Heterologous Expression, Purification, and Functional Analysis of the Plasmodium falciparum Phosphatidylinositol 4-Kinase IIIß.

Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe2+-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative IIIß isoform (PfPI4KIIIß)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIIIß. We therefore codon optimized the pfpi4kIIIß gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIIIß protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, Km,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIIIß activity is most consistent with the class IIIß designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIIIß, we titrated enzyme inhibition for these candidates versus purified PfPI4KIIIß and PfVps34. We also analyzed the activity versus purified PfPI4KIIIß mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIIIß inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIIIß. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.

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.

Definition and expression in E. coli of large fragments from the human lipid kinase phosphatidylinositol 4-kinase type III alpha, and purification of a 1100-residue N-terminal module.

The eukaryotic lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KA in higher eukaryotes) is a ubiquitous enzyme that synthesizes the plasma membrane pool of phosphatidylinositol 4-phosphate. This important phosphoinositide has key roles in different signalization pathways, vesicular traffic and cellular compartment identity. Moreover, human PI4K4A is an essential factor for hepatitis C virus replication. PI4KA is a large protein (2102 residues for human PI4KA) with the kinase domain making up the ca 400 C-terminal residues. There is essentially no structural information about the 1500N-terminal residues and no clue as to the function of most of this region of PI4KA. In this report, we use computational methods in order to delineate fragments of human PI4KA amenable to soluble production in Escherichia coli. We clone and express these fragments as GST-fusions and evaluate the soluble fraction of each protein. Finally, we produce and purify to homogeneity a 1100-residue PI4KA N-terminal fragment. Our results further suggest that PI4KA can be described as a two-module protein. They open the way to structural characterization of the N-terminal regulatory module of PI4KA.

A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases.

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.

Quantitative measurement of phosphatidylinositol 3,4,5-trisphosphate.

The activation of class I phosphoinositide 3-kinases (PI(3)Ks) by cell surface receptors represents the initiation of a large and complex signaling network that couples many growth factors, antigens, and inflammatory stimuli to important cellular responses, such as cell growth, survival, and movement. The most direct measurement of class I PI(3)K activity in cells is the rate of production of its lipid product, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. This chapter describes in detail two approaches used to estimate the levels of PtdIns(3,4,5)P(3) in cells. One approach uses radiotracer labeling of cells, lipid extraction, deacylation, and subsequent quantitation of phosphoinositides by anion-exchange high-performance liquid chromatography. The second approach uses a novel, nonradioactive assay in which the cellular lipids are extracted, phosphoinositides are enriched through binding to a neomycin matrix, dried onto a nitrocellulose membrane, and PtdIns(3,4,5)P(3) quantified by a protein-lipid overlay approach using a GRP(1) PH domain probe.

Identification and characterization of differentially active pools of type IIalpha phosphatidylinositol 4-kinase activity in unstimulated A431 cells.

The seven known polyphosphoinositides have been implicated in a wide range of regulated and constitutive cell functions, including cell-surface signalling, vesicle trafficking and cytoskeletal reorganization. In order to understand the spatial and temporal control of these diverse cell functions it is necessary to characterize the subcellular distribution of a wide variety of polyphosphoinositide synthesis and signalling events. The predominant phosphatidylinositol kinase activity in many mammalian cell types involves the synthesis of the signalling precursor, phosphatidylinositol 4-phosphate, in a reaction catalysed by the recently cloned PI4KIIalpha (type IIalpha phosphatidylinositol 4-kinase). However the regulation of this enzyme and the cellular distribution of its product in different organelles are very poorly understood. This report identifies the existence, in unstimulated cells, of two major subcellular membrane fractions, which contain PI4KIIalpha possessing different levels of intrinsic activity. Separation of these membranes from each other and from contaminating activities was achieved by density gradient ultracentrifugation at pH 11 in a specific detergent mixture in which both membrane fractions, but not other membranes, were insoluble. Kinetic comparison of the purified membrane fractions revealed a 4-fold difference in K (m) for phosphatidylinositol and a 3.5-fold difference in V (max), thereby indicating a different mechanism of regulation to that described previously for agonist-stimulated cells. These marked differences in basal activity and the occurrence of this isozyme in multiple organelles emphasize the need to investigate cell signalling via PI4KIIalpha at the level of individual organelles rather than whole-cell lysates.

Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro.

A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses.

Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase.

The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.

Signalling and non-caveolar rafts.

Rafts are small membrane domains containing discrete subsets of lipids and proteins. Although microscopic raft structures termed 'caveolae' were described nearly 50 years ago, the importance of rafts, particularly signalling within rafts, is only beginning to be understood. Our studies focus on receptor-dependent phosphoinositide signalling. Using their characteristic buoyancy in density gradients, we and others found that the epidermal growth factor (EGF) receptor, phosphatidylinositol 4-kinase and phosphoinositides are localized within a caveolin-rich fraction of A431 carcinoma cells. We subsequently found that membrane fragments containing the EGF receptor and most cellular phosphoinositides can be separated from caveolae. Consequently, components of EGF-dependent phosphoinositide signalling localize to one or more novel types of raft, the composition of which we are currently determining. A key component is the type II phosphatidylinositol 4-kinase, which, for many years, has proven difficult to purify and clone. We describe our recent purification from rafts and cloning of this elusive enzyme, and discuss how the structure sheds light on the rafting of this enzyme.

Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain.

Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a PtdIns 4-kinase from lymphocytes.

A PtdIns 4-kinase from rat spleen particulate fraction was purified to homogeneity and its molecular properties were compared with a PtdIns 4-kinase from splenic lymphocytes. The enzyme activity was solubilized from spleen particulate fraction with Triton X-100 and chromatographed sequentially on phosphocellulose, DEAE-sephacel, heparin acrylamide and hydroxyapatite columns. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with silver staining. Renaturation of the enzyme activity from SDS-PAGE showed that it comigrated with the 55 kDa protein. Characterization of the enzyme showed that it was a type II PtdIns 4-kinase. Polyclonal antibodies raised against PtdIns 4-kinase inhibited the enzyme activity in in vitro assays. Analysis of adult rat tissue particulate fractions on immunoblots showed restricted immunoreactivity among PtdIns 4-kinases. However, the immunoreactivity is conserved in lymphoid tissues from mouse to human, suggesting that lymphoid tissue has a distinct PtdIns 4-kinase. Activation of rat splenocytes with Con A showed two fold increase in PtdIns 4-kinase activity. Comparison of PtdIns 4-kinases from spleen and splenic lymphocytes showed identical chromatographic behaviour, molecular mass, immunoreactivity, K(m) values for PtdIns and inhibition by adenosine.

Phosphatidylinositol 4-kinase of Torpedo californica electrocytes: physico-chemical characterization and regulation by calcium and vicinal molecules of phosphatidylinositol.

A phosphatidylinositol 4-kinase (Ptdlns 4-kinase, M(r) approximately 95,000) from the membranes of the electric organ of Torpedo californica was purified to apparent homogeneity. The Michaelis constant for ATP (KM = 280 +/- 60 microM at 20 degrees C) and the inhibition constant for adenosine (Ki = 0.4 mM at 20 degrees C) qualify the electrocyte Ptdlns 4-kinase as a type III kinase. The Ptdlns 4-kinase phosphorylates preferentially exogenous Ptdlns, added in the form of mixed Ptdlns/Triton X-100 micelles, whereas endogenously bound Ptdlns in the membrane fragments of electrocytes is a very poor substrate. It is important that the enzyme and the substrate Ptdlns are situated in different lipid bilayers. The catalytic turnover constant for exogenous Ptdlns is k = 55.3 +/- 6 min-1 at 20 degrees C and the molar Triton X-100/Ptdlns ratio of 16:1. For the substrate Ptdlns in the 'micellar solvent' Triton X-100, steady state kinetics were analysed in terms of the mole fraction X = n(Ptdlns)/[n(Ptdlns) + n(Triton X)] yielding the characteristic Michaelis mole fraction XM = 0.019 +/- 0.005 at 20 degrees C. The activity of the enzyme was enhanced about 5-fold in the presence of Triton X-114, yielding k = 277 +/- 30 min-1 at 20 degrees C. Triton X-114 has a shorter head-group, indicating that the vicinity of the Ptdlns head group in the mixed micelles should not be screened by bulky neighbours. The inhibition of the enzyme activity by Ca2+ is highly cooperative yielding the Hill inhibition constant Ki = 0.47 +/- 0.1 mM and the Hill coefficient h = 3.6 +/- 0.5. The enthalpy of activation is 100 +/- 10 kJ/mol between 0 degree C and 20 degrees C. Although the Ptdlns 4-kinase can be affinity-chromatographically copurified with the nicotinic acetylcholine (AcCho) receptor, suggesting tight association between the two proteins. AcCho does not affect the activity of the Ptdlns 4-kinase in the presence of the AcCho receptor.

Identification of 55 kDa phosphatidylinositol 4-kinase in HaCaT cells: comparison with the epithelial cell line A431.

Phosphatidylinositol kinases play a crucial role in signal transduction in many cell types. The 55 kDa isoform of phosphatidylinositol 4-kinases is a key enzyme in the metabolism of phosphoinositides, which work as regulators of cell function itself or as precursors of signal molecules. The experiments with HaCaT cells presented suggest that the phosphatidylinositol 4-kinase activity in this cell line corresponds to the 55 kDa isoform concerning kinetic parameters and specific activity in comparison with the malignant cell line A431. Km (for ATP and phosphatidyl-inositol) and Ki values (for Ca2+ and adenosine) are in good agreement with the parameters described for other cells. The findings support the idea that the 55 kDa phosphatidylinositol 4-kinase represents a key enzyme in the inositide signal transduction pathway.