Measuring Activity of Phosphoinositide Lipid Kinases Using a Bioluminescent ADP-Detecting Assay.

Phosphatidylinositol (PI) and its phosphorylated derivatives, collectively called phosphoinositides, are important second messengers involved in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Due to the central role of these kinases in signaling pathways, assays for measuring their activity are often used for drug development. Lipid kinase substrates are present in unique membrane environments in vivo and are insoluble in aqueous solutions. Therefore the most important consideration in developing successful lipid kinase assays is the physical state of lipid kinase substrates. Here we describe the preparation of lipid substrates for two major classes of lipid kinases, phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 4-kinases (PI4Ks). Using PI4Ks as an example, we also provide a detailed protocol for small-scale kinase expression and affinity purification from transiently transfected mammalian cells. For measuring lipid kinase activity we apply a universal bioluminescent ADP detection approach. The approach is compatible with diverse lipid substrates and can be used as a single integrated platform for measuring all classes of lipid and protein kinases.

Regulation of phosphoinositide-metabolizing enzymes by clathrin coat proteins.

Clathrin plays key roles in endocytic and endo-lysosomal membrane dynamics by facilitating the formation of coated vesicles at the plasma membrane and at the trans-Golgi network (TGN)/endosomal boundary. Assembly of the clathrin lattice critically depends on adaptor proteins and accessory proteins, which connect the clathrin scaffold to the membrane and to transmembrane cargo including receptors, transporters, channels, and SNARE proteins. The recruitment of adaptor proteins to membrane surfaces is triggered by coincidence-detection mechanisms involving phosphoinositides (PIs), cargo proteins, and in many cases small GTPases. To tightly regulate coat formation, there is extensive cross-talk between PI-metabolizing enzymes and adaptor proteins. One of the best studied examples is the endocytic clathrin adaptor complex AP-2, which binds plasma membrane-enriched PI(4,5)P(2). In neurons, PI(4,5)P(2) is synthesized from PI(4)P primarily by the γ-isoform of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIPKIγ), whose enzymatic activity is regulated by direct binding to, amongst others, the small GTPase Arf6 and AP-2. Cargo-bound AP-2 potently stimulates PIPK1γ activity and thereby drives AP-2-membrane interactions. This feed-forward loop is thought to facilitate membrane translocation of additional AP-2 molecules and concomitantly clathrin, but also of endocytic accessory proteins, many of which directly associate with PI(4,5)P(2). It is likely that similar mechanisms support the formation of coated vesicles at the trans-Golgi network (TGN) and on endosomes, involving PI(4)P and PI(3)P respectively, but detailed knowledge is lacking to date. To explore how coat proteins regulate kinase activity, assays are needed to sensitively detect subtle changes in PI synthesis and to discriminate between the various PI species. Here we describe a sensitive and specific radioactivity-based assay to measure PI kinase activity.

Gbetagamma-copurified lipid kinase impurity from Sf9 cells.

G-protein betagamma dimers are prime regulators transmitting extracellular signals to wide-ranging cellular effectors including phosphoinositide 3-kinase (PI3K) isoforms beta and gamma. Recombinant Gbetagamma purified from Sf9 cells via metal-affinity and anion exchange chromatography exhibited a wortmannin-insensitive phospholipid kinase activity that copurified from the insect cells. To exclude false-positive results of Gbetagamma-dependent lipid kinase activity, the elimination of insect phospholipid kinase from Gbetagamma protein samples is necessary to avoid interference with the intrinsic lipid kinase activity of PI3K isoforms in reconstitution experiments. Here we describe an improved procedure of Gbeta(1)gamma(2) purification from cell membranes that separates the contaminating phospholipid kinase.

Regulation of Beclin 1 in autophagy.

Class III phosphatidylinositol 3-kinase (PI3KC3) plays a pleiotropic role in autophagy and protein sorting pathways. The human core complex of PI3KC3 consists of three major components including PI3KC3/hVps34, p150 and Beclin 1. How the specificity of PI3KC3 complex is derived towards autophagy is not clear. Utilizing a sequential affinity purification coupled with mass spectrometry approach, we have successfully purified a human Beclin 1 complex and cloned a novel protein we called Barkor (Beclin 1-associated autophagy-related key regulator). The function of Barkor in autophagy has been manifested in several assays, including stress-induced LC3 lipidation, autophagosome formation and Salmonella typhimurium amplification. Mechanistically, Barkor competes with UV radiation resistance associated gene product (UVRAG) for interaction with Beclin 1, and orients Beclin 1 to autophagosomes. Barkor shares considerable sequence homology with Atg14 in yeast, representing an evolutionary conserved autophagy specific regulatory step in early autophagosome formation.

Expression and purification of PI3 kinase alpha and development of an ATP depletion and an alphascreen PI3 kinase activity assay.

Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal His-tag and the p85alpha regulatory domain in Sf9 insect cells. The complex consisting of p110alpha and p85alpha was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110alpha/p85alpha. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110alpha/p85alpha and determined the IC(50) for wortmannin, a known PI3 kinase inhibitor. The IC(50) for wortmannin was determined to be 7 nM. From a selection of substrates, phosphatidylinositol-4, 5-biphosphate turned out to be the best substrate at a concentration of 50 microM. p110alpha/p85alpha underwent autophosphorylation most prominently at the p85alpha subunit. However, in the presence of lipid substrate, the autophosphorylation was negligible. In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors.

Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms.

The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.

The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.

Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.

A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning and characterization.

A phosphatidylinositol (PI) 3-kinase gene (CaVPS34) of the human pathogenic yeast Candida albicans was cloned by a PCR-based homology approach. The open reading frame encodes a 1020 amino acid protein with a calculated molecular weight of 118 kDa and a relative isoelectric point of 6.9. It shares 47% sequence identity with Saccharomyces cerevisiae Vps34p. Southern pattern indicated that CaVPS34 is probably present as a single copy gene per haploid genome in C. albicans. We localized the CaVPS34 gene on chromosome 1. Under all conditions tested a major CaVPS34 transcript of approximately 3. 5 kb could be detected. CaVPS34 mRNA levels increased during exponential growth up to 12-fold followed by a decline upon entry into stationary phase. The size of a 6xHis tag-CaVps34p fusion protein purified from Escherichia coli is in agreement with the calculated molecular mass of CaVps34p. It exhibits in vitro PI 3-kinase activity and produces only phosphatidylinositol 3-phosphate. The CaVPS34 gene under the control of its own promoter were not able to complement the temperature-sensitive growth of S. cerevisiae vps34. However, overexpression of CaVPS34 was sufficient to rescue the temperature-sensitive vps34 phenotype, suggesting a functional conservation in C. albicans.

Biotinylated phosphatidylinositol 3,4,5-trisphosphate as affinity ligand.

Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a primary output signal of phosphoinositide (PI) 3-kinase, plays a crucial role in diverse cellular processes. Evidence indicates that PIP(3) exerts downstream signaling, in part, by recruiting effector proteins to plasma membranes. Consequently, identification of signaling enzymes with PIP(3)-binding motifs represents a viable approach to understand the mechanism by which specificity of the PI 3-kinase-mediated signaling network is maintained. To address this issue, we have developed biotinylated derivatives of PIP(3) as affinity probes for the purification and characterization of PIP(3)-binding proteins. Considering the relaxed requirement for the acyl moiety in PIP(3) recognition, these biotinylated PIP(3) analogues display two structural features. First, they contain short acyl side chains (C(4) and C(8)), allowing them to be soluble in aqueous milieu. This desirable feature avoids the formation of lipid aggregates, which minimizes nonspecific hydrophobic interactions with proteins. Second, the appended biotin is located at the terminus of the sn-1 acyl side chain, thereby maintaining the integrity of the phosphoinositol head group essential for selective recognition. The utility of these affinity ligands is validated by the purification of recombinant PIP(3)-binding proteins, expressed as GST fusion proteins, to homogeneity from bacterial lysates. These include the C-terminal SH2 domain of the p85 subunit of PI 3-kinase and the N-terminal PH domain of PLCgamma1. The efficiency of biotinylated PIP(3) analogues in the purification of these recombinant proteins was approximately 20% of that of glutathione beads

Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor.

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.

The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol.

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.

Separation of IRS-1 and PI3-kinase from GLUT4 vesicles in rat skeletal muscle.

In fat and muscle tissues, insulin stimulates cellular glucose uptake by initiating a phosphorylation cascade which ultimately results in the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular storage pool(s) to the plasma membrane in fat and to t-tubules in skeletal muscle. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) are known to be involved in cellular responses to insulin such as GLUT4 translocation, but the biochemical mechanism(s) connecting IRS-1 and PI3-kinase to GLUT4-containing intracellular membranes remains unclear. Here, in control and insulin-stimulated rat skeletal muscle, the intracellular localization of these two proteins was compared to that of GLUT4 using subcellular fractionation by sucrose velocity gradients followed by immunoblotting. Our data show that insulin-sensitive GLUT4-containing vesicles are present in fractions 1 through 10, whereas IRS-1 and PI3-kinase are found in fractions 16 through 24. These results indicate that in intracellular fractions derived from skeletal muscle, IRS-1 and PI3-kinase are excluded from membranes harboring GLUT4.

Extracellular HIV-1 Tat protein activates phosphatidylinositol 3- and Akt/PKB kinases in CD4+ T lymphoblastoid Jurkat cells.

The biological basis for the pleiotropic activity of extracellular human immunodeficiency virus (HIV)-1 Tat protein on lymphoid T cell survival is not well understood. We have here demonstrated that the addition in culture of 0.1-10 nM Tat protein to 36-h serum-starved lymphoblastoid Jurkat T cells rapidly stimulates the catalytic activity of phosphatidylinositol 3-kinase (PI 3-K). The peak of activation was observed 30 min after Tat addition. Extracellular Tat also stimulated the catalytic activity of the Akt/PKB kinase, a major target of PI 3-K lipid products. Pretreatment of serum-starved Jurkat cells with 100 nM wortmannin (WT) or 10 microM LY294002, two unrelated pharmacological inhibitors of PI 3-K, markedly suppressed the catalytic activity of both PI 3-K and Akt/PKB in Jurkat cells. Moreover, at low concentrations (0.1-1 nM), extracellular Tat showed a small but reproducible protection of Jurkat cells from apoptosis induced by serum deprivation (p < 0.05), while the combination of Tat plus 100 nM WT significantly (p < 0.05) increased the percentage of apoptosis with respect to cells left untreated or treated with Tat alone. Taken together, these data suggest that the anti-apoptotic activity of low concentrations of Tat protein on Jurkat cells is mediated by a PI 3-kinase/Akt pathway.