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Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells
Plant Physiol. Biochem., 1999, 37 (6), 473−480
Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells Camellia Moses Okpodu Department of Biological Sciences, Hampton University, Hampton, Virginia 23668, USA (fax +1 757 727 5961; e-mail [email protected])
(Received September 6, 1998; accepted March 13, 1999) Abstract — We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 µM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 µM inhibited 100 % of the nuclear-associated enzyme activity. The Km for ATP was estimated to be between 36 and 40 µM. The nuclear-associated PI kinase activity was inhibited by both 50 µM ADP and 10 µM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A2 and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast. © Elsevier, Paris Lipid kinase / nuclei / phosphatidylinositol / phosphatidylinositol kinase / phosphatidylinositol monophosphate / Daucus carota CDTA, trans-1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid hydrate / LPI, lysophosphatidylinositol / LPIP, lysophosphatidylinositol monophosphate / NBD-PA, (1-acyl-2-N-4-nitrobenzo-2-oxa-1,3-diazol)-aminocaproyl phosphatidic acid / NBD-PC, (1-acyl-2-N-4-nitrobenzo-2-oxa-1,3-diazol)-aminocaproyl phosphatidylcholine / PA, phosphatidic acid / PI, phosphatidylinositol / PIP, phosphatidylinositol 4-monophosphate / PIP2, phosphatidylinositol 4,5-bisphosphate / PLA2, phospholipase A2 / PolyPI, polyphosphoinositides / PS, phosphatidylserine
1. INTRODUCTION Because of the importance of phosphoinositide lipid signaling systems for the control of various physiological processes in eukaryotic metabolism, these lipids have been studied as potential regulators of cellular events in plants. The enzyme, phosphatidylinositol4-kinase(ATP,phosphatidylinositol-4-phosphotransferase, EC 2.7.1.67), catalyzes the first committed step in the formation of PI-4-P from PI and is therefore a critical regulatory step in the turnover of the second messenger, PIP2. Although this metabolic process is associated with the plasma membrane, evidence that inositol lipids are found in other cellular compartments (e.g. the Golgi apparatus [19], lysosomes [8] and nuclei in animals [32]; and the cytoskeleton [35, 40], a solublefraction [24] and isolated nuclei in plants [17]) adds complexity and suggests that the process is not rePlant Physiol. Biochem., 0981-9428/99/6/© Elsevier, Paris
stricted exclusively to the plasma membrane. If stimuli are transduced via polyPI turnover in the nucleus (a) the polyphosphoinositides must be located in the nucleus and (b) the enzymes responsible for the synthesis and degradation of the polyPIs are likely to be associated with the nuclear membrane. The enzyme that catalyzes the first committed step in the synthesis of the second messenger has been well characterized in some systems. The PI kinase activity has been classified into three subclasses (type I, II and III) (for review see [28]). The type I enzyme, PI 3-kinase, phosphorylates the D-3 position of the inositol ring. The enzyme is a heterodimer consisting of a 100-kDa catalytic subunit and an 85-kDa regulatory subunit [18]. The regulatory subunit (85 kDa) was cloned and it contained two scr homologous domains (ie, SH domains) [26]. SH domains are potential phosphorylation sites as well as sites that allow the enzyme to associate with many membrane tyrosine kinases [5].
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A gene encoding for the 110-kDa catalytic subunit has been isolated from bovine brain [18]. Sequence analysis of the 110-kDa subunit showed similarities to a yeast lysosomal-like vacuolar protein, VSP34 [31]. The type I enzyme has an apparent Km for ATP in the range of 16 to 120 µM [22, 23, 33, 38] and is sensitive to non-ionic detergents. It is not involved in the classical signal transduction pathway that produces inositol-1,4,5-trisphosphate. Type II and type III enzymes phosphorylate inositol in the D-4 position (i.e. both are PI-4-kinases). They are classified according to their biochemical properties (for review see [28]). The type II enzyme molecular mass averages 43 ± 6 kDa [15, 36]. The enzyme is stimulated by non-ionic detergents and is inhibited by low levels of adenosine (Ki 20–100 µM) and ADP. The apparent Km for ATP is in the range of 30 to 100 µM and divalent cations are required for maximal activity. Calcium concentrations ranging from 300 to 400 µM inhibit the enzyme activity. Type III PI kinases are stimulated by Triton X-100, have a higher Km for ATP (250–742 µM) than the type II PI kinase, and are relatively insensitive to adenosine (Ki 1.5 mM, [28]). The molecular mass of the purified type III enzyme ranges between 76 and 125 kDa [10]. These molecular masses are considerably smaller than those first reported by Endemann et al. [9]. The discrepancy in molecular mass could be a preparation artifact since Endemann et al. [9] reported the molecular mass of a crude preparation versus the purified enzyme. In higher plant systems, the plasma membrane PI kinase has been well characterized but never purified. In previous work [25], we characterized and partially purified a soluble (i.e. non-membrane associated) form of the PI kinase from carrot suspension culture cells. The PI kinase associated with the nucleus has been identified [17] but never characterized biochemically. If there is to be a parallel signaling pathway in the nucleus as has been suggested by York and Phillip [39], then the regulation of the nuclear-associated PI kinase is important. In this paper, we have used biochemical properties to further characterize the nuclear-associated PI kinase activity from carrot suspension culture cells.
ATPase for the plasma membrane, latent IDPase for Golgi, cytochrome c oxidase for the mitochondria and cytochrome c reductase for the endoplasmic reticulum [12, 29, 30]. Both the vandate-sensitive K+ATPase activity and latent IDPase activity were undetectable. The cytochrome c oxidase and reductase activities were respectively, 0.008 ± 0.005 and 0.121 ± 0.08 µmol⋅mg–1 protein⋅min–1 [24]. The results are in accordance with data presented by Hendrix et al. [17] and support the statement that the nuclei were not contaminated by plasma membrane.
2.2. PI kinase activity It has been reported that a PI 3-kinase activity is present in higher plants and, because the nuclear enzyme had never been characterized, we wondered if the PI kinase activity associated with isolated nuclei in carrot suspension culture cells was a PI 3-kinase. To address this question, we took isolated nuclei and assayed using conditions which had been reported to be optimum for either PI 3- or 4-kinase (respectively, 1 mM PI with 400 µM PS as is shown in (figure 1, lane 1) and 530 µM PI in 0.25 % (v/v) Triton X-100 as in (figure 1, lane 2)). After 10 min, the reaction was stopped by adding 1.5 mL ice cold CHCl3/MeOH (1/2, v/v). The lipids were extracted and separated in a boric acid system (lipid standards migrated as noted). Separation of the products via conditions that have been reported to separate PI-3-P from PI-4-P [37] suggested that the nuclear PI kinase was a PI 4-kinase. Subse-
2. RESULTS 2.1. Isolation of nuclei Plasma membrane-depleted nuclei from wild carrot suspension culture cells were isolated as described by Hendrix et al. [17]. Purity of the isolated nuclei was analyzed using different enzyme marker assays: K+Plant Physiol. Biochem.
Figure 1. Separation of phospholipids from isolated nuclei using different substrates. Isolated nuclei were assayed for the presence of PI 3-kinase (lane 1) and PI 4-kinase (lane 2) as outlined in Methods. The same amount of nuclear protein was used for each assay.
Type II nuclear phosphatidylinositol 4-kinase
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quently, head group analysis of the products showed that under all assay conditions, the nuclear-associated PI kinase produced 99.5 % PI-4-P (data not shown).
2.3. Characterization of the nuclear-associated PI 4-kinase Because the nuclear-associated PI kinase phosphorylated inositol on the 4th position of the inositol ring, we then wished to further characterize the PI 4-kinase. The nuclear-associated enzyme had a distinct pH optimum dependent on the conditions under which the substrate was added (figure 2). Endogenous nuclear-associated PI kinase had a broad pH optimum between 6.5 to 7.5. The pH optimum of the nuclear-associated enzyme was 7.0 when PI was added in the form of Triton X-100 mixed micelles. The pH optimum was 6.5 with PI and PS as the substrate. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at 0.5 % (v/v) Triton X-100 (figure 3). The apparent Km for ATP was 40 µM when the data was analyzed by a LineweaverBurke plot for the nuclear-associated PI kinase (figure 4 A). An Eadie-Hofstee plot indicated that the Km for ATP was 48 µM (figure 4 B). Adenosine, which has been shown to be an inhibitor of type II PI 4-kinase activity [38], decreased the activity of the nuclearassociated PI kinase (figure 5 A). ADP (10 µM) decreased the specific activity of the nuclear PI kinase by 50 % (figure 5, B). Calcium at 10 µM inhibited the nuclear enzyme by 90 % (figure 5, C).
Figure 3. Effects of Triton X-100 on the nuclear-associated PI kinase activity. Isolated nuclei were assayed for PI kinase activity with increasing concentrations of Triton X-100. The values are the average of duplicates. The experiment has been repeated three times and the trends were consistent (representative data are shown).
Figure 4. Kinetics for the utilization of ATP by the nuclearassociated PI kinase. A, Lineweaver-Burke plot. B, Eadie-Hofstee plot. Isolated nuclei (10 µg) were assayed in duplicate as described in Methods. Activity in the presence of varying concentrations of ATP is shown. (V is measure in nmol⋅mg–1protein⋅min–1).
2.4. The nuclear-associated enzyme could not be solubilized
Figure 2. pH Optimum. PI kinase activity was measured at the indicated pH values with 30 mM MES-Tris or Tris-MES buffer in the absence, i.e. endogenous substrate (●), or presence of exogenous substrate. Exogenous substrate was added either as PI/Triton X-100 mixed micelles (´) or PI/PS micelles (_). The numbers are averages of triplicate values. The deviation from the mean is indicated when it is larger than the symbol. The experiment was repeated three times and the trends were consistent.
Attempts to remove or solubilize the enzyme from the nucleus with either Triton X-100 (0.5–1.5 %) or Zwittergen 3-14 (0.25 %) were unsuccessful (figure 6). When nuclei were treated with 0.5 % Triton X-100 and centrifuged, the PI kinase activity was associated with the pellet and suggested that the solubilized nuclear PI kinase was associated with the nuclear matrix proteins and/or chromatin. It is quite possible that the enzyme is tightly associated with nuclear matrix proteins or the nucleic acids as suggested by Frederiks et al. [11]. However, when Triton X-100 solubilized pellets were treated with DNase or RNase, there was no significant vol. 37 (6) 1999
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Figure 6. Solubilization of PI kinase from isolated nuclei. Isolated nuclei were treated with PLA2, DNase, RNase, or Triton X-100 (0.5 % v/v). Equal aliquots (20 µL) of all supernatants (30 µg protein) were used for phosphorylation as described in Methods. The 0.01 % Triton X-100 control is 6 ± 0.5 pmol⋅mg–1 protein⋅min–1 for the supernatant ( ) and 12 ± 2 pmol⋅mg-1 protein⋅min–1 for the pellet (´).
Figure 5. Effects of adenosine, ADP and calcium on the nuclearassociated PI kinase activity. A, Isolated nuclei were assayed in the presence of increasing concentrations of adenosine; B, isolated nuclei were assayed in the presence of increasing concentrations of ADP; C, CaCl2-EGTA buffer was present to give the indicated free calcium concentration. The calcium concentrations were calculated using the method of Buckhout [4]. In C, results are presented as percent control. The control contained 2.5 mM EGTA and the specific activity was 20.0 ± 0.4 pmol [32P]-phosphatidylinositol monophosphate⋅mg–1 protein⋅min–1. In A–C, the numbers are the average of duplicate values. The experiments were repeated three times and the trends were similar (standard deviation is shown when it is larger than the symbol).
increase in PI kinase activity in the supernatant (figure 6). The PI kinase activity remained in the pellet suggesting that the PI kinase was not binding to nucleic acids. Because it has been shown that the PI kinase activity can be associated with actin [27, 35], we wondered if F-actin was associated with the nuclei. Our results show that we could not detect actin in isolated nuclei as determined by western blotting (figure 7, lane 1). If the nuclear enzyme is a membrane-bound PI kinase, as has been shown with the plasma membrane enzyme, it differs from the plasma membrane with response to positive effectors (Okpodu and Boss, unpubl. results). The PI kinase associated with the nucleus is not sensitive to spermidine or spermine at concentrations that have been shown to be effective for the plasma membrane enzyme (Yang and Boss, unpubl. results). In addition, Plant Physiol. Biochem.
Figure 7. Detection of actin in isolated nuclei. Nuclei were obtained as described in Methods and separated on 12 % SDS-PAGE. The proteins were blotted and tested with cross-reactivity with antibodies against F-actin. Lane 1, isolated nuclei (33 µg protein); arrow denotes the migration of actin (43 kDa); lane 2, F-actin fraction from microsomal membranes (∼15 µg protein).
treatment of the intact nuclei with phospholipase A2, a treatment which has been shown to be effective in releasing the enzyme from the plasma membrane [16], did not release PI kinase activity from the nuclear fraction (figure 6).
3. DISCUSSION Pools of phosphoinositides are known to exist in various cellular compartments other than the plasma membrane. The nucleus has a lipid pattern that differs significantly from that of the plasma membrane and the cytoplasm [7, 17, 21], and changes in the nuclear lipid composition have been correlated with differentiation
Type II nuclear phosphatidylinositol 4-kinase
and a mitogenic stimulation. Stimulation of a variety of receptors at the cell surface can activate one or two different signaling pathways that use PI derivatives as second messengers. One pathway involves the PI 3-kinase which phosphorylates the third position of the inositol ring (see [5] for review). PI 3-kinase mediates the transmission of the mitogenic signal of receptor or non-receptor kinases. The second, well characterized signaling pathway, involves the hydrolysis of PIP2 to DAG and IP3 via a phosphoinositide-specific PLC. Phosphatidylinositol (PI) kinase activity of isolated nuclei from carrot suspension culture cells was characterized. The nuclear-associated PI kinase had a broad pH optimum, which was dependent on the form of substrate added. The enzyme had an apparent Km for ATP between 40 and 48 µM. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). ADP (10 µM) decreased the nuclear enzyme activity. Calcium at 10 µM inhibited 100 % of the nuclearassociated enzyme activity. Adenosine (50 µM) decreased the activity. The nuclear-associated PI kinase was not easily solubilized. The isolated nuclei were not contaminated with actin as determined by antibodies. Because we could not remove the nuclear PI kinase from the nuclear fraction, we used antibodies against a yeast PI kinase to see if we could determine the molecular mass. This antibody recognized a 38kDa polypeptide (data not shown). This molecular mass is considerably different from the 119 922 Da PI 4-kinase, PIK1, associated with yeast nuclei [13]; however, there is a discrepancy in the molecular mass since the monoclonal antibody which was used to differentially screen for the PI kinase also recognized an additional protein of approximately 100 kDa. In carrot suspension culture cells, we could not detect PI 3-kinase activity associated with isolated nuclei based on analysis of products formed under several assay conditions. However, the enzyme may be present in vivo and activity for the PI 3-kinase was lost during the isolation procedure. The isomer PI-3-P has been reported to be present in other eukaryotic cells including Saccharomyces cerevisae [1]. Although we have used conditions that were optimal for the PI 3-kinase in mammalian systems, the conditions may not be optimal for the carrot suspension culture cells. Thus, we cannot conclude that the PI 3-kinase was not present in carrot suspension culture, but that under the conditions which we used, the nuclear-associated enzyme had no PI 3-kinase activity.
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Previously, we have described a PI kinase associated with isolated nuclei [17]. With the discovery of a soluble form of the PI kinase [25], we asked if the soluble PI kinase and the nuclear enzyme were the same enzyme? The soluble PI 4-kinase has a Km for ATP of 400 µM, is insensitive to increasing concentration of ADP, and is activated by Triton X-100. The molecular mass of the soluble enzyme is 80 kDa [25]. The soluble enzyme was not significantly affected at 100 µM free calcium. The soluble PI kinase was similar to the type III enzyme, except for its sensitivity to adenosine. The plant soluble PI kinase was sensitive to adenosine with a IC50 of 50 µM; therefore, we classified the enzyme as a mixed type or plant type III PI 4-kinase [24]. Type III kinases are reported to be relatively insensitive to inhibition by adenosine (Ki = 1.52 mM). The nuclear-associated PI 4-kinase was inhibited by slightly lower concentrations of adenosine (IC50 = 10 µM). The nuclear-associated PI kinase has a Km for ATP of 40 µM, and ADP (IC50 = 10 µM) decreased the nuclear enzyme activity. Based on these criteria, the nuclear-associated PI kinase is a type II PI 4-kinase. The nuclear-associated enzyme is not released with detergent treatment or PLA2, methods that have been successful in removing the enzyme from the plasma membrane. Results from the DNase and RNase experiments suggest that the nuclear PI 4-kinase is not associated with nucleic acids. This is consistent with other findings for the nuclear PI kinase. In nuclei isolated from rat liver, Manzoli et al. [21] reported that a significant amount of the enzyme remained associated to the nuclear matrix after extensive extraction with nucleases and detergents. The different phospholipids observed with the nucleus may play a structural role affecting the organization of the chromatin as well as facilitating the binding of the enzyme to the nucleoskeletal structure. Association of the PI kinase with the cytoskeleton has been shown for the plasma membrane PI kinase [35]. Western blots using antibodies to actin indicated that isolated nuclei did not contain detectable actin. It appears that the nuclear PI kinase is tightly associated with the nuclear matrix. In addition, when the nuclear matrix was isolated (as described by Payrastre et al. [27]) the PI 4-kinase was associated with the peripheral matrix or the lamina-pore complex fraction (Okpodu and Boss, unpubl. data). Compartmentalization of the PI kinase activity to the peripheral nuclear matrix has been shown in nuclear matrices isolated from NIH 3T3-fibroblasts and rat liver cells [27]. Our results are consistent with vol. 37 (6) 1999
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the hypothesis that the periphery of the nucleus is a crucial site for key regulatory events to take place and represents an important site of processing and exchange of information with the cytoplasm. Changes in inositol metabolism have been reported by Cocco et al. [7] to be correlated with cell differentiation. Thus, an interaction may exist between the phosphoinositide cycle in the nucleus and signal perceptions within the cell. Further studies will be necessary to determine the role that phosphoinositides play in nuclear signaling and cell division. Recently, both a type II and III PI 4-kinase have been enzymatically identified from bovine brain by partial protein and cDNA sequencing. Based only on sequence information, the type III isoform has been localized to the nucleus [14]. In summary, we have characterized a nuclearassociated PI 4-kinase that differs biochemically from the soluble PI kinase we described previously [25]. It is possible that the soluble enzyme is translocated to the nucleus upon stress, facilitating ‘cross-talk’ between the compartment’s microdomains; thus, it is conceivable that the soluble enzyme and nuclear PI kinase are the same enzyme. However, if this is the case, when the PI kinase becomes associated with the nucleus its biochemical properties change. While the roles of these new sources of PI 4-kinase remain to be elucidated, PIP and IP2 have been shown to activate DNA polymerase [34]. Thus, changes in nuclear PIP metabolism brought about by the PI 4-kinase could potentially affect gene expression.
4. METHODS 4.1. Materials Carrot cells (Daucus carota L.) were grown in suspension culture according to Boss and Ruesink [2]. Cells were transferred serially every 7 d and used for experiments at d 4. 4.2. Chemicals CDTA was purchased from Aldrich; PI, PIP and PS were from Sigma; NBD-PA and NBD-PC were from Avanti Polar Lipids. The anti-mouse antibody conjugated with alkaline phosphatase, DNase-free RNase and RNase-free DNase were purchased from Promega. Triton X-100 was purchased from Pierce. [32P]-ATP and monoclonal anti-chicken muscle actin antibody (clone C4) were purchased from ICN. Prestained molecular mass markers (low range) were purchased from Bio-Rad. 4.3. Protoplast isolation Protoplasts, isolated from 1.2 g cells as described by Hendrix et al. [17], were divided into two aliquots. Plant Physiol. Biochem.
Each aliquot was washed twice with 4 mL 2 mM MES-KOH (pH 6.0) and 0.45 molal sorbitol and centrifuged at 35 × g for 3 min. The protoplasts were resuspended in 4 mL ice cold 0.7 molal sorbitol in nuclear isolation buffer (NIB) (25 mM MES-KOH pH 5.2, 2 mM EGTA, 10 mM KCl, 10 mM NaCl); placed on ice (4 min), and centrifuged 35 × g for 3 min. All subsequent steps of the nuclei isolation were performed at 4 °C.
4.4. Nuclei isolation Nuclei were isolated using the method of Hendrix et al. [17]. The isolated nuclei were used for the PI kinase assay. Protein was determined by the methods of Lowry et al. [20] and Bradford [3] using bovine, serum albumin (BSA) as a standard.
4.5. Lipid kinase assay The PI 4-kinase activity (EC 2.7.1.67) was assayed using 20 lL nuclei preparation or the soluble enzyme (10–30 lg protein). The enzyme was added to 30 lL reaction mixture to give a final concentration of 50 mM Tris-HCl (pH 7.0), 15 mM MgCl2, 0.5 mM EGTA, 0.53 mM PI, 0.26 % (v/v) Triton X-100, 1.35 mM ATP containing [32P]-ATP containing (0.18 Bq⋅nmol–1). After 10 min, the reaction was stopped by the addition of 1.5 mL CHCl3/MeOH (1/2, v/v). Lipids were extracted and separated on thin layer chromatography plates as previously described [6, 37]. The PI 3-kinase activity (EC 2.7.1.137) was assayed using 20 lL preparation from isolated nuclei or the soluble fraction (10–30 lg protein). The enzyme was added to 30 lL reaction mixture to give a final concentration of 50 mM Tris-HCl (pH 6.5), 15 mM MgCl2, 0.5 mM EGTA, 100 µM ATP, 1 mM PI and 400 µM PS containing [32P]-ATP (0.18 Bq⋅nmol–1). The PI/PS substrate was stored in chloroform/ methanol/water (1/2/1, v/v/v). The solvent was evaporated under nitrogen and the lipids were resuspended in reaction mixture and sonicated for 5 min in a batch sonicator. After 10 min, the reaction was stopped by the addition of 1.5 mL CHCl3/MeOH (1/2, v/v). The lipids were separated on thin layer chromatography plates. Lipids were resolved either by the method of Cho et al. [6] or by Walsh et al. [37].
4.6. Chromatography of phosphatidylinositol monophosphates To separate PI-3-P from PI-4-P by thin layer chromatography, we used the boric acid solvent system described by Walsh et al. [37]. Briefly, LK5D thin layer plates were soaked for 10 s in a CDTA solution.
Type II nuclear phosphatidylinositol 4-kinase
The solution was made by mixing 4.55 g disodium CDTA.H2O, 165 mL H20, 330 mL ethanol and 3.0 mL NaOH until the CDTA was dissolved. The CDTAsoaked plates were air-dried (1 h) and baked (100 °C for 10 min) before use. Lipids were separated in a solvent consisting of a chloroform (160 mL), pyridine (45 mL), boric acid (12 g), water (7.5 mL) and 88 % (v/v) formic acid (3.0 mL). The plates were developed in a saturated tank and were removed from the tank when NBD-PC had migrated 14 cm (z3 h).
4.7. Solubilization of the nuclear-associated PI kinase Isolated nuclei were resuspended in buffer I (30 mM Tris-MES pH 7.5, 0.4 molal sorbitol, 15 mM MgCl2 in 0.01 % (v/v) Triton X-100) with 10 units⋅mL–1 PLA2 and incubated for 10 min at 25 °C. The reaction was stopped with the addition of EGTA (final concentration 5 mM). The samples were centrifuged at 14 000 × g for 5 min. The supernatant was removed and the pellet resuspended in 30 lL buffer I. The total protein was determined in all samples. The final concentration of protein was adjusted to (30 lg⋅lL–1) for all samples prior to assaying for PI kinase activity. For treatment with DNaseI and RNase A, the nuclei were resuspended in buffer II (30 mM Tris-MES pH 7.5, 15 mM MgCl2, 0.4 molal sorbitol and 1 mM DTT) and incubated for 2 h at 25 °C with 100 units⋅mL–1 DNase or RNase. The samples were centrifuged at 10 000 × g and the supernatant was removed and assayed for PI kinase activity. The pellet was resuspended in buffer II and centrifuged at 10 000 × g for 5 min. The supernatant was discarded and the pellet assayed for PI kinase activity. The total protein was adjusted to 45 lg⋅lL–1 in all samples.
4.8. Enzyme assays Vanadate-sensitive K+/ATPase activity (EC 3.6.1.3) was used as a marker for plasma membranes [12] and latent IDPase activity (EC 3.6.1.6) was used to identify the presence of Golgi apparatus [29]. Mitochondria were assayed by measuring cytochrome c oxidase activity (EC 1.9.3.1) and NADH-dependent cytochrome c reductase (EC 1.6.99.3) was used as a measure of the presence of endoplasmic reticulum [30].
Acknowledgments The authors wish to thank Drs Donald Lyons, Nicholas Kenny (Hampton University, Hampton, VA), Evan Keller (Eastern Virginia Medical School, Norfolk, VA) and Marc Jacobs (Swarthmore College,
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Swarthmore, PA) for helpful comments on the manuscript, and Mr Burrell (Hampton University, Hampton, VA) for assistance with photography. This research was supported in part by grant No. DCB-8812580 from the National Science Foundation and in part by the North Carolina Agricultural Research Service.
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[13] Garcia-Bustos J.F., Marini F., Stevenson I., Christian F., Michael N.H., PIK1, an essential phosphatidylinositol 4-kinase associated with the yeast nucleus, EMBO J. 13 (1994) 2352–2361. [14] Gehrmann T., Vereb G., Schmidt M., Klix D., Meyer H.E., Varsanyi M., Heilmeyeer Jr L.M.G., Identification of a 200-kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequence, Biochim. Biophys. Acta 1311 (1996) 53–63. [15] Graziani A., Ling L.E., Endemann G., Carpenter C.L., Cantley L.C., Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes, Biochem. J. 284 (1992) 39–45. [16] Gross W., Yang W., Boss W.F., Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70-kDa protein, Biochim. Biophys. Acta 1134 (1992) 73–80. [17] Hendrix W., Assefa H., Boss W.F., The polyphosphoinositides, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate, are present in nuclei isolated from carrot protoplast, Protoplasma 51 (1989) 62–72. [18] Hiles I.D., Otsu M., Volinia S., Fry M.J., Gout I., Dhand R., Panayotou G., Ruiz-Larrea F., Thompson A., Totty N.F., Hsuan J.J., Courtneidge S.A., Parker P.J., Waterfield M.D., Phosphatidylinositol 3-kinase: Structure and expression of the 110-kDa catalytic subunit, Cell 70 (1992) 419–429. [19] Jergil G., Sundler R., Phosphorylation of phosphatidylinositol in rat liver Golgi, J. Biol. Chem. 258 (1983) 7968–7973. [20] Lowry O.H., Rosebrogh N.J., Farr A.L., Randall R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [21] Manzoli F.A., Capitani S., Cocco L., Maraldi N.M., Mazzotti G., Barnabei O., Lipid mediated signal transduction in the cell nucleus, Adv. Enzyme Regul. 27 (1992) 83–92. [22] Morgan S.J., Smith A.D., Parker P.J., Purification and characterization of bovine brain type I phosphatidylinositol kinase, Eur. J. Biochem. 191 (1990) 761–767. [23] Okada T., Sakuma L., Fukui Y., Hazeki O., Ui M., Blockage of chematatic peptide-induced stimulation of neutrophils by Wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase, J. Biol. Chem. 269 (1994) 3563–3567. [24] Okpodu C.M., Characterization of two distinct phosphatidylinositol (PI) 4-kinases in carrot suspension culture cells, Ph.D. thesis, North Carolina State University, 1994, 186 p. [25] Okpodu C.M., Gross W., Burkhart W., Boss W.F., Purification and characterization of a soluble phosphatidylinositol 4-kinase from carrot suspension culture cells, Plant Physiol. 107 (1995) 491–500. [26] Otsu M., Hiles I., Gout I., Fry M.J., Ruiz-Larrea F., Panaotou G., Thompson A., Dhand R., Hsuan J., Totty N., Smith A.D., Morgan S.J., Courtneidge S.A., Parker Plant Physiol. Biochem.
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P.J., Waterfield M.D., Characterization of two 85 kDa proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI 3-kinase, Cell 65 (1991) 91–104. Payrastre B., Niever M., Boonstra J., Breton M., Verkleij A.J., van Bergen en Henegouwen P.M.P., A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix, J. Biol. Chem. 267 (1992) 5978–5084. Pike L.J., Phosphatidylinositol 4-kinase and the role of polyphosphoinositides in cellular regulation, Endocr. Rev. 13 (1992) 692–706. Ray P.M., Auxin-binding sites of maize coleoptiles are localized on membranes of the endoplasmic reticulum, Plant Physiol. 59 (1977) 594–599. Ray P.M., Shiniger T.L., Ray M.M., Isolation of β-glucan synthetase particles from plant cells and identification with Golgi membranes, Proc. Natl. Acad. Sci. USA 64 (1969) 605–612. Schu P.V., Takegawa K., Fry M.J., Stack J.H., Waterfield M.D., Emr S.D., Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting, Science 260 (1993) 88–91. Smith C.D., Wells W.W., Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation, J. Biol. Chem. 258 (1983) 9368–9373. Stephens L., Cooke F.T., Walters R., Jackson T., Volinia S., Gout I., Waterfield M.D., Hawkins P.T., Characterization of a phosphatidylinositol-specific phosphoinositde 3-kinase from mammalian cells, Curr. Biol. 4 (1994) 203–214. Sylvia V., Curtin G., Norman J., Stec J., Busbee D., Activation of a low specific activity form of DNA polymerase by inositol-1,4-bisphosphate, Cell 54 (1988) 651–658. Tan Z., Boss W.F., Association of phosphatidylinositol kinase, phosphatidylinositol monophosphate kinase, and diacylglycerol kinase with the cytoskeleton and F-actin fractions of carrot (Daucus carota L.) cells grown in suspension culture, Plant Physiol. 100 (1992) 2116–2120. Walker D.H., Dougherty N., Pike L.J., Purification and characterization of a phosphatidylinositol kinase from A431 cells, Biochemistry 27 (1988) 6504–6511. Walsh J.P., Caldwell K.K., Majerus P.W., Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate, Proc. Natl. Acad. Sci. USA 88 (1991) 9184–9187. Whitman M., Kaplan D., Roberts T., Cantley L., Evidence for two distinct types of phosphatidylinositol kinase, Biochemistry 26 (1987) 6845–6852. York J.D., Phillip W.M., Nuclear phosphatidylinositols decrease during s-phase of the cell cycle in HeLa cells, J. Biol. Chem. 269 (1994) 7847–7850. Xu P., Lloyd C.W., Staiger C.J., Drobak B.K., Association of phosphatidylinositol 4-kinase with the plant cytoskeleton, Plant Cell 4 (1992) 941–951.
Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells Camellia Moses Okpodu Department of Biological Sciences, Hampton University, Hampton, Virginia 23668, USA (fax +1 757 727 5961; e-mail [email protected])
(Received September 6, 1998; accepted March 13, 1999) Abstract — We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 µM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 µM inhibited 100 % of the nuclear-associated enzyme activity. The Km for ATP was estimated to be between 36 and 40 µM. The nuclear-associated PI kinase activity was inhibited by both 50 µM ADP and 10 µM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A2 and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast. © Elsevier, Paris Lipid kinase / nuclei / phosphatidylinositol / phosphatidylinositol kinase / phosphatidylinositol monophosphate / Daucus carota CDTA, trans-1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid hydrate / LPI, lysophosphatidylinositol / LPIP, lysophosphatidylinositol monophosphate / NBD-PA, (1-acyl-2-N-4-nitrobenzo-2-oxa-1,3-diazol)-aminocaproyl phosphatidic acid / NBD-PC, (1-acyl-2-N-4-nitrobenzo-2-oxa-1,3-diazol)-aminocaproyl phosphatidylcholine / PA, phosphatidic acid / PI, phosphatidylinositol / PIP, phosphatidylinositol 4-monophosphate / PIP2, phosphatidylinositol 4,5-bisphosphate / PLA2, phospholipase A2 / PolyPI, polyphosphoinositides / PS, phosphatidylserine
1. INTRODUCTION Because of the importance of phosphoinositide lipid signaling systems for the control of various physiological processes in eukaryotic metabolism, these lipids have been studied as potential regulators of cellular events in plants. The enzyme, phosphatidylinositol4-kinase(ATP,phosphatidylinositol-4-phosphotransferase, EC 2.7.1.67), catalyzes the first committed step in the formation of PI-4-P from PI and is therefore a critical regulatory step in the turnover of the second messenger, PIP2. Although this metabolic process is associated with the plasma membrane, evidence that inositol lipids are found in other cellular compartments (e.g. the Golgi apparatus [19], lysosomes [8] and nuclei in animals [32]; and the cytoskeleton [35, 40], a solublefraction [24] and isolated nuclei in plants [17]) adds complexity and suggests that the process is not rePlant Physiol. Biochem., 0981-9428/99/6/© Elsevier, Paris
stricted exclusively to the plasma membrane. If stimuli are transduced via polyPI turnover in the nucleus (a) the polyphosphoinositides must be located in the nucleus and (b) the enzymes responsible for the synthesis and degradation of the polyPIs are likely to be associated with the nuclear membrane. The enzyme that catalyzes the first committed step in the synthesis of the second messenger has been well characterized in some systems. The PI kinase activity has been classified into three subclasses (type I, II and III) (for review see [28]). The type I enzyme, PI 3-kinase, phosphorylates the D-3 position of the inositol ring. The enzyme is a heterodimer consisting of a 100-kDa catalytic subunit and an 85-kDa regulatory subunit [18]. The regulatory subunit (85 kDa) was cloned and it contained two scr homologous domains (ie, SH domains) [26]. SH domains are potential phosphorylation sites as well as sites that allow the enzyme to associate with many membrane tyrosine kinases [5].
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A gene encoding for the 110-kDa catalytic subunit has been isolated from bovine brain [18]. Sequence analysis of the 110-kDa subunit showed similarities to a yeast lysosomal-like vacuolar protein, VSP34 [31]. The type I enzyme has an apparent Km for ATP in the range of 16 to 120 µM [22, 23, 33, 38] and is sensitive to non-ionic detergents. It is not involved in the classical signal transduction pathway that produces inositol-1,4,5-trisphosphate. Type II and type III enzymes phosphorylate inositol in the D-4 position (i.e. both are PI-4-kinases). They are classified according to their biochemical properties (for review see [28]). The type II enzyme molecular mass averages 43 ± 6 kDa [15, 36]. The enzyme is stimulated by non-ionic detergents and is inhibited by low levels of adenosine (Ki 20–100 µM) and ADP. The apparent Km for ATP is in the range of 30 to 100 µM and divalent cations are required for maximal activity. Calcium concentrations ranging from 300 to 400 µM inhibit the enzyme activity. Type III PI kinases are stimulated by Triton X-100, have a higher Km for ATP (250–742 µM) than the type II PI kinase, and are relatively insensitive to adenosine (Ki 1.5 mM, [28]). The molecular mass of the purified type III enzyme ranges between 76 and 125 kDa [10]. These molecular masses are considerably smaller than those first reported by Endemann et al. [9]. The discrepancy in molecular mass could be a preparation artifact since Endemann et al. [9] reported the molecular mass of a crude preparation versus the purified enzyme. In higher plant systems, the plasma membrane PI kinase has been well characterized but never purified. In previous work [25], we characterized and partially purified a soluble (i.e. non-membrane associated) form of the PI kinase from carrot suspension culture cells. The PI kinase associated with the nucleus has been identified [17] but never characterized biochemically. If there is to be a parallel signaling pathway in the nucleus as has been suggested by York and Phillip [39], then the regulation of the nuclear-associated PI kinase is important. In this paper, we have used biochemical properties to further characterize the nuclear-associated PI kinase activity from carrot suspension culture cells.
ATPase for the plasma membrane, latent IDPase for Golgi, cytochrome c oxidase for the mitochondria and cytochrome c reductase for the endoplasmic reticulum [12, 29, 30]. Both the vandate-sensitive K+ATPase activity and latent IDPase activity were undetectable. The cytochrome c oxidase and reductase activities were respectively, 0.008 ± 0.005 and 0.121 ± 0.08 µmol⋅mg–1 protein⋅min–1 [24]. The results are in accordance with data presented by Hendrix et al. [17] and support the statement that the nuclei were not contaminated by plasma membrane.
2.2. PI kinase activity It has been reported that a PI 3-kinase activity is present in higher plants and, because the nuclear enzyme had never been characterized, we wondered if the PI kinase activity associated with isolated nuclei in carrot suspension culture cells was a PI 3-kinase. To address this question, we took isolated nuclei and assayed using conditions which had been reported to be optimum for either PI 3- or 4-kinase (respectively, 1 mM PI with 400 µM PS as is shown in (figure 1, lane 1) and 530 µM PI in 0.25 % (v/v) Triton X-100 as in (figure 1, lane 2)). After 10 min, the reaction was stopped by adding 1.5 mL ice cold CHCl3/MeOH (1/2, v/v). The lipids were extracted and separated in a boric acid system (lipid standards migrated as noted). Separation of the products via conditions that have been reported to separate PI-3-P from PI-4-P [37] suggested that the nuclear PI kinase was a PI 4-kinase. Subse-
2. RESULTS 2.1. Isolation of nuclei Plasma membrane-depleted nuclei from wild carrot suspension culture cells were isolated as described by Hendrix et al. [17]. Purity of the isolated nuclei was analyzed using different enzyme marker assays: K+Plant Physiol. Biochem.
Figure 1. Separation of phospholipids from isolated nuclei using different substrates. Isolated nuclei were assayed for the presence of PI 3-kinase (lane 1) and PI 4-kinase (lane 2) as outlined in Methods. The same amount of nuclear protein was used for each assay.
Type II nuclear phosphatidylinositol 4-kinase
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quently, head group analysis of the products showed that under all assay conditions, the nuclear-associated PI kinase produced 99.5 % PI-4-P (data not shown).
2.3. Characterization of the nuclear-associated PI 4-kinase Because the nuclear-associated PI kinase phosphorylated inositol on the 4th position of the inositol ring, we then wished to further characterize the PI 4-kinase. The nuclear-associated enzyme had a distinct pH optimum dependent on the conditions under which the substrate was added (figure 2). Endogenous nuclear-associated PI kinase had a broad pH optimum between 6.5 to 7.5. The pH optimum of the nuclear-associated enzyme was 7.0 when PI was added in the form of Triton X-100 mixed micelles. The pH optimum was 6.5 with PI and PS as the substrate. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at 0.5 % (v/v) Triton X-100 (figure 3). The apparent Km for ATP was 40 µM when the data was analyzed by a LineweaverBurke plot for the nuclear-associated PI kinase (figure 4 A). An Eadie-Hofstee plot indicated that the Km for ATP was 48 µM (figure 4 B). Adenosine, which has been shown to be an inhibitor of type II PI 4-kinase activity [38], decreased the activity of the nuclearassociated PI kinase (figure 5 A). ADP (10 µM) decreased the specific activity of the nuclear PI kinase by 50 % (figure 5, B). Calcium at 10 µM inhibited the nuclear enzyme by 90 % (figure 5, C).
Figure 3. Effects of Triton X-100 on the nuclear-associated PI kinase activity. Isolated nuclei were assayed for PI kinase activity with increasing concentrations of Triton X-100. The values are the average of duplicates. The experiment has been repeated three times and the trends were consistent (representative data are shown).
Figure 4. Kinetics for the utilization of ATP by the nuclearassociated PI kinase. A, Lineweaver-Burke plot. B, Eadie-Hofstee plot. Isolated nuclei (10 µg) were assayed in duplicate as described in Methods. Activity in the presence of varying concentrations of ATP is shown. (V is measure in nmol⋅mg–1protein⋅min–1).
2.4. The nuclear-associated enzyme could not be solubilized
Figure 2. pH Optimum. PI kinase activity was measured at the indicated pH values with 30 mM MES-Tris or Tris-MES buffer in the absence, i.e. endogenous substrate (●), or presence of exogenous substrate. Exogenous substrate was added either as PI/Triton X-100 mixed micelles (´) or PI/PS micelles (_). The numbers are averages of triplicate values. The deviation from the mean is indicated when it is larger than the symbol. The experiment was repeated three times and the trends were consistent.
Attempts to remove or solubilize the enzyme from the nucleus with either Triton X-100 (0.5–1.5 %) or Zwittergen 3-14 (0.25 %) were unsuccessful (figure 6). When nuclei were treated with 0.5 % Triton X-100 and centrifuged, the PI kinase activity was associated with the pellet and suggested that the solubilized nuclear PI kinase was associated with the nuclear matrix proteins and/or chromatin. It is quite possible that the enzyme is tightly associated with nuclear matrix proteins or the nucleic acids as suggested by Frederiks et al. [11]. However, when Triton X-100 solubilized pellets were treated with DNase or RNase, there was no significant vol. 37 (6) 1999
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Figure 6. Solubilization of PI kinase from isolated nuclei. Isolated nuclei were treated with PLA2, DNase, RNase, or Triton X-100 (0.5 % v/v). Equal aliquots (20 µL) of all supernatants (30 µg protein) were used for phosphorylation as described in Methods. The 0.01 % Triton X-100 control is 6 ± 0.5 pmol⋅mg–1 protein⋅min–1 for the supernatant ( ) and 12 ± 2 pmol⋅mg-1 protein⋅min–1 for the pellet (´).
Figure 5. Effects of adenosine, ADP and calcium on the nuclearassociated PI kinase activity. A, Isolated nuclei were assayed in the presence of increasing concentrations of adenosine; B, isolated nuclei were assayed in the presence of increasing concentrations of ADP; C, CaCl2-EGTA buffer was present to give the indicated free calcium concentration. The calcium concentrations were calculated using the method of Buckhout [4]. In C, results are presented as percent control. The control contained 2.5 mM EGTA and the specific activity was 20.0 ± 0.4 pmol [32P]-phosphatidylinositol monophosphate⋅mg–1 protein⋅min–1. In A–C, the numbers are the average of duplicate values. The experiments were repeated three times and the trends were similar (standard deviation is shown when it is larger than the symbol).
increase in PI kinase activity in the supernatant (figure 6). The PI kinase activity remained in the pellet suggesting that the PI kinase was not binding to nucleic acids. Because it has been shown that the PI kinase activity can be associated with actin [27, 35], we wondered if F-actin was associated with the nuclei. Our results show that we could not detect actin in isolated nuclei as determined by western blotting (figure 7, lane 1). If the nuclear enzyme is a membrane-bound PI kinase, as has been shown with the plasma membrane enzyme, it differs from the plasma membrane with response to positive effectors (Okpodu and Boss, unpubl. results). The PI kinase associated with the nucleus is not sensitive to spermidine or spermine at concentrations that have been shown to be effective for the plasma membrane enzyme (Yang and Boss, unpubl. results). In addition, Plant Physiol. Biochem.
Figure 7. Detection of actin in isolated nuclei. Nuclei were obtained as described in Methods and separated on 12 % SDS-PAGE. The proteins were blotted and tested with cross-reactivity with antibodies against F-actin. Lane 1, isolated nuclei (33 µg protein); arrow denotes the migration of actin (43 kDa); lane 2, F-actin fraction from microsomal membranes (∼15 µg protein).
treatment of the intact nuclei with phospholipase A2, a treatment which has been shown to be effective in releasing the enzyme from the plasma membrane [16], did not release PI kinase activity from the nuclear fraction (figure 6).
3. DISCUSSION Pools of phosphoinositides are known to exist in various cellular compartments other than the plasma membrane. The nucleus has a lipid pattern that differs significantly from that of the plasma membrane and the cytoplasm [7, 17, 21], and changes in the nuclear lipid composition have been correlated with differentiation
Type II nuclear phosphatidylinositol 4-kinase
and a mitogenic stimulation. Stimulation of a variety of receptors at the cell surface can activate one or two different signaling pathways that use PI derivatives as second messengers. One pathway involves the PI 3-kinase which phosphorylates the third position of the inositol ring (see [5] for review). PI 3-kinase mediates the transmission of the mitogenic signal of receptor or non-receptor kinases. The second, well characterized signaling pathway, involves the hydrolysis of PIP2 to DAG and IP3 via a phosphoinositide-specific PLC. Phosphatidylinositol (PI) kinase activity of isolated nuclei from carrot suspension culture cells was characterized. The nuclear-associated PI kinase had a broad pH optimum, which was dependent on the form of substrate added. The enzyme had an apparent Km for ATP between 40 and 48 µM. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). ADP (10 µM) decreased the nuclear enzyme activity. Calcium at 10 µM inhibited 100 % of the nuclearassociated enzyme activity. Adenosine (50 µM) decreased the activity. The nuclear-associated PI kinase was not easily solubilized. The isolated nuclei were not contaminated with actin as determined by antibodies. Because we could not remove the nuclear PI kinase from the nuclear fraction, we used antibodies against a yeast PI kinase to see if we could determine the molecular mass. This antibody recognized a 38kDa polypeptide (data not shown). This molecular mass is considerably different from the 119 922 Da PI 4-kinase, PIK1, associated with yeast nuclei [13]; however, there is a discrepancy in the molecular mass since the monoclonal antibody which was used to differentially screen for the PI kinase also recognized an additional protein of approximately 100 kDa. In carrot suspension culture cells, we could not detect PI 3-kinase activity associated with isolated nuclei based on analysis of products formed under several assay conditions. However, the enzyme may be present in vivo and activity for the PI 3-kinase was lost during the isolation procedure. The isomer PI-3-P has been reported to be present in other eukaryotic cells including Saccharomyces cerevisae [1]. Although we have used conditions that were optimal for the PI 3-kinase in mammalian systems, the conditions may not be optimal for the carrot suspension culture cells. Thus, we cannot conclude that the PI 3-kinase was not present in carrot suspension culture, but that under the conditions which we used, the nuclear-associated enzyme had no PI 3-kinase activity.
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Previously, we have described a PI kinase associated with isolated nuclei [17]. With the discovery of a soluble form of the PI kinase [25], we asked if the soluble PI kinase and the nuclear enzyme were the same enzyme? The soluble PI 4-kinase has a Km for ATP of 400 µM, is insensitive to increasing concentration of ADP, and is activated by Triton X-100. The molecular mass of the soluble enzyme is 80 kDa [25]. The soluble enzyme was not significantly affected at 100 µM free calcium. The soluble PI kinase was similar to the type III enzyme, except for its sensitivity to adenosine. The plant soluble PI kinase was sensitive to adenosine with a IC50 of 50 µM; therefore, we classified the enzyme as a mixed type or plant type III PI 4-kinase [24]. Type III kinases are reported to be relatively insensitive to inhibition by adenosine (Ki = 1.52 mM). The nuclear-associated PI 4-kinase was inhibited by slightly lower concentrations of adenosine (IC50 = 10 µM). The nuclear-associated PI kinase has a Km for ATP of 40 µM, and ADP (IC50 = 10 µM) decreased the nuclear enzyme activity. Based on these criteria, the nuclear-associated PI kinase is a type II PI 4-kinase. The nuclear-associated enzyme is not released with detergent treatment or PLA2, methods that have been successful in removing the enzyme from the plasma membrane. Results from the DNase and RNase experiments suggest that the nuclear PI 4-kinase is not associated with nucleic acids. This is consistent with other findings for the nuclear PI kinase. In nuclei isolated from rat liver, Manzoli et al. [21] reported that a significant amount of the enzyme remained associated to the nuclear matrix after extensive extraction with nucleases and detergents. The different phospholipids observed with the nucleus may play a structural role affecting the organization of the chromatin as well as facilitating the binding of the enzyme to the nucleoskeletal structure. Association of the PI kinase with the cytoskeleton has been shown for the plasma membrane PI kinase [35]. Western blots using antibodies to actin indicated that isolated nuclei did not contain detectable actin. It appears that the nuclear PI kinase is tightly associated with the nuclear matrix. In addition, when the nuclear matrix was isolated (as described by Payrastre et al. [27]) the PI 4-kinase was associated with the peripheral matrix or the lamina-pore complex fraction (Okpodu and Boss, unpubl. data). Compartmentalization of the PI kinase activity to the peripheral nuclear matrix has been shown in nuclear matrices isolated from NIH 3T3-fibroblasts and rat liver cells [27]. Our results are consistent with vol. 37 (6) 1999
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the hypothesis that the periphery of the nucleus is a crucial site for key regulatory events to take place and represents an important site of processing and exchange of information with the cytoplasm. Changes in inositol metabolism have been reported by Cocco et al. [7] to be correlated with cell differentiation. Thus, an interaction may exist between the phosphoinositide cycle in the nucleus and signal perceptions within the cell. Further studies will be necessary to determine the role that phosphoinositides play in nuclear signaling and cell division. Recently, both a type II and III PI 4-kinase have been enzymatically identified from bovine brain by partial protein and cDNA sequencing. Based only on sequence information, the type III isoform has been localized to the nucleus [14]. In summary, we have characterized a nuclearassociated PI 4-kinase that differs biochemically from the soluble PI kinase we described previously [25]. It is possible that the soluble enzyme is translocated to the nucleus upon stress, facilitating ‘cross-talk’ between the compartment’s microdomains; thus, it is conceivable that the soluble enzyme and nuclear PI kinase are the same enzyme. However, if this is the case, when the PI kinase becomes associated with the nucleus its biochemical properties change. While the roles of these new sources of PI 4-kinase remain to be elucidated, PIP and IP2 have been shown to activate DNA polymerase [34]. Thus, changes in nuclear PIP metabolism brought about by the PI 4-kinase could potentially affect gene expression.
4. METHODS 4.1. Materials Carrot cells (Daucus carota L.) were grown in suspension culture according to Boss and Ruesink [2]. Cells were transferred serially every 7 d and used for experiments at d 4. 4.2. Chemicals CDTA was purchased from Aldrich; PI, PIP and PS were from Sigma; NBD-PA and NBD-PC were from Avanti Polar Lipids. The anti-mouse antibody conjugated with alkaline phosphatase, DNase-free RNase and RNase-free DNase were purchased from Promega. Triton X-100 was purchased from Pierce. [32P]-ATP and monoclonal anti-chicken muscle actin antibody (clone C4) were purchased from ICN. Prestained molecular mass markers (low range) were purchased from Bio-Rad. 4.3. Protoplast isolation Protoplasts, isolated from 1.2 g cells as described by Hendrix et al. [17], were divided into two aliquots. Plant Physiol. Biochem.
Each aliquot was washed twice with 4 mL 2 mM MES-KOH (pH 6.0) and 0.45 molal sorbitol and centrifuged at 35 × g for 3 min. The protoplasts were resuspended in 4 mL ice cold 0.7 molal sorbitol in nuclear isolation buffer (NIB) (25 mM MES-KOH pH 5.2, 2 mM EGTA, 10 mM KCl, 10 mM NaCl); placed on ice (4 min), and centrifuged 35 × g for 3 min. All subsequent steps of the nuclei isolation were performed at 4 °C.
4.4. Nuclei isolation Nuclei were isolated using the method of Hendrix et al. [17]. The isolated nuclei were used for the PI kinase assay. Protein was determined by the methods of Lowry et al. [20] and Bradford [3] using bovine, serum albumin (BSA) as a standard.
4.5. Lipid kinase assay The PI 4-kinase activity (EC 2.7.1.67) was assayed using 20 lL nuclei preparation or the soluble enzyme (10–30 lg protein). The enzyme was added to 30 lL reaction mixture to give a final concentration of 50 mM Tris-HCl (pH 7.0), 15 mM MgCl2, 0.5 mM EGTA, 0.53 mM PI, 0.26 % (v/v) Triton X-100, 1.35 mM ATP containing [32P]-ATP containing (0.18 Bq⋅nmol–1). After 10 min, the reaction was stopped by the addition of 1.5 mL CHCl3/MeOH (1/2, v/v). Lipids were extracted and separated on thin layer chromatography plates as previously described [6, 37]. The PI 3-kinase activity (EC 2.7.1.137) was assayed using 20 lL preparation from isolated nuclei or the soluble fraction (10–30 lg protein). The enzyme was added to 30 lL reaction mixture to give a final concentration of 50 mM Tris-HCl (pH 6.5), 15 mM MgCl2, 0.5 mM EGTA, 100 µM ATP, 1 mM PI and 400 µM PS containing [32P]-ATP (0.18 Bq⋅nmol–1). The PI/PS substrate was stored in chloroform/ methanol/water (1/2/1, v/v/v). The solvent was evaporated under nitrogen and the lipids were resuspended in reaction mixture and sonicated for 5 min in a batch sonicator. After 10 min, the reaction was stopped by the addition of 1.5 mL CHCl3/MeOH (1/2, v/v). The lipids were separated on thin layer chromatography plates. Lipids were resolved either by the method of Cho et al. [6] or by Walsh et al. [37].
4.6. Chromatography of phosphatidylinositol monophosphates To separate PI-3-P from PI-4-P by thin layer chromatography, we used the boric acid solvent system described by Walsh et al. [37]. Briefly, LK5D thin layer plates were soaked for 10 s in a CDTA solution.
Type II nuclear phosphatidylinositol 4-kinase
The solution was made by mixing 4.55 g disodium CDTA.H2O, 165 mL H20, 330 mL ethanol and 3.0 mL NaOH until the CDTA was dissolved. The CDTAsoaked plates were air-dried (1 h) and baked (100 °C for 10 min) before use. Lipids were separated in a solvent consisting of a chloroform (160 mL), pyridine (45 mL), boric acid (12 g), water (7.5 mL) and 88 % (v/v) formic acid (3.0 mL). The plates were developed in a saturated tank and were removed from the tank when NBD-PC had migrated 14 cm (z3 h).
4.7. Solubilization of the nuclear-associated PI kinase Isolated nuclei were resuspended in buffer I (30 mM Tris-MES pH 7.5, 0.4 molal sorbitol, 15 mM MgCl2 in 0.01 % (v/v) Triton X-100) with 10 units⋅mL–1 PLA2 and incubated for 10 min at 25 °C. The reaction was stopped with the addition of EGTA (final concentration 5 mM). The samples were centrifuged at 14 000 × g for 5 min. The supernatant was removed and the pellet resuspended in 30 lL buffer I. The total protein was determined in all samples. The final concentration of protein was adjusted to (30 lg⋅lL–1) for all samples prior to assaying for PI kinase activity. For treatment with DNaseI and RNase A, the nuclei were resuspended in buffer II (30 mM Tris-MES pH 7.5, 15 mM MgCl2, 0.4 molal sorbitol and 1 mM DTT) and incubated for 2 h at 25 °C with 100 units⋅mL–1 DNase or RNase. The samples were centrifuged at 10 000 × g and the supernatant was removed and assayed for PI kinase activity. The pellet was resuspended in buffer II and centrifuged at 10 000 × g for 5 min. The supernatant was discarded and the pellet assayed for PI kinase activity. The total protein was adjusted to 45 lg⋅lL–1 in all samples.
4.8. Enzyme assays Vanadate-sensitive K+/ATPase activity (EC 3.6.1.3) was used as a marker for plasma membranes [12] and latent IDPase activity (EC 3.6.1.6) was used to identify the presence of Golgi apparatus [29]. Mitochondria were assayed by measuring cytochrome c oxidase activity (EC 1.9.3.1) and NADH-dependent cytochrome c reductase (EC 1.6.99.3) was used as a measure of the presence of endoplasmic reticulum [30].
Acknowledgments The authors wish to thank Drs Donald Lyons, Nicholas Kenny (Hampton University, Hampton, VA), Evan Keller (Eastern Virginia Medical School, Norfolk, VA) and Marc Jacobs (Swarthmore College,
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Swarthmore, PA) for helpful comments on the manuscript, and Mr Burrell (Hampton University, Hampton, VA) for assistance with photography. This research was supported in part by grant No. DCB-8812580 from the National Science Foundation and in part by the North Carolina Agricultural Research Service.
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