Phosphorylation of ATP-citrate lyase by nucleoside diphosphate kinase.

Rat liver nucleoside diphosphate kinase (NDPK) and PC12 cell cytosol were used to determine whether NDPK could function as a protein kinase. NDPK was phosphorylated on its catalytic histidine using ATP, and the phosphorylated NDPK separated from [γ-32P]ATP. The addition of phosphorylated NDPK to dialyzed PC12 cell cytosol resulted in the phosphorylation of a protein with a subunit molecular mass of about 120 kDa. This phosphorylation appeared to occur by a direct transfer of a phosphoryl group from the catalytic histidine of NDPK to a histidine on the 120-kDa protein. The 120-kDa protein was partially purified and shown by peptide sequencing to be ATP-citrate lyase. ATP-citrate lyase is the primary source of cytosolic acetyl-CoA. NDPK phosphorylated the histidine at the catalytic site of ATP-citrate lyase. This histidine can also be phosphorylated by ATP, and its phosphorylation is the first step in the conversion of citrate and CoA to oxaloacetate and acetyl-CoA by ATP-citrate lyase. The level of phosphorylation of PC12 cell ATP-citrate lyase by phosphorylated NDPK was comparable with that by ATP. Thus, in addition to its nucleoside diphosphate kinase activity, NDPK can function as a protein kinase.

The high energy phosphate is usually supplied by ATP, and NDPK is thought to be responsible for maintaining nucleoside triphosphate pools. Most NDPKs autophosphorylate on a histidine residue at their catalytic sites (2)(3)(4)(5)(6). NDPKs from Myxococcus xanthus (4) and humans (5) have been reported to autophosphorylate on both histidine and serine residues. NDPK from rat mast cells has been reported to contain a phosphorylated aspartic or glutamic acid at its catalytic site (7).
The Drosophila awd gene and the murine and human nm23 genes encode NDPKs (8). The awd gene is essential for normal Drosophila development. Mutations in this gene cause severe developmental defects and result in death of the larvae (9). nm23 genes have been implicated in control of tumor metastasis. For some, but not all types, of tumor, there is an inverse relationship between the level of nm23 expression and metastatic potential (10 -19). nm23 genes are also thought to be involved in cellular proliferation (20) and differentiation (21). The simple maintenance of nucleoside 5Ј-triphosphate pools does not appear to explain the involvement of NDPK in these various cellular processes, and it seems possible that NDPK might have other activities. NDPK has been reported to be associated with GTP-binding proteins (22)(23)(24)(25), and it has been suggested that NDPK might be involved in regulating GTP binding to these proteins. NDPK has also been reported to bind to DNA and stimulate c-myc transcription in vitro (26,27).
We have reported previously that a GTP-binding protein that regulates exocytosis in rat pheochromocytoma PC12 cells may interact with NDPK (25). While investigating this interaction, we observed what appeared to be phosphorylation of NDPK and became interested in both the autophosphorylation of NDPK and whether NDPK might function as a protein kinase. To examine the latter possibility, rat liver NDPK containing 32 P at its catalytic histidine was used to phosphorylate an extract of PC12 cells. NDPK appeared to directly transfer a phosphate from its catalytic histidine to a histidine on another protein. This protein was isolated and shown to be ATP-citrate lyase. This enzyme is the primary source of cytosolic acetyl-CoA which is used in a number of biosynthetic pathways, including lipogenesis and cholesterogenesis. The phosphorylation of ATP-citrate lyase by NDPK suggests that NDPK may have a role in the regulation of membrane biosynthesis. It also seems possible that NDPK can phosphorylate proteins other than ATP-citrate lyase.

MATERIALS AND METHODS
PC12 Cell Cytosol-PC12 cells were cultured as described previously (28). A cytosolic extract of PC12 cells was prepared by suspending 0.3 g of cells in 1.5 ml of 20 mM PIPES, pH 7.0, 140 mM potassium glutamate, 2 mM MgCl 2 , 5 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 g/ml leupeptin, and 20 g/ml soybean trypsin inhibitor on ice. The cells were lysed by 20 passages through a 25-gauge needle and centrifuged for 2 min at 12,000 ϫ g to remove nuclei and intact cells. The supernatant was centrifuged for 1 h at 100,000 ϫ g to give cytosolic and membrane fractions. The cytosolic fraction was dialyzed against 140 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, and 20 mM Tris, pH 7.5, at 4°C, for 3 days with two buffer changes.
Partial Purification of the 120-kDa Phosphoprotein-For isolation of the 120-kDa phosphoprotein 4 -5 g of PC12 cells were suspended in 25 ml of cold 20 mM HEPES, pH 7.4, 1 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 5 g/ml leupeptin, 10 g/ml soybean trypsin inhibitor, and 1 mM PMSF. The cells were lysed with a glass Teflon homogenizer. The extract was clarified by centrifugation at 100,000 ϫ g for 1 h and fractionated with (NH 4 ) 2 SO 4 . The 25-45% (NH 4 ) 2 SO 4 pellet was dialyzed overnight against 50 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 0.2 mM PMSF, and 20 mM Tris, pH 7.5 at 4°C, clarified by centrifugation at 100,000 ϫ g, and loaded onto a Mono Q HR 5/5 column (Pharmacia Biotech Inc.) equilibrated in 50 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, and 20 mM Tris, pH 7.5 at 4°C. Proteins were eluted using a 20-ml linear gradient of 50 -500 mM NaCl at a flow rate of 0.5 ml/min, and 2-ml fractions were collected. Most of the 120-kDa protein phosphorylated by [␥-32 P]ATP in EDTA and by [ 32 P]NDPK in MgCl 2 eluted in a single tube in about 200 mM NaCl. This Mono Q fraction was concentrated with a Centricon (Amicon) to 200 l and loaded onto a Superose 12 HR 10/30 column (Pharmacia) equilibrated in 100 mM NaCl, 1 mM EGTA, 1 mM DTT, and 20 mM Tris, pH 7.5 (Buffer A), containing 1 mM MgCl 2 at 4°C. The column was eluted at 0.4 ml/min, and 0.5-ml fractions were collected. The 120-kDa protein phosphorylated by [␥-32 P]ATP in EDTA and by [ 32 P]NDPK in MgCl 2 eluted near the void volume of the column and corresponded to the elution of a 120-kDa protein observed by Coomassie Blue staining (see Fig. 3).
Purification and Phosphorylation of Rat Liver NDPK-Rat liver NDPK was purified as described by Kimura and Shimada (29), except Pharmacia S200 and Mono Q columns were used in place of Ultrogel AcA 34 and DEAE-Sepharose columns. The isolated NDPK was more than 99% pure. [ 32 P]NDPK was made by incubating 20 M NDPK with 100 M [␥-32 P]ATP for 10 min at room temperature in Buffer A containing 5 mM MgCl 2 . [ 32 P]NDPK was separated from free [␥-32 P]ATP by gel filtration on a PD10 column (Pharmacia) equilibrated in 100 mM NaCl, 1 mM DTT, and 20 mM Tris, pH 7.5. The extent of NDPK phosphorylation and the fraction of 32 P remaining bound to NDPK after various treatments (acid, base, hydroxylamine, or nucleoside diphosphates) were determined by filtration on nitrocellulose filters; 20 -40-l aliquots containing phosphorylated NDPK were diluted with 0.5 ml of 20 mM Tris, pH 7.5, and filtered through a 25-mm diameter nitrocellulose filter (7). The filters were washed four times with 2 ml of 20 mM Tris, pH 7.5, and counted.
Basic SDS-PAGE-This procedure is a modification of Laemmli SDS-PAGE (30). The gel was cast as usual except there was no stacking gel. The running buffer was adjusted to pH 8.6 at 4°C. The SDS-sample buffer was 5% SDS, 25% glycerol, 180 mM Tris, pH 8.8, 0.01% bromphenol blue, 50 mM DTT, 50 g/ml leupeptin, and 4 mM PMSF. Protein samples were mixed with 0.5-1 volume of SDS-sample buffer and, unless indicated otherwise, incubated for 10 min at room temperature before being loaded onto a cold gel. The gel was electrophoresed in the cold room for 16 h at 80 volts. The gels were fixed in 18.5% formaldehyde and 50 mM sodium phosphate, pH 9.6 (31), for 1 h, washed in 25% isopropanol and 0.5% NaCO 3 , and dried. Prestained molecular weight markers were used to estimate the molecular weights of the phosphoproteins.
Nondenaturing Gel Electrophoresis-Polyacrylamide slab gels (14 ϫ 12 cm) containing 4.5% acrylamide and 0.12% bisacrylamide were prerun at 50 mA for 2 h at 4°C in 10 mM ␤-mercaptoethanol and 500 mM Tris, pH 9.2. The sample wells were rinsed with running buffer (25 mM Tris, 192 mM glycine, and 10 mM ␤-mercaptoethanol), and protein samples containing 15% glycerol and 0.01% bromphenol blue were loaded. The gels were electrophoresed at 4°C for 16 h at 16 mA. The proteins were transferred to nitrocellulose by electrophoresis in 25 mM Tris and 192 mM glycine at 4°C. Following transfer of the proteins, the nitrocellulose membrane was rinsed at room temperature with wash buffer, 100 mM NaCl, 1 mM DTT, and 20 mM Tris, pH 7.5. The membrane was blocked by incubation for 30 min at room temperature in wash buffer containing 5% bovine serum albumin. After blocking, the membrane was rinsed twice for 5 min with wash buffer containing 0.05% Tween 20 and incubated for 1 h at room temperature with either 200 nM [ 32 P]NDPK or 200 nM [␥-32 P]GTP in wash buffer containing 0.05% Tween 20 and 5 mM MgCl 2 . The membrane was then washed five times for 5 min with cold 100 mM NaCl, 1 mM DTT, and 20 mM Tris, pH 9.0. The membrane was air-dried and phosphorylated proteins detected by autoradiography.

Isolation and Characterization of Phosphorylated NDPK-
Rat liver NDPK incubated with [␥-32 P]ATP incorporated a maximum of 0.5-0.6 mol of 32 P/mol of NDPK 18-kDa subunit. When 4 M NDPK was used, 5 M [␥-32 P]ATP gave half-maximum phosphorylation, and 50 M [␥-32 P] ATP gave maximum phosphorylation. The time course of phosphorylation of NDPK was rapid with maximum phosphorylation occurring in less than 5 s at room temperature. When 100 M [␣-32 P]ATP was used, there was no incorporation of 32 P into NDPK. When the phosphorylation reaction was carried out with 50 M [␥-32 P]ATP in 10 mM EDTA instead of 5 mM MgCl 2 , NDPK still incorporated approximately 0.6 mol of 32 32 P. This indicates that most of the 32 P bound to NDPK was on a histidine as phosphohistidines are acid-labile and base-stable (33,34). In contrast, phosphoserine, phosphothreonine, and phosphotyrosine are acid-stable, and phosphoglutamic acid and phosphoaspartic acid are both acid-and baselabile. Hydroxylamine hydrolyzes phosphohistidines, phosphoglutamic acids, and phosphoaspartic acids (33). Incubation of [ 32 P]NDPK in hydroxylamine resulted in the loss of 98% of the bound 32 P. Phosphoglutamic and phosphoaspartic acids, but not phosphohistidine, can be readily cleaved by treatment with sodium borohydride (32,33). Incubation of [ 32 P]NDPK with 25 mM sodium borohydride did not cause any release of 32 P. Phosphohistidines are thermolabile (6). Incubation of [ 32 P]NDPK for 30 min at 37°C in a neutral buffer resulted in a loss of about 30% of the bound 32 P, and a 2-min incubation in boiling water resulted in the loss of more than 95% of the bound 32 P.
The addition of GDP, UDP, or ADP to [ 32 P]NDPK resulted in the 32 P being transferred to the nucleoside diphosphates to give 32 P-labeled triphosphates (data not shown). After incubation with 50 M GDP, UDP, or ADP, less than 2% of the 32 P remained bound to NDPK, indicating that most of the 32 P bound to NDPK was at the catalytic site.
Phosphorylation of Cytosolic Proteins by [ 32 P]NDPK-Initially the phosphorylation of cytosolic PC12 cell proteins by [ 32 P]NDPK was analyzed using Laemmli SDS-PAGE and fixing the gel in 10% acetic acid. Protein phosphorylation was detected by autoradiography. Incubation of dialyzed PC12 cell cytosol with 1 M [ 32 P]NDPK resulted in a very low level of protein phosphorylation compared to that obtained with 1 M [␥-32 P] ATP (data not shown), and there was no convincing evidence for a direct transfer of 32 P from [ 32 P]NDPK to other proteins. However, phosphohistidine, phosphoarginine, and phospholysine are unstable under conditions used for Laemmli SDS-PAGE. For example, less than 1% of 32 P bound to NDPK remained after Laemmli SDS-PAGE and acid fixing. Conditions used for SDS-PAGE were modified as described under "Materials and Methods" to stabilize phosphohistidines, phospholysines, and phosphoarginines. This method is referred to as basic SDS-PAGE.
PC12 cell cytosol was incubated in MgCl 2 with 1 M [␥-32 P]ATP or 1 M [ 32 P]NDPK and the products analyzed by basic SDS-PAGE and autoradiography (Fig. 1). While the sam-ple incubated in [␥-32 P]ATP contained many 32 P-labeled proteins (lane 6), the sample incubated with [ 32 P]NDPK contained only two major 32 P-labeled proteins, NDPK and a protein of about 120 kDa (lane 5). This 120-kDa phosphoprotein was not detected when [ 32 P]NDPK was boiled prior to its addition to the cytosol (lane 2), nor was it observed when [ 32 P]NDPK was incubated with buffer and no cytosol (lane 1). In some samples there was a phosphoprotein of about 60 kDa. It was most prominent when [ 32 P]NDPK was incubated with just buffer (lane 1). When samples containing [ 32 P]NDPK were boiled or heated to 37°C, the 60-kDa phosphoprotein band was not observed. This 60-kDa phosphoprotein appears to be an oligomer of NDPK resulting from the incomplete denaturation of NDPK. Immunoblots with anti-NDPK antibodies also indicated that this 60-kDa protein was an aggregate of NDPK.
The phosphorylation pattern obtained when cytosol was incubated with 1 M [ 32 P]NDPK and 0.5 or 5 M ADP (Fig. 1,  lanes 7 and 8) was similar to that obtained when cytosol was incubated with 1 M [␥-32 P]ATP. A phosphoprotein of about 120 kDa was observed in these samples, but it contained much less 32 P than did the 120-kDa phosphoprotein obtained when cytosol was incubated with just [ 32 P]NDPK. When cytosol was incubated with 100 nM [␥- 32  abolished the phosphorylation of the 120-kDa protein (data not shown), indicating that the 32 P incorporated into the 120-kDa protein came from the phosphohistidine on NDPK.
When cytosol was incubated with [ 32 P]NDPK in 10 mM EDTA and the products analyzed by basic SDS-PAGE and autoradiography, only [ 32 P]NDPK was detected. However, when the cytosol was incubated with [␥-32 P]ATP in 10 mM EDTA, both NDPK and a 120-kDa protein were phosphorylated (Fig. 1, lane 3). GTP, dGTP, and UTP greatly reduced the phosphorylation of NDPK by [␥-32 P]ATP in EDTA, but not the phosphorylation of the 120-kDa protein by [␥-32 P]ATP in EDTA (data not shown).
Partial Purification of the 120-kDa Phosphoprotein-Isolation of the 120-kDa protein was monitored by phosphorylation with [␥-32 P]ATP in EDTA and by phosphorylation with [ 32 P]NDPK in MgCl 2 . Most of the 120-kDa protein precipitated between 25 and 45% saturated (NH 4 ) 2 SO 4 . The 25-45% (NH 4 ) 2 SO 4 pellet was fractionated on a Mono Q column. The 120-kDa protein(s) phosphorylated by [␥-32 P] ATP in EDTA and by [ 32 P]NDPK in MgCl 2 eluted in about 200 mM NaCl. The fraction from the Mono Q column most enriched in the 120-kDa phosphoprotein was fractionated on a Superose 12 column (Fig.  3). The elution of a 120-kDa phosphoprotein observed by phosphorylation with [␥-32 P]ATP in EDTA and with [ 32 P]NDPK in MgCl 2 (Fig. 3B) corresponded with the elution of a 120-kDa protein observed by Coomassie Blue staining (Fig. 3A). The 120-kDa protein may be an oligomer as it eluted from the Superose 12 column with an apparent molecular mass greater than 200 kDa. About 100 g of partially purified 120-kDa protein was obtained from 4.5 g of PC12 cells.
Phosphorylation of the 120-kDa Protein-Incubation of the  8 -14). The samples were then treated as follows: placed on ice (lanes 1 and 8), incubated for 10 min at 37°C in SDS-sample buffer containing 1 M HCl (lanes 2 and 9), incubated for 10 min at 37°C in SDS-sample buffer containing 1 M NaOH (lanes 3 and 10), incubated for 10 min at 37°C in SDS-sample buffer pH 8.8 (lanes 4 and 11), incubated for 10 min at 37°C in 100 mM NaCl at pH 7.5 (lanes 5 and 12), incubated for 10 min at 37°C in 0.8 M hydroxylamine at pH 5.4 (lanes 6 and 13), incubated for 10 min at 37°C in 0.8 M NaCl at pH 5.4 (lanes 7 and 14). After these incubations, the samples were neutralized and analyzed by basic SDS-PAGE and autoradiography.
partially purified 120-kDa protein with [␥-32 P]ATP in either MgCl 2 or EDTA resulted in the incorporation of 0.4 -0.5 mol of 32 P/mol of 120-kDa protein. There was no detectable 32 P incorporation into other proteins. The 32 P incorporated into the 120-kDa protein was acid-labile and base-stable, which is consistent with a histidine residue being phosphorylated in both MgCl 2 and in EDTA. A comparison of the autoradiograms of the 120-kDa protein phosphorylated with [␥-32 P]ATP with those of the 120-kDa protein phosphorylated with [ 32 P]NDPK (Fig. 3) indicate that the 120-kDa protein phosphorylated by [ 32 P]NDPK also contained about 0.4 mol of 32 P/mol of 120-kDa protein.
Thin layer chromatography was used to directly show the presence of phosphohistidine. 32 P-Labeled 120-kDa protein was separated from free [␥-32 P]ATP and hydrolyzed in KOH. The resulting products were analyzed by thin layer chromatography. About half of the 32 P remained at the origin as free phosphate, and the other half co-migrated with the N 1 -phosphohistidine standard (Fig. 4, lanes 1 and 2). An alkali digest of [ 32 P]NDPK gave a 32 P-labeled product that migrated at the solvent front (Fig. 4, lane 3). This is where N 1 -phosphohistidine should migrate. NDPK is thought to contain an N 1 -phosphohistidine (35). An alkali digest of a mixture of [ 32 P]NDPK and the 120-kDa protein phosphorylated by [ 32 P]NDPK gave both N 1 -and N 3 -phosphohistidines.
Direct Transfer of 32 P from [ 32 P]NDPK to the 120-kDa Protein-Glucose and hexokinase were used to examine the possibility that the phosphorylation of the 120-kDa protein by [ 32 P]NDPK was due to a small amount of [␥-32 P]ATP which was either present in the [ 32 P]NDPK or formed from contaminating ADP. The addition of 1.7 mM glucose and 1.5 units/ml hexokinase completely prevented the phosphorylation of the partially purified 120-kDa protein in MgCl 2 by 1 M [␥-32 P]ATP, but not its phosphorylation by 1 M [ 32 P]NDPK (Fig. 5). When five times as much glucose and hexokinase were used, there was a decrease in the phosphorylation of the 120-kDa protein by [ 32 P]NDPK.
Nondenaturing gel electrophoresis was also used to look for a direct transfer of 32 P from [ 32 P]NDPK to the 120-kDa protein.
NDPK and partially purified 120-kDa protein were electro-  (Fig. 6). To ensure that the [ 32 P]NDPK did not contain any [␥-32 P]ATP, the [ 32 P]NDPK used in this experiment was made by phosphorylation of NDPK with [␥-32 P]GTP. Incubation of the blots with either [ 32 P]NDPK or [␥-32 P]GTP resulted in the phosphorylation of both the 120-kDa protein and the electrophoresed NDPK. However, the relative levels of phosphorylation of NDPK and the 120-kDa protein were very different depending on whether [ 32 P]NDPK or [␥-32 P]GTP was used in the incubation. More 120-kDa protein was phosphorylated by [ 32 P]NDPK than by [␥-32 P]GTP, but more NDPK was phosphorylated by [␥-32 P]GTP than by [ 32 P]NDPK. This difference in substrate specificity indicates that the phosphorylation of the 120-kDa protein by the [ 32 P]NDPK was not due to the presence of a small amount of [␥-32 P] GTP, but rather that it resulted from direct transfer of 32 P from [ 32 P]NDPK to the 120 kDa protein.
[ 32 P]NDPK electrophoresed on a nondenaturing gel and transferred to nitrocellulose gave a doublet with the same mobilities as shown in Fig. 6 for NDPK phosphorylated after electrophoresis and blotting, and 32 P-labeled 120-kDa protein electrophoresed on a nondenaturing gel and transferred to nitrocellulose gave a single band with the same mobility as shown in Fig. 6 for partially purified 120-kDa protein phosphorylated after electrophoresis and blotting.
Identification of 120-kDa Phosphoprotein as ATP-Citrate Lyase-The partially purified 120-kDa protein (30 g) was separated by SDS-PAGE, transferred to a polyvinylidene membrane (Immobilon-P), and digested with 1 g of endoproteinase Lys-C (36,37). The resulting peptides were separated by high performance liquid chromatography, and five major well resolved peaks were sequenced using an Applied Biosystems gas phase sequencer. These peaks gave peptide sequences that matched sequences found in rat liver ATP-citrate lyase (38). These sequences were: S A T L F S R H T K, which is identical to residues 448 -457 of ATP-citrate lyase; F Y W G H K, which is identical to residues 540 -545 of ATP-citrate lyase; L I M G I G H R V K, which is identical to residues 968 -977 of ATPcitrate lyase; S I N N P D M R V Q I L K, which is identical to residues 978 -990 of ATP-citrate lyase; and Q X F P A X P L L, which matches residues 995-1003 of ATP-citrate lyase. ATPcitrate lyase is a tetramer composed of 120-kDa subunits (39), and it autophosphorylates on a histidine residue at its catalytic site (40,41).
ATP-citrate lyase catalyzes the formation of acetyl-CoA and oxaloacetate from citrate and CoA with the concomitant hydrolysis of ATP to ADP. ATP-citrate lyase activity of the 120-kDa protein was assayed by measuring oxaloacetate production as described for rat liver ATP-citrate lyase (42). The partially purified 120-kDa protein had a specific activity of 3 mol⅐min Ϫ1 ⅐mg Ϫ1 . Values for rat liver ATP-citrate lyase vary from 4 to 12 mol⅐min Ϫ1 ⅐mg Ϫ1 .
ATP-citrate lyase follows a double displacement mechanism with a phosphoenzyme intermediate (43). The first step is the phosphorylation of a histidine at the active site by ATP. Citrate then binds to the enzyme, and the phosphate is transferred to citrate to give citryl-phosphate. The phosphorylation of ATPcitrate lyase by ATP is reversible, and phosphate bound to ATP-citrate lyase can be removed by the addition of ADP (43,44). If the phosphate incorporated into the 120-kDa protein was at the active site histidine of ATP-citrate lyase, the addition of ADP or citrate to 32 P-labeled 120-kDa protein should result the loss of bound 32 P. As shown in Fig. 7, the addition of ADP or citrate to the 120-kDa protein phosphorylated with either [␥-32 P]ATP or [ 32 P]NDPK resulted in the removal of 32 P from the 120-kDa protein.
The rate of transfer of phosphate from NDPK to ATP-citrate lyase was determined using 0.1-0. was used, ATP-citrate lyase was maximally phosphorylated in less than 10 s. DISCUSSION Rat liver NDPK incorporated 0.5-0.6 mol of 32 P/mol of 18-kDa subunit. In agreement with most of the data in the literature (2,3,6), the phosphorylated residue was primarily histidine. As only 2% of the 32 P incorporated into rat liver NDPK was acid-stable, autophosphorylated rat liver NDPK appeared to contain less than 0.01 mol of phosphoserine or phosphothreonine per 18 kDa subunit. As reported for NDPK in extracts from human colon cancer tissues (45) and for NDPKs from Xenopus oocytes (46) and Myxococcus xanthus (4), rat liver NDPK autophosphorylated in EDTA. Oocyte NDPK and a commercial bovine liver NDPK preparation have low levels of nucleoside diphosphate kinase activity in EDTA (46). Rat liver NDPK had nucleoside diphosphate kinase activity in EDTA, but the rate at which it formed UTP from UDP and ATP in EDTA was only about 1% the rate in MgCl 2 (data not shown).
Incubation of PC12 cell cytosol with [ 32 P]NDPK resulted mainly in the phosphorylation of a single protein with a mobility on SDS gels of about 120 kDa. This phosphorylation appears to result from a direct transfer of 32 P from the histidine at the catalytic site of NDPK to a histidine on the 120-kDa protein.
Sequencing of peptides from the 120-kDa protein showed that it was ATP-citrate lyase. The only difference in the phosphorylation of ATP-citrate lyase and that of the 120-kDa protein is that the phosphorylation of ATP-citrate lyase is reported to require a divalent cation (44). The 120-kDa protein was phosphorylated in EDTA, but the rate of phosphorylation was slower in EDTA than in MgCl 2 and higher concentrations of ATP were required for phosphorylation in EDTA (data not shown). [ 32 P]NDPK appeared to phosphorylate the histidine at the active site of ATP-citrate lyase.
The amino acid sequence of residues 560 -800 of rat liver ATP-citrate lyase has 33% sequence identity with residues 60 -290 of the ␣ chain of E. coli succinyl-CoA synthetase (38). These enzymes catalyze similar reactions, and both autophosphorylate on a catalytic histidine (47). The sequence around the catalytic histidine of succinyl-CoA synthetase, GHAGA, is the same as that around the catalytic histidine of ATP-citrate lyase. NDPK from Pseudomonas aeruginosa associates and copurifies with succinyl-CoA synthetase (48). It was suggested that NDPK might either phosphorylate this succinyl-CoA synthetase or funnel ATP to its active site. It has also been suggested that in the mitochondrial matrix NDPK interacts with succinyl-CoA synthetase (49).
NDPK has been reported to co-purify with microtubules, and immunofluoresence suggests some NDPK is bound to microtubules in the cell (8). However, this interaction is not very strong as NDPK does not appear to bind to purified microtubules (50). Most tubulins contain the sequence FGQSGA, which is similar to the amino acid sequence FGHAGA around the catalytic histidine of ATP-citrate lyase. (Q for H and S for A are considered conservative replacements). This sequence similarity suggests that these residues might be the site where NDPK binds to microtubules.
While histidine kinases in eukaryotes are only just beginning to be identified, a number of bacterial histidine kinases have been extensively studied (51). Most of these kinases autophosphorylate on a histidine and then usually transfer the phosphate to an acyl group either on the same or different protein. One of the most extensively characterized processes in bacteria that involves histidine phosphorylation is the phosphoenolpyruvate:sugar phosphotransferase system (PTS). PTS transfers a phosphate from phosphoenolpyruvate to a sugar hydroxyl via a series of transfer proteins (52). A phosphoryl group is transferred sequentially from phosphoenolpyruvate to enzyme I, from enzyme I to the protein HPr, from HPr to enzyme IIA, from enzyme IIA to enzyme IIB, and from enzyme IIB to a sugar. Enzyme I, HPr, and enzyme IIA are all phosphorylated on histidines.
There are two examples of PTS proteins being phosphorylated on their active site histidines by kinases other than PTS transfer proteins (52). Enzyme I can be phosphorylated on its active site histidine by phosphoenolpyruvate and by acetate kinase, and HPr can be phosphorylated its active site histidine by phosphorylated enzyme I and by a glycerol kinase. These reactions are analogous to that reported here for the phosphorylation of ATP-citrate lyase by ATP and by phosphorylated NDPK.
The physiological significance of the phosphorylation of ATPcitrate lyase by NDPK is unclear as it is phosphorylated much more rapidly by ATP than by NDPK. However, in addition to binding ATP, ATP-citrate lyase binds citrate, CoA, ADP, oxaloacetate, and acetyl-CoA, and it is possible that the binding of one of these substrates or products may inhibit the phosphorylation of ATP-citrate lyase by ATP but not by phosphorylated NDPK. Alternatively, the binding of NDPK to the catalytic site ATP-citrate lyase might inhibit the phosphorylation of ATPcitrate lyase by ATP and, thereby, reduce the rate of acetyl-CoA formation.
ATP-citrate lyase is the primary source of cytosolic acetyl-CoA, which is used in a number of biosynthetic pathways, including fatty acid, cholesterol, and ganglioside biosynthesis. A change in the rate of acetyl-CoA production could affect the synthesis of one or more these molecules all of which have been implicated in tumorigenesis and/or cell growth (53)(54)(55). Recently, a prognostic molecule in tumor cells of breast cancer, OA-519, has been shown to be fatty acid synthetase (53). Tumors marked by OA-519 were nearly four times more likely to recur and metastasize as tumors not marked by this antigen. Inhibition of fatty acid synthesis inhibited the growth of tumor cells with high levels of fatty acid synthetase (53). An inhibition of ATP-citrate lyase could also result in a decrease in fatty acid synthesis.
The results presented here demonstrate that NDPK can phosphorylate ATP-citrate lyase. Whether this phosphorylation is relevant to the role of NDPK in differentiation and metastasis remains to be determined. While incubation of PC12 cell cytosol with [ 32 P]NDPK resulted mainly in the phosphorylation of ATP-citrate lyase, it is possible that NDPK can also phosphorylate other proteins, but these proteins are present in low amounts or they are nuclear or membrane proteins. The t1 ⁄2 for transfer of phosphate from NDPK to ATPcitrate lyase at room temperature was about 2 min. This rate is comparable with that of some prokaryotic histidine kinases (56). Escherichia coli nitrogen regulator II protein transfers a phosphate from its catalytic histidine to nitrogen regulator I protein.
In the presence of an excess of nitrogen regulator I, about 80% of the phosphate bound to nitrogen regulator II is transferred in 1 min at 37°C (32).