprecipitated from intact T cells. Far-Western blots of gel-purified, immunoprecipitated PI3-K with a recombinant 14-3-3 fusion protein revealed direct binding of 14-3-3 to the catalytic subunit (p110) of PI3-K. Finally, anti-phosphotyrosine immunoprecipitates from activated, 14-3-3-overexpressing cells contained lower PI3-K enzymatic activity than similar immunoprecipitates from control cells. These findings suggest that association of 14-3-3 with PI3-K in hematopoietic (and possibly other) cells regulates the enzymatic activity of PI3-K during receptor-initiated signal transduction.

Characterisation of phosphatidylinositol 3-kinase in PC12 cell extracts and isolation of the enzyme from rat brain extracts.

Baxter RM, Bramham J, Thomason PA, Downes CP, Carter AN
Biochem Soc Trans. 1993; 21: 359-359

Related GAP domains in inositol polyphosphate 5-phosphatase and the p85 subunit of phosphatidylinositol 3-kinase.

Baldwin GS, Zhang QX
Trends Biochem Sci. 1993; 18: 378-80

Association of phosphatidylinositol 3-kinase, via the SH2 domains of p85, with focal adhesion kinase in polyoma middle t-transformed fibroblasts.

Bachelot C, Rameh L, Parsons T, Cantley LC
Biochim Biophys Acta. 1996; 1311: 45-52

Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, becomes activated and phosphorylated on tyrosine in cells transformed with v-src. By cytoimmunofluorescence a sub-fraction of the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) localized in focal adhesion plaques. We examined the possibility that FAK associates with PI 3-kinase. In fibroblasts transformed with polyoma middle t, PI 3-kinase activity co-immunoprecipitated with pp125FAK using two different antibodies against this protein. PP125FAK from middle t-transformed cells associated with a glutathione-S-transferase fusion protein containing the 85-kDa subunit of phosphatidylinositol 3-kinase. Both of the SH2 domains and the SH3 domain of p85 also formed complexes with pp125FAK in vitro. Phosphopeptides that bind to the SH2 domains completely blocked the binding of full-length p85 to pp125FAK, while a peptide that binds to the SH3 domain was ineffective, indicating that the association between p85 and pp125FAK is mediated by the SH2 domains of p85.

Platelet-derived growth factor-induced formation of tensin and phosphoinositide 3-kinase complexes.

Auger KR, Songyang Z, Lo SH, Roberts TM, Chen LB
J Biol Chem. 1996; 271: 23452-7

Tensin is an SH2 domain-containing cytoskeletal protein that binds to and caps actin filaments. Investigation of signal transduction mechanisms associated with tensin revealed the presence of phosphoinositide 3-kinase (PI 3-kinase) activity in tensin immunoprecipitates from platelet-derived growth factor-treated cells. Association of PI 3-kinase activity with tensin was transitory, and the amount of activity was approximately 1% of the total PI 3-kinase activity found in anti-phosphotyrosine (anti-pY) immunoprecipitates. In vitro, PI 3-kinase activity associated with the SH2 domain of tensin in a platelet-derived growth factor-dependent manner. The optimal phosphopeptide binding specificity of the SH2 domain of tensin was determined to be phospho-Y (E or D), N, (I, V, or F). Synthetic phosphopeptides containing the sequence YENI could specifically block the association of PI 3-kinase activity with tensin in a dose-dependent manner. These results suggest that PI 3-kinase interacts with the cytoskeleton via the SH2 domain of tensin and may play an important role in platelet-derived growth factor-induced cytoskeletal reorganization that is concomitant with cell migration and proliferation.

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

Antonetti DA, Algenstaedt P, Kahn CR
Mol Cell Biol. 1996; 16: 2195-203

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.

Activation of phosphatidylinositol 3-kinase by prolactin in Nb2 cells.

al-Sakkaf KA, Dobson PR, Brown BL
Biochem Biophys Res Commun. 1996; 221: 779-84

In the present studies, using anti-phosphotyrosine (PY20) and PI3-kinase (p85) antibodies, we have shown that PRL causes activation of phosphatidyl inositol 3-kinase (PI3-kinase) in vitro in a dose- and time-dependent manner in Nb2 cells. PRL activated PI3-kinase was completely inhibited by LY294002 (1 microgram/ml). Stimulation of the cells with PRL also increased tyrosine phosphorylation of the 85-kDa regulatory subunit. Moreover, in vitro kinase assay followed by SDS-PAGE protein separation demonstrated the phosphorylation of several other proteins besides the p85. However, no direct association between p85 and JAK2 tyrosine kinase was observed. These results indicate, for the first time, the involvement of PI3-kinase in PRL-stimulated Nb2 cell growth.

Isolation and characterization of the droPIK57 gene encoding a new regulatory subunit of phosphatidylinositol 3-kinase from Drosophila melanogaster.

Albert S, Twardzik T, Heisenberg M, Schneuwly S
Gene. 1997; 198: 181-9

Mammalian phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in the regulation of various cellular, receptor tyrosine kinase-mediated processes, such as mitogenesis and transformation. PI 3-kinase is composed of a 110-kDa catalytic subunit and a regulatory subunit of 85 kDa or 55 kDa. We have cloned a gene for a regulatory subunit from Drosophila melanogaster, named droPIK57, from head-specific cDNA libraries. The droPIK57 gene encodes a protein containing two SH2 domains with significant sequence homology to those in p85 and p55. Like the p55 subunits, DroPIK57 is missing the SH3 domain and the bcr homology region of the p85 subunit. The short N-terminus as well as the C-terminus of the DroPIK57 protein show no identity to the known PI 3-kinase subunits, suggesting that it is a new member in the family of regulatory subunits. In-situ hybridization and Northern blot analysis indicate a widespread function of this gene during embryogenesis and in the CNS.