Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b.

We examined the function of two key DNA methyltransferase (DNMT) enzymes in epigenetic regulation of X-linked cancer/germline (CG-X) antigen genes in human cancer cells, using MAGE-A1, NY-ESO-1, and XAGE-1 as models. In HCT116 cells, genetic knockout of DNMT1 caused moderate activation of CG-X genes, DNMT3b knockout had a negligible effect, and double knockout of both enzymes caused robust gene induction. Similarly, dual DNMT knockout caused dramatic hypomethylation of the MAGE-A1 and NY-ESO-1 promoters, DNMT1 knockout showed moderate hypomethylation, and DNMT3b knockout elicited only slight methylation changes. In contrast, both single and double knockout cells showed significant hypomethylation of the XAGE-1 promoter. RNA interference (RNAi) targeting of DNMT1 in HCT116 cells validated the results seen using genetic knockout cells; however, RNAi targeting of DNMT1 in a different colorectal cancer cell line revealed a greater independent role for DNMT1 in mediating CG-X gene repression and promoter methylation in other cell types. Notably, the histone H3 modification pattern at CG-X promoters was altered following DNMT knockout. DNMT1 or DNMT3b knockout reduced dimethylated lysine-9 (diMe-H3K9) levels, but did not significantly affect dimethylated lysine-4 (diMe-H3K4) or acetylated lysine-9 (Ac-H3-K9) levels. In contrast, dual DNMT1/3b knockout reduced the level of diMe-H3K9 and dramatically increased the levels of diMe-H3K4 and Ac-H3K9 at CG-X gene loci. In summary, DNMT1 and DNMT3b were found to perform both redundant and independent functions in epigenetic regulation of CG-X antigen genes in human cancer cells.

Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha.

BACKGROUND: Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies 'downstream' of phosphoinositide 3-kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is required for maximal activation of PKB. The kinases that phosphorylate PKB are, however, unknown. RESULTS: We have purified 500 000-fold from rabbit skeletal muscle extracts a protein kinase which phosphorylates PKBalpha at Thr308 and increases its activity over 30-fold. We tested the kinase in the presence of several inositol phospholipids and found that only low micromolar concentrations of the D enantiomers of either phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) or PtdIns(3,4)P2 were effective in potently activating the kinase, which has been named PtdIns(3,4,5)P3-dependent protein kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKBalpha or induced its phosphorylation under the conditions used. PDK1 activity was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-kinase family. CONLCUSIONS: PDK1 is likely to be one of the protein kinases that mediate the activation of PKB by insulin and growth factors. PDK1 may, therefore, play a key role in mediating many of the actions of the second messenger(s) PtdIns(3,4, 5)P3 and/or PtdIns(3,4)P2.

Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein.

The eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments [1]. A key marker for the mammalian TGN is TGN38/46 [2]. This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN [2] [3]. In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors. Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments. Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals.

A model of activation of the protein tyrosine phosphatase SHP-2 by the human leptin receptor.

Signalling through the leptin receptor has been shown to activate the SH2 domain-containing tyrosine phosphatase SHP-2 through tyrosine phosphorylation. The human leptin receptor contains five tyrosine residues in the cytoplasmic domain that may become phosphorylated. We show here using BIAcore studies, wherein binding of peptides to SHP-2 was detected, that peptides corresponding to sequences containing phosphotyrosines 974 and 986 (LR974P and LR986P, respectively) from the leptin receptor cytoplasmic domain were the only two peptides that bound to the enzyme. Binding of LR974P to SHP-2 was inhibited in a dose-dependent fashion by orthovanadate, whereas binding of LY986P was not, indicating that the enzyme binds to these peptides through different sites. Only the leptin receptor-derived peptide corresponding to tyrosine 974 was dephosphorylated by recombinant purified SHP-2. Time courses of the reaction were complex, and fitted a two exponent rate equation. Preincubation of SHP-2 with LR986P markedly activated the enzyme at early time points and time courses of the activated enzyme fitted a single exponential first order rate equation. We propose that LR974P binds to the active site of SHP-2, whereas LR986P may bind to the N- and C-terminal SH2 domains of SHP-2, thus activating the phosphatase activity. These data support a model in which SHP-2 binds to phosphotyrosine 986 in the activated leptin receptor and is activated to dephosphorylate phosphotyrosine 974, downregulating signalling events emanating from SH2 domain-containing proteins that bind here.

Structural and mechanistic features of phospholipases C: effectors of inositol phospholipid-mediated signal transduction.

The production of the intracellular second messengers inositol (1,4,5)-trisphosphate (InsP3) and sn 1,2-diacylglycerol (DG) in response to a wide variety of extracellular primary messengers is achieved by an extended family of inositol phospholipid phosphodiesterases termed phospholipases C (PLC, E.C. 3.1.4.11). This family has been the subject of extensive research and it is clear that the different isoenzymes exhibit some common characteristics (e.g., interactions with substrates) and other distinctive features (e.g., modes of regulation). The recent description of the X-ray crystal structure of a mammalian PLC has served to clarify much about the behaviour of the PLCs, emphasising the "modular" structure of these enzymes. The main focus of this review will concern the specific adaptations of PLC molecules which make them efficient lipid-metabolising enzymes. We also describe what is known about how these enzymes interact with their lipid substrates, which will serve as a basis for considering how PLCs may be activated.

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial.

BACKGROUND: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. METHODS: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0.9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. FINDINGS: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3.7%, 95% CI 0.1-7.3; p=0.046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. INTERPRETATION: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials. FUNDING: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.

Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding.

The t(8;21) translocation fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape, we measured genome-wide RUNX1- and RUNX1/ETO-bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end, we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide redistribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal, and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML.

Monocarboxylate transporter 8 in neuronal cell growth.

Thyroid hormones are essential for the normal growth and development of the fetus, and even small alterations in maternal thyroid hormone status during early pregnancy may be associated with neurodevelopmental abnormalities in childhood. Mutations in the novel and specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) have been associated with severe neurodevelopmental impairment. However, the mechanism by which MCT8 influences neural development remains poorly defined. We have therefore investigated the effect of wild-type (WT) MCT8, and the previously reported L471P mutant, on the growth and function of human neuronal precursor NT2 cells as well as MCT8-null JEG-3 cells. HA-tagged WT MCT8 correctly localized to the plasma membrane in NT2 cells and increased T(3) uptake in both cell types. In contrast, L471P MCT8 was largely retained in the endoplasmic reticulum and displayed no T(3) transport activity. Transient overexpression of WT and mutant MCT8 proteins failed to induce endoplasmic reticular stress or apoptosis. However, MCT8 overexpression significantly repressed cell proliferation in each cell type in both the presence and absence of the active thyroid hormone T(3) and in a dose-dependent manner. In contrast, L471P MCT8 showed no such influence. Finally, small interfering RNA depletion of endogenous MCT8 resulted in increased cell survival and decreased T(3) uptake. Given that T(3) stimulated proliferation in embryonic neuronal NT2 cells, whereas MCT8 repressed cell growth, these data suggest an entirely novel role for MCT8 in addition to T(3) transport, mediated through the modulation of cell proliferation in the developing brain.

Intracellular free Ca2+ fluxes and responses to phorbol ester in T lymphocytes from healthy elderly subjects.

A group of healthy elderly subjects (greater than or equal to 75 years) was selected by the strict criteria of the SENIEUR protocol, and compared with healthy young (less than or equal to 35 years) volunteers. Mitogenic responses of peripheral blood mononuclear cells to phytohaemagglutinin and anti-CD3 were significantly reduced in the elderly (P less than 0.0002), thereby confirming that even though in perfect health, elderly individuals show impaired cell-mediated immunity. However, no abnormality of intracellular free Ca2+ fluxes could be detected in purified T cells from the elderly subjects when stimulated with anti-CD3 antibody. Nevertheless, both the proliferative responses of purified T cells to phorbol ester and calcium ionophore (Ionomycin) and the phorbol ester-induced inhibition of the Ca2+ response were defective in the elderly subjects (P less than 0.003 and P less than 0.0002, respectively). These data suggest that signal transduction and the generation of second messengers proceed normally in T cells from the elderly, but downstream events mediated by activation of protein kinase C are dysfunctional.

Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C.

The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.

Time-dependent inhibition of phospholipase C beta-catalysed phosphoinositide hydrolysis: a comparison of different assays.

The properties of three different beta-isoforms of phospholipase C (PLC) were analysed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles and phospholipid monolayers spread at an air-water interface. Phosphatidylinositol 4,5-bisphosphate hydrolysis went virtually to completion in monolayers, but inositol trisphosphate production was curtailed prematurely in vesicular and micellar assays. Assays were linear for less than 2 min with vesicles; the linear portion could be significantly extended in micelles by increasing the ratio of micelles to enzyme molecules. However, onset of a second lower rate of substrate hydrolysis always occurred when < or = 10% of PtdIns(4,5)P(2) had been utilized. This was not due to enzyme inactivation in the micellar interface, determined by addition of fresh substrate or fresh enzyme after the slow phase of activity had started, nor was it due to overt product inhibition of PLC or apparent entrapment of PLC at the micelle surface. These results are similar to those seen in assays using bacterial PLC and we suggest that the biphasic kinetics may be due to product-dependent changes in the presentation of substrate lipic to PLC in lamellar assays, leading to reduced activity.

The turkey erythrocyte beta-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein alpha subunits.

By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response.

Kinetic analysis of phospholipase C beta isoforms using phospholipid-detergent mixed micelles. Evidence for interfacial catalysis involving distinct micelle binding and catalytic steps.

Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)-P2) hydrolysis by three different beta-isoforms of phospholipase C (PLC) was examined to investigate the catalytic action of these extracellular signal-regulated enzymes. Depletion of phospholipase C from solution by incubation with sucrose-loaded vesicles of differing compositions followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles from which an equilibrium association constant of PLC with PtdIns (4,5)P2 could be determined. A mixed micellar system was established to assay PLC activity using dodecyl maltoside, which behaved as an essentially inert diluent of PtdIns (4,5)P2 with respect to PLC beta activity. Kinetic analyses were performed to test whether PLC beta activity was dependent on both bulk PtdIns (4,5)P2 concentration and surface concentration in the micelles as has been shown for other lipid metabolising enzymes. Each of the PLC beta isoforms behaved similarly in these analyses, which indicated the involvement of at least two binding events. Interfacial Michaelis constants were calculated to be between 0.1-0.2 mol fraction for all three enzymes, and Ks (the equilibrium dissociation constant of PLC for lipid) ranged between 100-200 microM. The apparent multiple interfacial binding events did not appear to result from lipid-induced PLC beta oligomerization implying that PLC beta monomers possess more than one lipid-binding site. Surface dilution of PLC-catalyzed PtdIns (4,5)P2 hydrolysis was assessed in the presence of increasing concentrations of various nonsubstrate phospholipids, which profoundly reduced PLC activity, suggesting that these lipids may inhibit enzyme action. The data indicate that G protein-regulated isoforms of PLC operate with separate lipid binding and catalytic steps and imply that under physiological conditions, PLC beta isoforms operate under first-order conditions. These findings may have implications for the mechanisms of regulation of PLC beta s by G protein subunits.

Dependence of the activity of phospholipase C beta on surface pressure and surface composition in phospholipid monolayers and its implications for their regulation.

We have examined the influence of surface pressure and phospholipid composition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C beta 1 (PLC beta 1) and PLC beta 2 in mixed composition phospholipid monolayers. Increasing the monolayer surface pressure from 15 to 36 mN/m reduced the rate at which PIP2 was hydrolyzed by PLC beta 1 and PLC beta 2 by 4-6-fold, although PLC beta 1 was more active than PLC beta 2, even at high surface pressure. Reduced enzyme activity was accompanied by an increase in reaction induction times, suggesting that increasing surface pressure reduced the penetration rate of the enzymes into the monolayer. Quantitation of interfacial enzyme concentration using 35S-labeled PLC beta 1 confirmed that less enzyme was associated with the monolayer at higher pressures. The relationship between PLC activity and substrate concentration was examined at a single surface pressure of 30 mN/m. This relationship was not hyperbolic, and increases in the mole percentage (mol %) of PIP2 in the monolayer resulted in an upwardly-curving increase in PLC activity. Thus, PLC beta 1 activity increased 7-fold and PLC beta 2 activity increased 4-fold when the mol % of PIP2 in the monolayer increased from 17.9% to 29%, increasing further thereafter. Paradoxically, increasing the mol % of PIP2 from 0 to 60% was accompanied by a 3-fold decrease in interfacial enzyme concentrations. Taken together, these data show that the catalytic activity of PLC beta involves some element of penetration of lipid interfaces, and suggest that the organization of the substrate facilitates PLC activity, giving credence to the substrate theory of interfacial activation of phospholipases. We present a hypothesis suggesting that PIP2 molecules coalesce into enriched lateral domains which favor PLC beta activity.

A G-protein beta gamma-subunit-responsive phosphoinositide 3-kinase activity in human platelet cytosol.

Thrombin activates phosphoinositide 3-kinase (PI 3-kinase) in platelets via a mechanism involving G-proteins, possibly of both the heterotrimeric and the low molecular weight families. We have investigated the regulation of PI 3-kinase present in platelet cytosol, and we show that this activity can be stimulated by a mixed preparation of G-protein beta gamma-subunits. This stimulation is reversed by preincubation of the beta gamma-subunits with GDP-liganded alpha-subunits. The beta gamma-stimulated activity is inhibited by wortmannin, a recently identified inhibitor of PI 3-kinase in other systems. In addition, the activity associates with PDGF receptor phosphotyrosyl peptide and monoclonal antibody designed to interact with the p85 subunit of PI 3-kinase. We suggest that this beta gamma-sensitive activity is related to previously identified forms of PI 3-kinase.

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1.

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.

Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation.

Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.