Essential and distinct roles of phosphatidylinositol 4-kinases, Pik1p and Stt4p, in yeast autophagy.

Autophagy is a degradative cellular pathway that protects eukaryotic cells from starvation/stress. Phosphatidylinositol 4-kinases, Pik1p and Stt4p, are indispensable for autophagy in budding yeast, but participation of PtdIns-4 kinases and their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is not understood. Nanoscale membrane lipid distribution analysis showed PtdIns(4)P is more abundant in yeast autophagosomes in the luminal leaflet than the cytoplasmic leaflet. PtdIns(4)P is confined to the cytoplasmic leaflet of autophagosomal inner and outer membranes in mammalian cells. Using temperature-conditional single PIK1 or STT4 PtdIns 4-kinase mutants, autophagic bodies in the vacuole of PIK1 and STT4 mutant cells dramatically decreased at restrictive temperatures, and the number of autophagosomes in the cytosol of PIK1 mutants cells was also decreased, whereas autophagosome levels of STT4 mutant cells were comparable to that of wild-type and STT4 mutant cells at permissive temperatures. Localization of PtdIns(4)P in the luminal leaflet in the biological membrane is a novel finding, and differences in PtdIns(4)P distribution suggest substantial differences between yeast and mammals. We also demonstrate in this study that Pik1p and Stt4p play essential roles in autophagosome formation and autophagosome-vacuole fusion in yeast cells, respectively.

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.

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Quantification of multiple phosphatidylinositol 4-kinase isozyme activities in cell extracts.

A wide spectrum of intracellular signaling events mediated by up to seven different phosphorylated forms of phosphatidylinositol (PtdIns) occurs in all eukaryotic cells. The activities of multiple, nondegenerate PI kinases and phosphatases control these signaling events. The PI 4-kinase isozymes account for the major PI kinase activity in many different cell types, and the activity of each isozyme is differentially regulated. The ability to measure and distinguish the activity of individual enzymes is therefore important and forms the subject of the methods in this chapter. We describe the use and application of a versatile radiometric assay to measuring PI 4-kinase activity in a variety of biochemical contexts, from purified enzymes to membrane preparations and permeabilized cells. Until a suitable nonradioactive reagent becomes available, this assay is destined to remain the most widely used method.

Platelet tetraspanin complexes and their association with lipid rafts.

Tetraspanins are a superfamily of integral membrane proteins that facilitate the organization of membrane and intracellular signaling molecules into dynamic signaling microdomains, tetraspanin-enriched microdomains (TEMs). Four tetraspanin family members have been identified in platelets: CD9, CD151 and TSSC6, which are constitutively associated with alphaIIbbeta3, and CD63, which is present on granule membranes in resting platelets and associates with alphaIIbbeta3-CD9 following platelet activation. CD63 and CD9 associate with a type II phosphatidylinositol 4-kinase, PI4K55, in both resting and activated platelets. Immunoelectron microscopic studies showed co-localization of CD63 and PI4K55 on internal membranes of resting platelets and on the filopodia of thrombin-activated platelets. Because TEMs in malignant cell lines appear to be distinct from prototypic lipid rafts, this study examined whether CD63-PI4K55 and CD9-PI4K55 complexes were resident in platelet-lipid rafts, or formed distinct microdomains. CD63, CD9 and PI4K55 were recovered from low-density membrane fractions (LDMFs) of sucrose gradients following platelet lysis in Brij 35, but unlike lipid-raft proteins were not insoluble in Triton X-100, being absent from LDMFs of platelets lysed with Triton. Incubation of platelets with methyl-beta-cyclodextrin, to deplete cholesterol and disrupt lipid rafts, shifted the complexes to higher density sucrose gradient fractions, but did not disrupt the tetraspanin-PI4K55 complexes. These results demonstrate that tetraspanin complexes in platelets form cholesterol-associated microdomains that are distinct from lipid rafts. It is probable that TEMs and lipid rafts associate under certain conditions, resulting in the close proximity of distinct sets of signaling molecules, facilitating signal transduction.

Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation.

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.

Determination of the catalytic activities of mTOR and other members of the phosphoinositide-3-kinase-related kinase family.

Members of the phosphoinositide-3-kinase-related kinase (PIKK) family, which includes mTOR, ATM, ATR, and hSMG-1, play important roles in regulating the cellular response to environmental stimuli. Despite the similarity of their catalytic domain to that of phosphoinositide-3-kinase, these extremely large (>250 kDa) polypeptides function as serine/threonine protein kinases. The catalytic activities of these PIKK family members can now be measured in immune-complex kinase assays. This assay involves isolation of the kinase by immunoprecipitation and the in vitro phosphorylation of a specific substrate in the presence of radio-labeled ATP. Here we describe, in detail, the determination of PIKK catalytic activity with a standardized immune-complex kinase assay protocol.

Overexpression of kidney phosphatidylinositol 4-kinasebeta and phospholipase C(gamma1) proteins in two rodent models of polycystic kidney disease.

Our studies of renal phosphoinositide levels and metabolism in the pcy mouse with polycystic kidney disease (PKD) suggest that phosphatidylinositol kinase (PtdInsK) and phospholipase C (PLC) are elevated in this renal disorder. Therefore, the steady-state levels of select isoforms of these enzymes were examined in renal cytosolic and particulate (detergent-soluble) fractions in male and female normal and CD1-pcy/pcy (pcy) mice at 60, 120 and 180 days of age, and in male and female normal and diseased (Han:SPRD-cy) rats at 28 and 70 days of age. Disease-related increases in phosphatidylinositol 4-kinasebeta (PtdIns4Kbeta) and PLC(gamma1) levels were present in both models. PtdIns4Kbeta levels were higher by as much as 233% in pcy mice and by 95% in diseased Han:SPRD-cy rats compared to normals of the same age and gender. Steady-state levels of PLC(gamma1) were as much as 74% and 35% higher in pcy mice and diseased Han:SPRD-cy rats, respectively, compared to their controls. The consistency of these alterations in two accepted models of PKD indicates the importance of the phosphoinositide signalling pathway in the evolution of this disorder, and represents a potential site for therapeutic intervention.

Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion-exchange high-performance liquid chromatography with suppressed conductivity detection.

Phosphatidylinositol 4,5-biphosphate (PIP(2)) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP(2) levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP(2) are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP(2) amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 micromol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP(2), whereas overexpression of Type II PI4-kinase can increase both PIP and PIP(2). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and the D3 isomers of PIP(2) are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP(2) remains low; exogenous PIP(2) is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.

Localization of phosphatidylinositol 4-kinase isoenzymes in rat liver plasma membrane domains.

The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.

Axonal transport of neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta in the adult rat sciatic nerve.

Neuronal calcium sensor-1 (NCS-1) and its putative substrate phosphatidylinositol 4-kinase beta (PtdIns 4-kinase beta) both indirectly regulate synaptic vesicle exocytosis and are located in DRG neurites. In this study we have tested whether NCS-1 and PtdIns 4-kinase beta are transported in axons using the analysis of double ligation approach in the adult rat sciatic nerve. We show that NCS-1 accumulates on both the distal and proximal side of the nerve ligation indicating that this protein undergoes bidirectional transport in axons. In contrast, PtdIns 4-kinase beta accumulated on the distal side which suggests that it undergoes retrograde axonal transport and unlike NCS-1 was also present in non-neuronal cells.

Reductions in brain phosphatidylinositol kinase activities in Alzheimer's disease.

BACKGROUND: Converging lines of evidence suggest that alterations in the intracellular trafficking of the amyloid precursor protein, its derivatives, and other relevant proteins may contribute to the pathophysiology of Alzheimer's disease (AD). Since phosphatidylinositol (PI) kinase plays a pivotal role in the sorting and transport of newly synthesized proteins to their final destinations, we explored the hypothesis that AD is associated with alterations in the specific activities of these enzymes in postmortem brain tissue. METHODS: The specific activities of soluble and particulate pools of PI 3-kinase and PI 4-kinase from the frontal cortex were compared between 11 cases with histopathologically confirmed AD and 11 nondemented controls matched for sex, race, age at death, and postmortem interval. Potential associations of these activities with sociodemographic and clinical features were also explored. RESULTS: AD was associated with 43-59% reductions in the specific activities of the soluble forms of both lipid kinases; but no significant change in the specific activities of the particulate species. Associations of these specific activities with sex, age at onset or death, duration of illness, postmortem interval, or densities of morphologic lesions in the frontal cortex were not observed among the 11 AD cases. CONCLUSIONS: In addition to regulating protein sorting and trafficking, PI kinases participate in a wide range of cellular processes including protection from apoptosis, differentiation and cell growth, regulation of the cytoskeleton, and glucose metabolism. The results of this study suggest that one or more of these alterations in AD may result from a common abnormality in PI kinase regulation.