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Vol. 19, Issue 2, 711-721, February 2008

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Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase III

Andras Balla^*,, Yeun Ju Kim^*, Peter Varnai^*,, Zsofia Szentpetery^*, Zachary Knight, Kevan M. Shokat, and Tamas Balla^*

*Section on Molecular Signal Transduction, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; Department of Physiology, Semmelweis University, School of Medicine, Budapest, Hungary H-1086; Program in Chemistry and Chemical Biology, University of California, San Francisco, CA 94158; and Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158

Submitted July 28, 2007; Revised October 25, 2007; Accepted November 27, 2007
Monitoring Editor: John York

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks ( or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the - and β-isoforms of type III PI4Ks, PI4KIII was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIII down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIII enzyme in maintaining plasma membrane phosphoinositides.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Activation of cell surface receptors by a variety of stimuli initiates a cascade of molecular events ultimately eliciting a response characteristic of the target cell. One of the most studied and best-characterized signal transduction pathways is initiated by the phospholipase C-mediated breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] to generate the Ca²⁺-mobilizing messenger inositol trisphosphate (InsP₃) and the protein kinase C activator diacylglycerol (Berridge and Irvine, 1984). It has long been recognized that the sustained production of these messengers requires continuous phosphorylation of phosphatidylinositol (PtdIns) to phosphatidylinositol 4-phosphate (PtdIns4P) and PtdIns(4,5)P₂ by phosphoinositide (PI) 4-kinase (PI4K) and PIP 5-kinase enzymes, due to the limited amount of PtdIns(4,5)P₂ present in the plasma membrane (Creba et al., 1983). Several isoforms within the PI 4-kinase and PIP 5-kinase families have been described previously (Doughman et al., 2003; Balla and Balla, 2006), and a recent study identified a splice variant of PIP 5-kinase as the enzyme responsible for PtdIns4P to PtdIns(4,5)P₂ conversion in agonist-induced Ca²⁺ signaling (Wang et al., 2004). In contrast, the PI 4-kinase that generates PtdIns4P for this process so far has eluded identification.

Four PI 4-kinase enzymes have been identified in mammalian cells that belong to either the type II or type III families (Balla and Balla, 2006). Type II PI 4-kinase enzymes (- and β-forms) are small, 56-kDa proteins that are abundant in almost all cellular membranes, and they are enriched in plasma membrane preparations. They are kept in the membrane by palmitoylation, and recent studies suggest that these enzymes have a role in post-trans-Golgi network trafficking (Wang et al., 2003, 2007) and the endocytic processing of the epidermal growth factor receptors (Minogue et al., 2006). Type III PI 4-kinases (- and β-forms), in contrast, are soluble enzymes structurally related to PI 3-kinases, and they can be inhibited by higher concentrations of PI 3-kinase inhibitors, such as wortmannin (Wm). We have reported over 10 years ago that production of the agonist-sensitive phosphoinositide pools required the activity of Wm-sensitive type III PI 4-kinase enzymes (Nakanishi et al., 1995). However, it is still not known which of the type III enzymes participates in the formation of the agonist-regulated PtdIns4P pools.

In the present study, we used both pharmacological and genetic approaches to interfere with the activity of all of the PI 4-kinase enzymes, and we monitored phosphoinositide changes both by conventional metabolic labeling of inositol lipids and -phosphates and by using green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains recognizing PtdIns4P and PtdIns(4,5)P₂. These results show that the PH domain of the yeast OSH2 protein is a valuable tool to monitor plasma membrane PtdIns4P pools and that PI 4-kinase III is required for sustained InsP₃ production and Ca²⁺ signaling. Although this conclusion was supported by small interfering RNA (siRNA)-mediated gene silencing of the individual PI4K enzymes, the present studies also highlight the difficulties in obtaining conclusive data with siRNA on enzymes whose functions are near essential and emphasize the value of inhibitors to dissect the roles of these proteins in cellular signaling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials
Wortmannin and ionomycin were purchased from Calbiochem (San Diego, CA). Phenylarsine oxide (PAO), β-mercaptoethanol, dithiothreitol, and poly-lysine were obtained from Sigma-Aldrich (St. Louis, MO). Fura-2/acetoxymethyl ester (AM) and Pluronic acid were from Invitrogen (Carlsbad, CA). PIK93 was synthesized as described previously (Knight et al., 2006). Myo-[³H]inositol (60 Ci/mmol) was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) and ortho-[³²P]phosphate (9000 Ci/mmol) was from General Electric (PerkinElmer Life and Analytical Sciences, Boston, MA). The polyclonal antibody against PI4KIIIβ was purchased from UBI (Lake Placid, NY), the polyclonal antibody against PI4KII was a kind gift from Dr. Pietro De Camilli (Yale School of Medicine, New Haven, CT), and the anti PI4KIII antibody was raised in New Zealand rabbits as described previously (Balla et al., 2005).

DNA Constructs and Transfections
The OSH2–2x-GFP construct was generated by amplifying the PH domain sequence of OSH2 (residues 256–424) from Saccharomyces cerevisiae cDNA (American Type Culture Collection, Manassas, VA) by using two primer pairs to obtain fragments flanked by XhoI/EcoRI and EcoRI/KpnI sites. These fragments were then cloned in tandem between the XhoI/KpnI sites of the pEGFP-C1 plasmid (Clontech, Mountain View, CA), with a linker (VNSKL) in between them following the design of Roy and Levine (2004). The single PH domain version of the PH domain also has been created as well as the cyan and yellow fluorescent versions of the tandem construct. The PLC₁PH-GFP construct (Várnai and Balla, 1998) and its color variants have been described previously (Varnai et al., 2002). The OSH1-PH-GFP was kindly provided by Dr. Mark Lemmon (University of Pennsylvania, Philadelphia, PA) (Yu et al., 2004), and the type IV phosphoinositide 5-phosphatase was a kind gift of Dr. Philip Majerus (Washington University, St. Louis, MO) (Kisseleva et al., 2000). The constructs used for the rapamycin-inducible translocation of the type-IV phosphoinositide 5-phosphatase have been described recently (Varnai et al., 2006).

The human embryonic kidney (HEK)-293AT1 cells used for these studies have been stably transfected with the hemagglutinin (HA)-AT1a and FLAG-AT1a angiotensin receptors in two rounds of selection by using G-418 and Zeocine (Zeo) (Invitrogen, Carlsbad, CA) for the two constructs, respectively. These cells were kindly provided by Drs. Alberto Jesus Olivares-Reyes and Kevin J. Catt (NICHD, NIH, Bethesda, MD). Cells were cultured in DMEM with Pen/Strep (Invitrogen) and 10% fetal bovine serum (FBS), and they have been subjected to a G-418/Zeo selection from time to time but not during cultures for the experiments.

For RNA interference (RNAi)-mediated knockdown, the cells were cultured either in 10-cm dishes (for suspension Ca²⁺ measurements), 12-well plates (for metabolic labeling studies), or 25-mm glass coverslips treated with poly-lysine (for single-cell Ca²⁺ or confocal studies). The duplexes used for treatment have been described previously (Balla et al., 2005). Cells were treated with 100 nM siRNA twice in consecutive days, and they were analyzed on the fourth day after the first treatment.

Analysis of Single Cells for Fluorescence Resonance Energy Transfer (FRET), Cytoplasmic Ca²⁺ Measurements, and Confocal Microscopy
HEK-293AT1 cells were cultured on glass coverslips (3 x 10⁵ cells/35-mm dish) pretreated with poly-lysine and transfected with the various constructs (0.5–2 µg DNA/dish) by using Lipofectamine 2000 (Invitrogen) for 24 h as described previously (Varnai et al., 2005). For calcium measurements, cells were loaded with 3 µM Fura-2/AM in medium 199/Earle's balanced salt solution containing 1.2 mM CaCl₂, 3.6 mM KCl, 25 mM HEPES containing 1 mg/ml bovine serum albumin (BSA), 0.06% Pluronic acid, and 200 µM sulfinpyrazone, for 45 min at room temperature. Calcium measurements were performed at room temperature in a modified Krebs-Ringer buffer containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl₂, 0.7 mM MgSO₄, 10 mM glucose, and Na-HEPES 10 mM, pH 7.4. An Olympus IX70 inverted microscope equipped with a Lamda-DG4 illuminator and a MicroMAX-1024BFT digital camera and the appropriate filter sets was used for Ca²⁺ analysis. The same microscope equipped with a beam-splitter (Optical Insights, Photometrics, Tucson, AZ) with a 505-nm dichroic mirror was used for FRET analysis with 435/25-nm excitation and 475/30- and 535/30-nm emission filters, respectively. Images acquired simultaneously in the two emission wavelengths in the two halves of the camera sensor chip were used to form the ratios. These ratio values were then normalized, taking their control initial values as 100 and the minimum values obtained after ionomycin (or agonist treatment if the latter was smaller) as zero.

Cells expressing the OSH2–2x-PH-GFP and PLC1PH-monomeric red fluorescent protein (mRFP) constructs were studied in the same modified Krebs-Ringer solution at 35°C by using a Zeiss 510Meta laser-scanning confocal microscope (Carl Zeiss, Thornwood, NY) in multitrack mode, and pictures were taken in time-series mode. Membrane localization was assessed by forming membrane/cytosol intensity ratio values from line-intensity histograms of cells in the whole series of images after acquisition. Data acquisitions for Ca²⁺ and FRET measurements and processing were performed by the MetaFluor software (Molecular Devices, Sunnyvale, CA). Postacquisition data analysis of the confocal images was performed with either the MetaView (Carl Zeiss) or the MetaMorph (Molecular Devices) software.

Cytoplasmic Ca²⁺ Measurements in Cell Suspensions
Cells grown on 10-cm culture plates were removed by mild trypsinization and loaded with 0.5 µM Fura-2/AM in the same solution described above for single Ca²⁺ measurements. Cells were then washed with the same medium without Fura-2/AM and stored at room temperature in the dark. Aliquots of cells (5 x 10⁵ cells) were centrifuged rapidly before the measurements and dispersed in 2.5 ml of the modified Krebs-Ringer buffer used for all other analysis. Ca²⁺ measurements were performed at 35°C in a PTI Deltascan spectrofluorometer (Photon Technology International, Princeton, NJ).

Analysis of Myo-[³H]Inositol- or [³²P]Phosphate-labeled Lipids and ³H-labeled Inositol Phosphates
Cultured cells were labeled with myo-[³H]inositol (20 µCi/ml) on 12-well culture plates in inositol-free DMEM supplemented with 2% dialyzed FBS and 1 mg/ml BSA for 24 h. For ³²P-phopshate labeling, the cells were labeled with 2 µCi/ml o-[³²P]phosphate for 3 h in phosphate-free DMEM supplemented with 1 mg/ml BSA. After myo-inositol labeling, the cells were washed twice with a medium without inositol, and after a 10-min preincubation, inhibitors were added for a further 10 min before stimulation with 100 nM angiotensin II (AngII) for the indicated times. Reactions were terminated by the addition of ice-cold perchloric acid (5% final concentration), and cells were kept on ice for 30 min. After scraping, and freezing/thawing, the cells were centrifuged, and the supernatant was processed for perchloric acid (PCA) extraction and analysis by high-performance liquid chromatography as described previously (Nakanishi et al., 1995). The cell pellet was also processed to extract the phosphoinositides by an acidic chloroform/methanol extraction followed by thin layer chromatography (TLC) analysis essentially as described previously (Nakanishi et al., 1995). ³²P-labeled cells were not washed after the labeling period, but they were treated in the same medium with inhibitors for 10 min followed by AngII (10^–7 M) stimulation. Reactions were terminated by PCA, and lipids were extracted and separated as with the inositol-labeled cells.

	RESULTS

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Pharmacological Manipulations of Type III PI 4-Kinase Activities Affect Agonist-regulated PtdIns4P Pools
High (micromolar) concentrations of Wm have been valuable tools to demonstrate a role of type III PI4Ks in InsP₃/Ca²⁺ signaling. However, it is not possible to discriminate between the - and β-forms of these PI4Ks based on their Wm sensitivities (Balla et al., 1997; Knight et al., 2006). Therefore, we took advantage of the recent characterization of isoform-specific PI 3-kinase inhibitors that revealed one of the inhibitors, PIK93, as being significantly more potent against PI4KIIIβ than PI4KIII (Knight et al., 2006). This inhibitor inhibits PI 3-kinases and clearly discriminates between the two PI 4-kinase enzymes as assessed by an in vitro PI kinase assay on the purified mammalian proteins (Knight et al., 2006). Unfortunately, none of the tested inhibitors showed the opposite selectivity, i.e., inhibition of PI4KIII more potently than the PI4KIIIβ enzyme. Phenylarsine oxide (PAO), a sulfhydryl (SH)-reactive agent has been shown earlier to inhibit PI4Ks (Wiedemann et al., 1996). PAO was found to inhibit type III PI 4-kinases, whereas it is relatively ineffective against the type II enzymes based on in vitro kinase assays of the purified proteins (Balla et al., 2002; Barylko et al., 2002). Among the type III enzymes, PI4KIII is significantly more sensitive to PAO than the type IIIβ form (Balla et al., 2002). This difference was also exploited to discriminate between the two enzymes in their involvement to generate the hormone-sensitive PtdIns4P pools. It is important to emphasize that PAO is a SH-reactive agent that probably has many targets in a cell. However, in the present study, the effects of PAO have only been tested on parameters that immediately reflect PI 4-kinase functions (see below); therefore, in this narrow context the effects of PAO on the purified PI 4-kinases and PtdIns4P formation in the intact cell can be correlated with somewhat higher confidence.

Several parameters were tested with these inhibitors in HEK-293 cells stably expressing AT_1a angiotensin receptors (HEK-293-AT1). Cellular phosphoinositides were monitored after [³²P]phosphate or myo-[³H]inositol labeling, and InsP₃ formation was followed from cells prelabeled with myo-[³H]inositol. Finally, the effects of the inhibitors on AngII-induced changes in cytoplasmic Ca²⁺ concentration [Ca²⁺]_i were measured in cell suspensions with Fura-2. Figure 1 shows the effects of inhibitors on InsP₃ levels analyzed from myo-[³H]inositol-labeled cells and on Ca²⁺ signaling. As shown earlier in AngII-stimulated adrenal glomerulosa cells (Nakanishi et al., 1995), 10 µM Wm pretreatment greatly reduced the size of AngII-induced InsP₃ elevation in HEK-293-AT1 cells, and it eliminated its sustained increase. This was also reflected in the cytoplasmic Ca²⁺ changes that became transient lacking the plateau phase of Ca²⁺ rise in Wm treated cells (Figure 1B). These effects of Wm were not observed at lower concentrations (<300 nM) that already inhibit PI 3-kinases (data not shown), and they were clearly caused by the limited supply of PtdIns4P and PtdIns(4,5)P₂, consistent with the inhibition of a PI4K that helps replenishing these pools during a sustained phospholipase C (PLC) activation. This is demonstrated in Figure 2, where the [³²P]phosphate-labeled PtdIns4P and PtdIns(4,5)P₂ were analyzed in AngII-stimulated cells. Without the inhibitors, AngII stimulation evokes a robust PLC activation, resulting in the rapid depletion of PtdIns(4,5)P₂ that slowly returns toward prestimulatory levels. A similar change is observed in PtdIns4P levels due to the conversion of this lipid to PtdIns(4,5)P₂ by the PIP 5-kinases and its relatively slower synthesis by the PI4Ks. Pretreatment of the cells with 10 µM Wm greatly reduced the ³²P-labeled PtdIns4P and, hence, the AngII-induced changes observed in HEK-293-AT1 cells (Figure 1A), but it only slightly reduced basal PtdIns(4,5)P₂ levels. However, this treatment significantly enhanced the AngII-induced decrease in PtdIns(4,5)P₂ and strongly inhibited its resynthesis (Figure 2B).

View larger version (16K): [in this window] [in a new window]   Figure 1. Effects of PI4K inhibitors on the kinetics of InsP3 and [Ca2+]i changes in HEK-293-AT1 cells stimulated with AngII. (A) HEK-293-AT1 cells were labeled with myo-[3H]inositol for 24 h in inositol-free medium as described under Materials and Methods. After washing, AngII (10–7 M) was added to the cells for the indicated times, and reactions were terminated by PCA. Labeled inositol phosphates were extracted from the soluble fraction and separated by high-performance liquid chromatography connected to a scintillation flow detector as described previously (Nakanishi et al., 1995). Data are shown are means ± range of duplicate determinations. The concentrations of the inhibitors added 10 min before AngII were 250 nM PIK93, 10 µM PAO with 1 mM β-mercaptoethanol (Mer), and 10 µM Wm. Two additional experiments were performed with the 10-min time points with similar results. (B) HEK-293-AT1 cells were loaded with Fura-2/AM, and their fluorescence monitored in a fluorescent spectrophotometer as described under Materials and Methods. AngII (10–7 M) was added at the indicated time. Pretreatment with the inhibitors for 10 min was as described in A. This result is a representative of three similar observations.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effects of PI4K inhibitors on the kinetics of phosphoinositide changes in HEK-293-AT1 cells stimulated with AngII. HEK-293-AT1 cells were labeled with [32P]phosphate for 3 h in a phosphate-free medium as described under Materials and Methods. AngII (10–7 M) was added to the cells at the indicated times, and reactions terminated by the addition of PCA. Lipids were extracted from the cell pellets, separated by TLC, and analyzed both by a PhosphorImager and scintillation counting of the spots cut out from the plates. Data shown are means ± range of duplicate determinations. The concentrations of the inhibitors added 10 min before AngII were 250 nM PIK93, 10 µM PAO with 1 mM Mer, and 10 µM Wm. This full time course experiment was repeated from myo-[3H]inositol-labeled cells with similar results, supporting the same conclusion.

Analysis of PtdIns(4,5)P₂ Changes in the Plasma Membrane with the PLC₁PH Domain
These metabolic data provided information on the overall inositide pools of the cells but only limited information on the question of which membranes contributed to these changes. In fact, when cells were labeled with myo-[³H]inositol, a smaller fraction of the inositides showed changes with AngII and the inhibitors (data not shown), suggesting the presence of additional metabolic pools that become labeled with myo-[³H]inositol in 24 h but not with [³²P]phosphate in 3 h. Therefore, further experiments were designed to monitor the phosphoinositide changes in single cells by using PH domains that recognize PtdIns4P and PtdIns(4,5)P₂. PtdIns(4,5)P₂ changes were followed in single cells by either confocal microscopy or by FRET analysis using yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-tagged versions of the PLC PH domain (van Der Wal et al., 2001).

Cells were stimulated with AngII for the indicated times followed by the addition of high concentration (10 µM) of ionomycin to activate PLC and to eliminate PLC₁PH-GFP localization and FRET (Várnai and Balla, 1998; Balla et al., 2005). Subsequent addition of 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA) (or EGTA) was used to reverse the Ca²⁺ change and allow the PtdIns(4,5)P₂ pools to recover. As shown in Figure 3, the PLC₁PH translocation was rapid and complete after AngII treatment, and the FRET signal rapidly returned close to the baseline in control cells (black trace). Ionomycin treatment then caused the complete translocation of the probe to the cytosol from which the cells recovered slowly after Ca²⁺ chelation. These changes were not affected by preincubation of the cells with 250 nM PIK93 (Figure 3, red trace), consistent with the previous data with metabolic labeling and suggesting that PI4KIIIβ plays no significant role in this process. In contrast, 10 µM PAO (with 1 mM β-mercaptoethanol) decreased the basal FRET by 40% and prevented the relocalization of the probe after the AngII-induced complete translocation (Figure 3, blue trace).

View larger version (21K): [in this window] [in a new window]   Figure 3. Effects of PI4K inhibitors on the kinetics of redistribution of PLC1PH domain in HEK-293-AT1 cells stimulated with AngII. HEK-293 cells stably transfected with the AT1a angiotensin receptors were cotransfected with the PLCd1PH-CFP and -YFP constructs for 24 h. The FRET signal from individual cells was then monitored in a wide-field fluorescent microscope equipped with an emission beam splitter using 430-nm excitation and 535- and 475-nm emissions as described under Materials and Methods. To minimize light exposure, after the first minute of stimulation, data were collected in longer intervals. AngII (10–7 M) was added at the indicated time followed by 10 µM inonomycin (Iono) and 5 mM BAPTA (or EGTA). The dark bar represents the exposure of the cells to the high (extracellular) Ca2+ concentration after ionomycin treatment. The concentrations of the inhibitors added 10 min before AngII were 250 nM PIK93 and 10 µM PAO with 1 mM Mer. FRET was expressed as fluorescent emission ratio values (535/475 nm), and it was normalized so that the ratio value recorded before the addition of inhibitors was taken as 100% and the lowest values (after AngII or ionomycin) were taken as 0%. Means ± SEM (or range) from five, two, and three cells are shown for control, PAO + Mer, and PIK93, respectively. Similar changes were observed in two additional experiments.

Evaluation of the OSH2 PH Domain as a Probe of Plasma Membrane PtdIns4P Levels
None of the type III PI4K enzymes can be detected by immunofluorescence at the plasma membrane; yet, PtdIns4P has to be present in the plasma membrane to be converted to PtdIns(4,5)P₂. To obtain information on the activity rather than the steady-state cellular distribution of the PI4K enzymes, it was highly desirable to use reporter probes that monitor PtdIns4P production in single cells. PH domain-GFP chimeras with high in vitro PtdIns4P binding specificities, such as the four phosphate adaptor protein (FAPP)1 or oxysterol binding protein (OSBP) PH domains have been used for this purpose, but these probes mostly report on PtdIns4P formed in the Golgi as they also require Arf1-GTP for their Golgi recruitment (Levine and Munro, 2002; Godi et al., 2004; Balla et al., 2005). Although both of these PH domains can detect PtdIns4P formation in the plasma membrane under special circumstances (Balla et al., 2005), they are not optimal for monitoring PtdIns4P in the plasma membrane. It has recently been shown by two independent studies that the PH domain of the yeast oxysterol binding protein homologue, OSH2 recognizes PtdIns4P in the plasma membrane, in spite of its limited specificity to bind PtdIns4P in vitro (Roy and Levine, 2004; Yu et al., 2004). Therefore, first, we wanted to determine whether the OSH2 PH domain could, in fact, be used to follow PtdIns4P changes in the plasma membrane of mammalian cells.

Expression of the single PH domain of OSH2 fused to GFP showed clear plasma membrane localization and a strong nuclear staining as described previously (Roy and Levine, 2004; Yu et al., 2004). Using two PH domains in tandem fused to GFP (GFP-OSH2-PH2x) greatly reduced the nuclear localization of the construct and clearly labeled the plasma membrane (Figure 4A). Importantly, unlike in yeast cells, neither the tandem nor the single OSH2 PH domain decorated the Golgi membrane in COS-7 or HEK-293 cells (Figure 4A). In contrast, the yeast OSH1 PH domain labeled both the plasma membrane (at high expression levels) and the Golgi (Figure 4A). Because the OSH2 PH domain has limited in vitro specificity to PtdIns4P (Yu et al., 2004), we were concerned that it may be recruited to the plasma membrane by PtdIns(4,5)P₂. The specificity question was examined with our recently developed method of acute depletion of PtdIns(4,5)P₂ in the plasma membrane. This approach is based on a drug induced plasma membrane recruitment of a truncated type-IV phosphoinositide 5-phosphatase (5-ptase) that was rendered cytoplasmic by mutations in its localization sequences (Varnai et al., 2006). Recruitment of this cytoplasmic 5-ptase to the plasma membrane rapidly eliminated PtdIns(4,5)P₂ as monitored by PLC₁PH-GFP localization (Figure 4B), without elimination of the GFP-OSH2-PH2x localization (Figure 4C). This question was further analyzed in total internal reflection fluorescence experiments where the release of the PH domain from the membrane can be better quantified. These experiments showed no effect of the phosphatase recruitment on GFP-OSH2-PH2x localization while eliminating PLC1PH-GFP localization in COS-7 cells (Korzeniowski and Balla, unpublished observations).

View larger version (44K): [in this window] [in a new window]   Figure 4. Localization of OSH1- and OSH2-PH domain-GFP fusion proteins in HEK-293-AT1 cells. (A) The PH domains of the yeast oxysterol-binding protein homologues OSH2 and OSH1 were fused to GFP as described under Materials and Methods. These constructs were transfected into HEK-293-AT1 cells and examined 24 h after transfection with live cell confocal microscopy. Note that the OSH2-PH domain binds to the plasma membrane and strongly accumulates in the nucleus but does not bind to the Golgi. A tandem PH domain of the OSH2 protein shows smaller signal in the nucleus and a strong plasma membrane binding. The OSH1-PH domain binds both the plasma membrane and the Golgi. (B) Elimination of the plasma membrane localization of PLC1PH-GFP [monitoring PtdIns(4,5)P2] by plasma membrane recruitment of a phosphoinositide 5-phosphatase. HEK-293-AT1 cells were transfected with a truncated type-IV phosphoinositide 5-phosphatase fused to mRFP and FKBP12, a plasma membrane-targeted FRB-CFP construct and the PLC1PH-YFP reporter described in Varnai et al. (2006). Addition of rapamycin for 3 min recruits the otherwise cytoplasmic 5-ptase construct to the plasma membrane (left) with a concomitant elimination of PtdIns(4,5)P2 and loss of PLC1PH-YFP localization (middle). (C) The same manipulations do not eliminate the plasma membrane localization of the OSH2-PH2x-GFP, suggesting that this construct is not kept at the membrane by PtdIns(4,5)P2.

View larger version (56K): [in this window] [in a new window]   Figure 5. Localization of OSH2-PH2x-GFP to the plasma membrane is wortmannin sensitive. (A) COS-7 cells were transfected with OSH2-PH2x-GFP together with PLC1PH-mRFP and the wild-type type IV phosphoinositide 5-phosphatase for 24 h. Cells were selected so that the PLC1PH-mRFP showed no localization, indicating the lack of PtdIns(4,5)P2 as a result of phosphatase expression. These cells still showed plasma membrane localization of OSH2-PH2x-GFP, indicating that the construct is kept in the membrane not by PtdIns(4,5)P2. Addition of 10 µM Wm to such cells caused a rapid translocation of the OSH2-PH2x-GFP domain construct from the membrane to the cytosol. (B) Release of the OSH2-PH2x-GFP construct from the membrane after Wm treatment is significantly slower in control cells where PtdIns(4,5)P2 is present in the membrane.

View larger version (48K): [in this window] [in a new window]   Figure 6. Angiotensin II-induced changes in membrane phosphoinositides simultaneously monitored by OSH2-PH2x-GFP and PLC1PH-mRFP. HEK-293 cells stably transfected with the AT1a angiotensin receptors were cotransfected with the PLC1PH-mRFP and OSH2-PH2x-GFP constructs for 24 h. Live cells were examined in a confocal microscope at 35°C during stimulation with AngII (10–7 M), and pictures were taken every 5 s. Note the faster response observed with the PLC1PH-mRFP. Quantification of membrane/cytosolic fluorescence intensities as function of time calculated from line-intensity histograms from five to seven cells obtained in two separate experiments (means ± SEM).

View larger version (12K): [in this window] [in a new window]   Figure 7. Effects of PI4K inhibitors on the membrane localization of the OSH2-PH2x-GFP. HEK-293 cells stably transfected with the AT1a angiotensin receptors were cotransfected with the OSH2-PH2x-GFP constructs for 24 h. Live cells were examined in a confocal microscope at 35°C. Calculation of membrane/cytosolic fluorescence intensities as function of time was performed from line-intensity histograms from images taken at every minute during a 10-min incubation with the inhibitors. The concentrations of the inhibitors were 250 nM PIK93, 10 µM PAO with 1 mM Mer, 10 µM PAO with 1 mM DTT, and 10 µM or 300 nM Wm. Means ± SEM from three to 25 cells from two to four separate experiments are shown.

View larger version (15K): [in this window] [in a new window]   Figure 8. Effects of AngII on the membrane localization of the OSH2-PH2x-GFP after treatment with PI4K inhibitors. HEK-293 cells stably transfected with the AT1a angiotensin receptors were cotransfected with the OSH2-PH2x-GFP constructs for 24 h. Live cells were examined in a confocal microscope at 35°C. Calculation of membrane/cytosolic fluorescence intensities as function of time was performed from line-intensity histograms from images taken at the indicated times after AngII addition. The concentrations of the inhibitors applied for 10 min before AngII were 250 nM PIK93, 10 µM PAO with 1 mM Mer, and 10 µM Wm. Means ± SEM from three to 25 cells from two to four separate experiments are shown. The initial ratio values of Wm-treated cells are higher in these experiments than those shown in Figure 7 at the end of Wm treatment, because cells with still measurable OSH2-PH2x-GFP localization after Wm treatment were selected for this analysis.

View larger version (20K): [in this window] [in a new window]   Figure 9. Effects of knockdown of the individual PI 4-kinases on [32P]phosphate labeling of phosphoinositides and their response to AngII stimulation in HEK-293-AT1 cells. HEK-293 cells stably transfected with the AT1a receptors were treated with siRNA for 3 d as described under Materials and Methods. The extent of knockdown was determined by Western blot analysis (A). On the fourth day, cells were labeled with [32P]phosphate for 3 h in phosphate-free medium. Cells were then either treated with AngII (10–7 M) or saline and incubated for an additional 10 min. Reactions were terminated by PCA, and lipids were extracted from the cell pellets, separated by TLC, and analyzed by a PhosphorImager (B). Due to cell loss in the PI4K-depleted cells, the individual PtdIns4P and PtdIns(4,5)P2 spots were normalized to the PtdA/PtdIns values measured in unstimulated samples of the respective groups. These ratios were then compared between the PI4K down-regulated or control siRNA-treated cells in each of the three experiments performed in duplicates (C). In each experiments, the effect of 250 nM PIK93 was also determined in the AngII group. Because PIK93-treated cells showed no difference compared with those treated with AngII only, these values were pooled in the statistical analysis. Means ± SEM are shown, and the p values obtained in one-sample t tests compared with control siRNA-treated cells are shown above the bars.

In cells labeled with myo-[³H]inositol, the picture was further complicated because we observed an increased labeling of PtdIns4P and PtdIns(4,5)P₂ (and the inositol phosphates) in cells in which PI4KIII was depleted. This paradoxical change was attributed to a significant decrease in the free inositol level of PI4KIII knockdown cells and hence an increased specific activity of the labeled inositol pool (data not shown). The reason for the consistent decrease in the level of inositol in cells depleted in PI4KIII is not known, and it is currently under further investigation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we asked the question of which of the PI4K enzymes is important for the generation of the plasma membrane pool of PtdIns4P, the precursor of PtdIns(4,5)P₂ during PLC activation in agonist-stimulated cells. This question has not been thoroughly addressed before for lack of appropriate tools to investigate it properly. In a previous study, we showed that Wm-sensitive type III PI4Ks are required for the maintenance of agonist-sensitive phosphoinositide pools and hence Ins(1,4,5)P₃ and calcium signaling (Nakanishi et al., 1995). These studies have been confirmed in other laboratories using different cell lines and different Ca²⁺-mobilizing receptors and agonists (Willars et al., 1998), but the identity of the enzyme(s) responsible for supplying PtdIns4P for the plasma membrane so far has eluded identification. The recent discovery of a PI 3-kinase inhibitor that discriminates between the type III PI4Ks has proven to be an invaluable tool to reinvestigate this question. Based on studies using ³²P-labeled phosphoinositides and myo-[³H]inositol-labeled inositol phosphates and cytoplasmic Ca²⁺ measurements, we were able to show that the effects of Wm are reproduced by inhibitors of PI4KIII but not PI4KIIIβ. It was also important to demonstrate that the PtdIns4P pool that is affected by the PI4KIII enzyme is in fact in the plasma membrane. This question was especially relevant, because none of the type III PI4Ks can be found in detectable amounts in the plasma membrane by immunofluorescence analysis of COS-7 or HEK-293 cells. Based on enzymatic characterization of the plasma membrane-associated PI4K activity performed two decades ago, it was concluded that the smaller and wortmannin-insensitive type II PI4Ks are present in the plasma membrane. This is supported by more recent immunocytochemical analysis that finds only the type II enzymes partially in the plasma membrane. Of the type III enzymes, PI4KIIIβ is found in the Golgi, and PI4KIII in the ER/Golgi compartment (see Balla and Balla, 2006 for citation of the original studies). So, it was of particular interest that the PH domain of the OSH2 protein, one of the yeast homologues of oxysterol binding proteins, is capable of reporting on changes in the plasma membrane that are consistent with changes of PtdIns4P levels (see also Yeung et al., 2006).

These studies using PH domains of PLC₁ and OSH2-PH2x to monitor PtdIns(4,5)P₂ and PtdIns4P, respectively, confirmed the conclusions of the labeling and Ca²⁺ experiments that PI4KIII is the enzyme necessary for the maintenance of the plasma membrane pool of these lipids. The exact mechanism how this is achieved by the enzyme that is primarily ER/Golgi localized remains to be answered, but its is quite possible that a small fraction of the enzyme is in fact found at the plasma membrane regulated by a unique mechanism, whereas the larger fraction of the protein has additional functions in the ER/Golgi compartments. It is interesting to note that in S. cerevisiae the Stt4p PI4K, a homologue of PI4KIII, was reported to generate the plasma membrane PtdIns4P pool, but unlike in mammalian cells, the yeast enzyme is primarily found in the plasma membrane or a tightly associated compartment (Audhya and Emr, 2002). Paradoxically, there are well-documented ER and vacuolar functions of Stt4p in yeast; yet, the protein has not been detected in those locations (Trotter et al., 1998; Audhya et al., 2000).

RNAi-mediated depletion of the individual PI4Ks has produced only small effects on ³²P-labeled PtdIns4P and PtdIns(4,5)P₂ but confirming the identity of the PI4KIII enzyme as one responsible for PtdIns4P and PtdIns(4,5)P₂ production at the plasma membrane during AngII stimulation. Although the levels of the enzymes can be knocked down to 15–30% of their original values, this level of knockdown produced only small effects in the labeled lipids and none on AngII-induced Ca²⁺ signaling. This probably reflects the fact that small amounts of these enzymes are sufficient to support the production of this signaling pool of the lipids and cells with more severe knockdown could simply be eliminated or developed adaptive mechanisms to cope with the lack of the protein. This highlights the difficulties with knockdown studies of enzymes of crucial importance and the value of inhibitors that can be tested in short-term experiments.

In recent studies, we were able to show the role of PI4Ks in other aspects of cellular PtdIns4P production where the knockdown data gave as clear results as the pharmacological approach. These included the recruitment of the FAPP1PH domain to the Golgi being mediated by both the PI4KIIIβ and PI4KII enzymes (Balla et al., 2005), and the role of PI4KIIIβ in the ER-to-Golgi transport of ceramide (Toth et al., 2006). In fact, in the former study we found that PI4KIII knockdown was able to decrease the plasma membrane recruitment of the FAPP1PH domain during recovery from a large Ca²⁺-mediated PLC activation (Balla et al., 2005). These studies collectively suggest that PI4Ks probably have multiple functions within the cells that are affected at different level of enzyme depletion.

Last, the experiments using the FAPP1PH, OSBP-PH, and OSH2-PH domains highlight the values and the limitations of PH domain reporters. They clearly demonstrate that specific pools of PtdIns4P are detected by the individual probes and that one reporter may not detect PtdIns4P in all locations within the cells. The FAPP1 and OSBP PH domains detect primarily the Golgi pool, although under special circumstances such as during recovery from massive Ca²⁺ induced PLC activation they can also detect PtdIns4P in the plasma membrane (Balla et al., 2005). In contrast, the OSH2–2x-PH-GFP reporter detects PtdIns4P in the plasma membrane but not in the Golgi compartment in mammalian cells. Only the yeast OSH1-PH decorates both the Golgi and the plasma membrane. Importantly, the plasma membrane localization of all of the PtdIns4P reporters depends on the activity of Wm-sensitive, type III PI4Ks, and based on the current studies, specifically of the PI4KIII enzyme.

In summary, the present results establish PI4KIII as an important component of the generation of the plasma membrane pool of phosphoinositides. This conclusion is based on pharmacological studies and confirmed by RNAi-mediated gene silencing, although the latter could not decrease the enzyme levels sufficiently to appreciably impair Ca²⁺ signaling. These results also establish the OSH2-PH2x domain as a useful reporter to follow PtdIns4P changes in the plasma membrane. Further studies are in progress to identify the mechanism by which the apparently ER/Golgi localized PI4KIII can affect the supply of plasma membrane PtdIns4P.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Roger Y. Tsien for the monomeric red fluorescent protein, Dr. Philip W. Majerus for the human type-IV 5-ptase clone, and Drs. Alberto-Jesus Olivares-Reyes and Kevin J. Catt for the HEK-293-AT1 cells. The confocal imaging was performed at the Microscopy and Imaging Core of the National Institute of Child Health and Human Development, National Institutes of Health (NIH), with the kind assistance of Drs. Vincent Schram and James T. Russell. This research was supported in part by the Intramural Research Program of the National Institute of Child Health and Human Development of the National Institutes of Health, and by an appointment of P.V. to the Senior Fellowship Program at the NIH. This latter program is administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the National Institutes of Health. A.B and P.V. are also Bolyai Fellows of the Hungarian Academy of Science and were supported by the Hungarian Scientific Research fund (OTKA NF-68563 and PF 63893) and the Medical Research Council (ETT 440/2006).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-07-0713) on December 12, 2007.

¹ Because AngII treatment increases the labeling of PtdIns/PtdA, and the knockdown of the individual enzymes could also affect this response, the correction for cell loss due to knockdown of the enzymes was based on the PtdIns/PtdA values obtained in the unstimulated cells also for the in AngII-stimulated samples. We assumed that AngII treatment does not cause cell loss.

Address correspondence to: Tamas Balla (ballat{at}mail.nih.gov)

Abbreviations used: 5-ptase, type-IV phosphoinositide 5-phosphatase; AngII, angiotensin II; DTT, dithiothreitol; ER, endoplasmic reticulum; FAPP, four phosphate adaptor protein; FRET, fluorescence resonance energy transfer; GFP, enhanced green fluorescent protein; InsP₃, inositol trisphosphate; Mer, β-mercaptoethanol; mRFP, monomeric red fluorescent protein; OSBP, oxysterol binding protein; PAO, phenylarsine oxide; PI4K, phosphatidylinositol 4-kinase; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PtdA, phosphatidic acid; PLC, phospholipase C; PH, pleckstrin homology; siRNA, small interfering RNA; Wm, wortmannin.

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