Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.     

Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.     

Interleukin 2- and polyomavirus middle T antigen-induced modification of phosphatidylinositol 3-kinase activity in activated T lymphocytes.

Stimulation of activated T lymphocytes with interleukin 2 (IL-2) results in rapid increases in intracellular protein tyrosine phosphorylation. Both the identity of the protein tyrosine kinase (PTK) activated by IL-2 receptor ligation and the identities of the critical target proteins for this PTK remain largely undefined. In this article, we demonstrate that stimulation of activated murine or human T cells with IL-2 for 10 to 30 min induces two- to threefold increases in the level of phosphatidylinositol (PtdIns) 3-kinase activity present in antiphosphotyrosine (p-Tyr) antibody immunoprecipitates from these cells. Furthermore, substantial levels of PtdIns 3-kinase activity were coprecipitated from IL-2-deprived T cells by antibodies to the src-related PTK p59fyn. Cellular stimulation with IL-2 induced a two- to threefold increase in the level of p59fyn-associated PtdIns 3-kinase activity. To examine the effect of a constitutive increase in PtdIns 3-kinase activity on the growth factor responsiveness of activated T cells, murine CTLL-2 cells were transfected with a polyomavirus middle T antigen (MTAg) expression vector. Anti-p-Tyr and anti-p59fyn immunoprecipitates from MTAg-transfected CTLL-2 cells contained three- to sixfold higher levels of PtdIns 3-kinase activity than wild-type cells. Immune complex kinase assays revealed that MTAg expression concomitantly induced a constitutive threefold increase in the PTK activity of p59fyn in these cells. However, stable MTAg expression did not abrogate the dependence of CTLL-2 cells on exogenous IL-2 for continued growth and proliferation.     

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.     

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.     

Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and rho GTPases in NIH 3T3 fibroblasts involvement of both phosphatidylinositol-4-phosphate 5-kinase and phospholipase C.

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4, 5)P2-producing enzyme, PtdIns4P 5-kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30-50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1. Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C.     

The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.     

Phosphoinositide hydrolysis by guanosine 5''-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes.

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen.

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinct phosphatidylinositol 3-kinase lipid products accumulate upon oxidative and osmotic stress and lead to different cellular responses

Signaling by phosphatidylinositol (PI) 3-kinases is mediated by 3-phosphoinositides, which bind to Pleckstrin homology (PH) domains that are present in a wide spectrum of proteins. PH domains can be classified into three groups based on their different lipid binding specificities. Distinct 3-phosphoinositides can accumulate upon PI 3-kinase activation in cells in response to different stimuli and mediate specific cellular responses. In Swiss 3T3 mouse fibroblasts, oxidative stress induced by 1 mM H(2)O(2) caused almost exclusive accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3, 4)P(2)), whereas osmotic stress increased both phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and PtdIns(3,4)P(2) levels. The increase in PtdIns(3,4)P(2) levels, caused by oxidative stress, correlated with the activation of protein kinase B, which has a promiscuous PH domain that binds both PtdIns(3,4,5)P(3) and PtdIns(3, 4)P(2). p70 S6 kinase, another signaling component downstream of PI 3-kinase, however, was not activated by this oxidative stress-induced increase in PtdIns(3,4)P(2) levels. Increased PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) levels in response to osmotic stress did not correlate with protein kinase B activation, because of concomitant activation of an inhibitory pathway, but p70 S6 kinase was activated by osmotic stress. These results demonstrate that PtdIns(3,4)P(2) can accumulate independently of PtdIns(3,4, 5)P(3) and exerts a pattern of cellular responses that is distinct from that induced by accumulation of PtdIns(3,4,5)P(3).     

The pathway for the production of inositol hexakisphosphate in human cells

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.     

Phosphoinositide 3-kinase: A new effector in signal transduction?

Interest in phosphopinositide 3-kinase (PI 3-kinase) has been fuelled by its identification as a major phosphotyrosyl protein detected in cells following growth factor stimulation and oncogenic transformation. It is found complexed with activated growth factor receptors and non-receptor tyrosine kinases, thus suggesting that it participates in the signal transduction pathways initiated by the activation of tyrosine kinases. PI 3-kinase phosphorylates the 3-position in the inositol ring of the well known inositol phospholipids in vitro giving phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [PtdIns3P, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃], respectively. The cellular levels of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ rapidly increase in circumstances where PI 3-kinase becomes complexed with tyrosine kinases. Accumulation of the same lipids also occurs in platelets and neutrophils following stimulation of G-protein linked -thrombin and chemotactic peptide receptors, respectively, leading to speculation that one or both of these lipids is a new second messenger whose function is not yet known. This review brings together recent information on the isolation, characterization and regulation of PI 3-kinase, the cellular occurrence of 3-phosphorylated inositol phospholipids and possible functions of the PI 3-kinase pathway in cell signalling.     

Thrombin stimulates the production of a novel polyphosphoinositide in human platelets

The sequential actions of phosphoinositide 4-kinase and 5-kinase and hydrolysis of phosphatidylinositol (PtdIns) 4,5-P2 are stimulated during platelet activation. Recently, a phosphoinositide 3-kinase has been implicated in signal transduction in several cell types. Stimulation of PtdIns(3,4)P2 synthesis has been shown in polyoma middle T-transformed and platelet-derived growth factor-stimulated cells, and this novel lipid has been implicated in signal transduction and regulation of cell proliferation. We demonstrate the formation of PtdIns(3,4)P2 in human platelets and show that the synthesis of this lipid (and of PtdIns(4,5)P2) is stimulated during activation of platelets by thrombin. This indicates the presence of phosphoinositide 3-kinase activity in platelets. We postulate that PtdIns(3,4)P2 is involved in signal transduction in platelets and discuss the possibility that this novel lipid is a substrate for phospholipase C.     

The type Ialpha inositol polyphosphate 4-phosphatase generates and terminates phosphoinositide 3-kinase signals on endosomes and the plasma membrane

Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Ialpha inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P2 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.     

Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants.

A phosphoinositide kinase specific for the D-3 position of the inositol ring, phosphatidylinositol (PI) 3-kinase, associates with activated receptors for platelet-derived growth factor, insulin, and colony-stimulating factor 1, with products of the oncogenes src, fms, yes, crk, and with polyomavirus middle T antigen. Efficient fibroblast transformation by proteins of the abl and src oncogene families requires activation of their protein-tyrosine kinase activity and membrane association via an amino-terminal myristoylation. We have demonstrated that the PI 3-kinase directly associates with autophosphorylated, activated protein-tyrosine kinase variants of the abl protein. In vivo, this association leads to accumulation of the highly phosphorylated products of PI 3-kinase, PI-3,4-bisphosphate and PI-3,4,5-trisphosphate, only in myristoylated, transforming abl protein variants. Myristoylation thus appears to be required to recruit PI 3-kinase activity to the plasma membrane for in vivo activation and correlates with the mitogenicity of the abl protein variants.     

Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions

Inositol phosphates are well-known signaling molecules, whereas the inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7/IP7) and bis-diphosphoinositol tetrakisphosphate (InsP8/IP8), are less well characterized. We demonstrate physiologic regulation of Dictyostelium chemotaxis by InsP7 mediated by its competition with PtdIns(3,4,5)P3 for binding pleckstrin homology (PH) domain-containing proteins. Chemoattractant stimulation triggers rapid and sustained elevations in InsP7/InsP8 levels. Depletion of InsP7 and InsP8 by deleting the gene for InsP6 kinase (InsP6K/IP6K), which converts inositol hexakisphosphate (InsP6/IP6) to InsP7, causes rapid aggregation of mutant cells and increased sensitivity to cAMP. Chemotaxis is mediated by membrane translocation of certain PH domain-containing proteins via specific binding to PtdIns(3,4,5)P3. InsP7 competes for PH domain binding with PtdIns(3,4,5)P3 both in vitro and in vivo. InsP7 depletion enhances PH domain membrane translocation and augments downstream chemotactic signaling activity.     

Role of TAPP1 and TAPP2 adaptor binding to PtdIns(3,4)P2 in regulating insulin sensitivity defined by knock-in analysis

Insulin sensitivity is critically dependent on the activity of PI3K (phosphoinositide 3-kinase) and generation of the PtdIns(3,4,5)P(3) second messenger. PtdIns(3,4,5)P(3) can be broken down to PtdIns(3,4)P(2) through the action of the SHIPs (Src-homology-2-domain-containing inositol phosphatases). As PtdIns(3,4)P(2) levels peak after those of PtdIns(3,4,5)P(3), it has been proposed that PtdIns(3,4)P(2) controls a negative-feedback loop that down-regulates the insulin and PI3K network. Previously, we identified two related adaptor proteins termed TAPP [tandem PH (pleckstrin homology)-domain-containing protein] 1 and TAPP2 that specifically bind to PtdIns(3,4)P(2) through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P(2). These homozygous TAPP1(R211L/R211L) TAPP2(R218L/R218L) double knock-in mice are viable and exhibit significantly enhanced activation of Akt, a key downstream mediator of insulin signalling. Consistent with increased PI3K and Akt activity, the double knock-in mice display enhanced whole body insulin sensitivity and disposal of glucose uptake into muscle tissues. We also generated wild-type and double TAPP1(R211L/R211L) TAPP2(R218L/R218L) knock-in embryonic fibroblasts and found that insulin triggered enhanced production of PtdIns(3,4,5)P(3) and Akt activity in the double knock-in fibroblasts. These observations provide the first genetic evidence to support the notion that binding of TAPP1 and TAPP2 adap-tors to PtdIns(3,4)P(2) function as negative regulators of the insulin and PI3K signalling pathways.     

Phosphatidylinositol 4,5-bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent upon Ca2+ influx.

Hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate is thought to be intimately involved in agonist-induced changes in intracellular Ca2+ levels. Recently we have shown that human preovulatory follicular fluid, which induces exocytosis in human sperm, can stimulate a rapid, transient increase in sperm cytosolic [Ca2+] [Thomas & Meizel (1988) Gamete Res. 20, 397-411]. We report here that both a Sephadex G-75 column fraction, derived from follicular fluid, and progesterone (a component of both the G-75 fraction and whole follicular fluid) stimulate rapid hydrolysis of PtdIns(4,5)P2 and PtdIns4P in human sperm. We also report that progesterone stimulates a rapid influx of Ca2+ in human sperm. Human spermatozoa were labelled for 24 h with myo-[3H]inositol and then treated with either the G-75 fraction or progesterone. A 30-65% loss of label was detected in PtdIns(4,5)P2 and PtdIns4P within 15 s of stimulus addition; no changes were observed in PtdIns during 2 min of treatment. The loss of label from both lipids was accompanied by an increase in water-soluble inositol phosphates. Production of both InsP3 and InsP2 was seen within 10 s; however, InsP3 was rapidly removed and had reached control levels by 1 min. Similarly, formation of InsP2 reached a peak by 30 s and then began a decline accompanied by a corresponding increase in InsP. No increases in InsP4 were seen in sperm treated in this fashion. Stimulated hydrolysis of the phosphoinositides and release of inositol phosphates were both blocked by the Ca2+ antagonist La3+. Likewise, the progesterone-induced increase in intracellular Ca2+ was inhibited by La3+, and phosphoinositide hydrolysis stimulated by this hormone was dependent upon the presence of extracellular Ca2+.     

Phosphatidylinositol 3-kinase is activated by nerve growth factor and epidermal growth factor in PC12 cells.

The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the regulation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity were assessed in rat pheochromocytoma (PC12) cells. Both NGF and EGF induced a rapid activation of PtdIns 3-kinase as assessed by a dramatic rise in growth factor-induced PtdIns 3-kinase activity found in antiphosphotyrosine immunoprecipitates. The intracellular levels of two of the lipid products of PtdIns 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), also rose dramatically, exhibiting time courses very similar to the appearance of PtdIns 3-kinase in immunoprecipitates. The activation of PtdIns 3-kinase is, therefore, a common event in the signal transduction processes elicited by growth factors stimulating distinct cellular end points in PC12 cells, namely the NGF-induced neuronal differentiation and EGF-stimulated mitogenesis. Thus the intracellular products of this enzyme may function in early biochemical events that are common components of the pathways controlling both differentiation and proliferation.