(Received for publication, May 2, 1995; and in revised form, June 26, 1995)
From the
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 [-
P]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.
Nucleoside diphosphate kinase (NDPK) ()catalyzes the
phosphorylation of nucleoside 5`-diphosphates to triphosphates by a
ping pong mechanism involving a high energy phosphorylated enzyme
intermediate(1) .
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, 11, 12, 13, 14, 15, 16, 17, 18, 19) . nm23 genes are also thought to be involved in cellular proliferation (20) and differentiation(21) . The simple maintenance of nucleoside 5`-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 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.
Figure 3:
Elution of the 120-kDa protein from a
Superose 12 column. The Mono Q column fraction most enriched in the
120-kDa protein was concentrated and fractionated on a Superose 12
column. A, 50-µl aliquots of the column fractions were
analyzed by SDS-PAGE. The gel was stained with Coomassie Blue. B, 15-µl aliquots of the column fractions were
phosphorylated for 10 min at room temperature in 10 mM EDTA
with 1 µM [-
P]ATP or in 5
mM MgCl
with 1 µM
[
P]NDPK. After these incubations, the samples
were analyzed by basic SDS-PAGE and
autoradiography.
Gel filtration was used to separate phosphorylated NDPK,
[P]NDPK, from
[
-
P]ATP. The concentrations of
[
P]NDPK given in this paper refer to the
concentration of
P bound to NDPK after gel filtration.
Thin layer chromatography showed that 1 µM [
P]NDPK (0.4-0.5 mol of
P/mol of NDPK 18-kDa subunit) contained less than 25
nM [
-
P]ATP.
Incubation of
[P]NDPK for 30 min at 37 °C in 1 M KOH resulted in no loss of bound
P, but incubation of
[
P]NDPK for 30 min at 37 °C in 1 M HCl resulted in loss of 98% of the bound
P. This
indicates that most of the
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
base-labile. Hydroxylamine hydrolyzes phosphohistidines,
phosphoglutamic acids, and phosphoaspartic acids(33) .
Incubation of [
P]NDPK in hydroxylamine resulted
in the loss of 98% of the bound
P. Phosphoglutamic and
phosphoaspartic acids, but not phosphohistidine, can be readily cleaved
by treatment with sodium borohydride(32, 33) .
Incubation of [
P]NDPK with 25 mM sodium
borohydride did not cause any release of
P.
Phosphohistidines are thermolabile(6) . Incubation of
[
P]NDPK for 30 min at 37 °C in a neutral
buffer resulted in a loss of about 30% of the bound
P, and
a 2-min incubation in boiling water resulted in the loss of more than
95% of the bound
P.
The addition of GDP, UDP, or ADP
to [P]NDPK resulted in the
P being
transferred to the nucleoside diphosphates to give
P-labeled triphosphates (data not shown). After incubation
with 50 µM GDP, UDP, or ADP, less than 2% of the
P remained bound to NDPK, indicating that most of the
P bound to NDPK was at the catalytic site.
PC12 cell cytosol was incubated in
MgCl with 1 µM [
-
P]ATP or 1 µM [
P]NDPK and the products analyzed by basic
SDS-PAGE and autoradiography (Fig. 1). While the sample
incubated in [
-
P]ATP contained many
P-labeled proteins (lane 6), the sample incubated
with [
P]NDPK contained only two major
P-labeled proteins, NDPK and a protein of about 120 kDa (lane 5). This 120-kDa phosphoprotein was not detected when
[
P]NDPK was boiled prior to its addition to the
cytosol (lane 2), nor was it observed when
[
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 [
P]NDPK was
incubated with just buffer (lane 1). When samples containing
[
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.
Figure 1:
Basic
SDS-PAGE of PC12 cell cytosol incubated with
[P]NDPK or [
-
P]ATP.
Samples were incubated for 10 min at room temperature in Buffer A
containing 5 mM MgCl
, except that in lane
3, which contained 10 mM EDTA. The sample shown in lane 1 contained 1 µM
[
P]NDPK and no cytosol. Samples shown in lanes 2-8 contained dialyzed PC12 cell cytosol (3 mg/ml)
and the following additions: 1 µM boiled
[
P]NDPK (lane 2), 1 µM [
-
P] ATP (lane 3), 0.1
µM [
-
P]ATP (lane 4),
1 µM [
P]NDPK (lane 5), 1
µM [
-
P] ATP (lane 6),
1 µM [
P]NDPK and 0.5 µM ADP (lane 7), and 1 µM [
P]NDPK and 5 µM ADP (lane
8). After these incubations, the samples were analyzed by basic
SDS-PAGE and autoradiography.
The
phosphorylation pattern obtained when cytosol was incubated with 1
µM [P]NDPK and 0.5 or 5 µM ADP (Fig. 1, lanes 7 and 8) was similar
to that obtained when cytosol was incubated with 1 µM [
-
P]ATP. A phosphoprotein of about 120
kDa was observed in these samples, but it contained much less
P than did the 120-kDa phosphoprotein obtained when
cytosol was incubated with just [
P]NDPK. When
cytosol was incubated with 100 nM
[
-
P]ATP, the 120-kDa phosphoprotein was not
observed (lane 4), nor was it observed when cytosol was
incubated with 100 nM [
-
P]ATP and
1 µM unlabeled NDPK. As 1 µM [
P]NDPK contained less than 25 nM [
-
P]ATP, the 120-kDa phosphoprotein
obtained when cytosol was incubated with 1 µM [
P]NDPK does not appear to be due to
residual [
-
P] ATP.
The addition of GDP,
dGDP, or UDP, which react with [P]NDPK to form
the corresponding nucleoside triphosphates, greatly reduced the
phosphorylation of 120-kDa protein by [
P]NDPK
(data not shown). Pretreatment of [
P]NDPK with
hydroxylamine at pH 5.4, but not with NaCl at pH 5.4, abolished the
phosphorylation of the 120-kDa protein (data not shown), indicating
that the
P incorporated into the 120-kDa protein came from
the phosphohistidine on NDPK.
When cytosol was incubated with
[P]NDPK in 10 mM EDTA and the products
analyzed by basic SDS-PAGE and autoradiography, only
[
P]NDPK was detected. However, when the cytosol
was incubated with [
-
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 [
-
P]ATP in
EDTA, but not the phosphorylation of the 120-kDa protein by
[
-
P]ATP in EDTA (data not shown).
The P bound to the 120-kDa protein(s) obtained by
phosphorylation of cytosol either with [
P]NDPK
in MgCl
(Fig. 2, lanes 1-7) or with
[
-
P]ATP in EDTA (Fig. 2, lanes
8-14) was acid-labile (lanes 2 and 9),
base-stable (lanes 3 and 10), and removed by
incubation in hydroxylamine at pH 5.4 (lanes 6 and 13), indicating that the 120-kDa protein(s) was phosphorylated
on a histidine.
Figure 2:
Stability of phosphorylated 120-kDa
protein. Dialyzed PC12 cytosol (3 mg/ml) was incubated at room
temperature for 10 min in Buffer A with either 5 mM MgCl and 1 µM [
P]NDPK (lanes
1-7) or 10 mM EDTA and 1 µM [
-
P]ATP (lanes 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.
Thin layer chromatography was
used to directly show the presence of phosphohistidine. P-Labeled 120-kDa protein was separated from free
[
-
P]ATP and hydrolyzed in KOH. The
resulting products were analyzed by thin layer chromatography. About
half of the
P remained at the origin as free phosphate,
and the other half co-migrated with the N
-phosphohistidine standard (Fig. 4, lanes 1 and 2). An alkali digest of
[
P]NDPK gave a
P-labeled product
that migrated at the solvent front (Fig. 4, lane 3).
This is where N
-phosphohistidine should migrate.
NDPK is thought to contain an N
-phosphohistidine(35) . An alkali digest
of a mixture of [
P]NDPK and the 120-kDa protein
phosphorylated by [
P]NDPK gave both N
- and N
-phosphohistidines.
Figure 4:
Phosphoamino acid analysis of
phosphorylated NDPK and phosphorylated 120-kDa protein. Partially
purified 120-kDa protein (100 µg/ml) was incubated for 10 min at
room temperature with 10 µM
[-
P]ATP in buffer A containing either 10
mM EDTA (lane 1) or 5 mM MgCl
(lane 2). Rat liver NDPK was phosphorylated with
[
-
P]ATP under standard conditions (lane
3). The phosphorylated proteins were separated from
[
-
P]ATP, hydrolyzed in 3 M KOH,
and analyzed by thin layer chromatography and autoradiography. The
positions of phosphoamino acid standards are
indicated.
Figure 5:
Glucose and hexokinase do not block the
phosphorylation of the 120-kDa protein by
[P]NDPK. Partially purified 120-kDa protein (50
µg/ml) was incubated in Buffer A containing 5 mM MgCl
and the following: lane 1, 1
µM [
P]NDPK; lane 2, 1
µM [
P]NDPK, 1.7 mM glucose, and 1.5 units/ml dialyzed yeast hexokinase; lane
3, 1 µM [
P]NDPK, 8.5 mM glucose, and 7.5 units/ml dialyzed yeast hexokinase; lane
4, [
-
P]ATP, 8.5 mM glucose,
and 7.5 units/ml dialyzed yeast hexokinase; lane 5, 1
µM [
-
P]ATP, 1.7 mM glucose, and 1.5 units/ml dialyzed yeast hexokinase; lane
6, 1 µM [
-
P]ATP. After 10
min at room temperature, the samples were analyzed by basic SDS-PAGE
and autoradiography.
Nondenaturing gel electrophoresis
was also used to look for a direct transfer of P from
[
P]NDPK to the 120-kDa protein. NDPK and
partially purified 120-kDa protein were electrophoresed on a
nondenaturing gel and transferred to nitrocellulose. The blotted
proteins were then incubated either with 200 nM [
-
P] GTP or with 200 nM [
P]NDPK (Fig. 6). To ensure that the
[
P]NDPK did not contain any
[
-
P]ATP, the [
P]NDPK
used in this experiment was made by phosphorylation of NDPK with
[
-
P]GTP. Incubation of the blots with
either [
P]NDPK or
[
-
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 [
P]NDPK or
[
-
P]GTP was used in the incubation. More
120-kDa protein was phosphorylated by [
P]NDPK
than by [
-
P]GTP, but more NDPK was
phosphorylated by [
-
P]GTP than by
[
P]NDPK. This difference in substrate
specificity indicates that the phosphorylation of the 120-kDa protein
by the [
P]NDPK was not due to the presence of a
small amount of [
-
P] GTP, but rather that
it resulted from direct transfer of
P from
[
P]NDPK to the 120 kDa protein.
Figure 6:
Phosphorylation of NDPK and the 120-kDa
protein after native gel electrophoresis. The following were
electrophoresed on nondenaturing polyacrylamide gels and transferred to
nitrocellulose; lanes 1 and 4, 2.5 µg of NDPK; lanes 2 and 5, 7 µg of protein from Superose 12
column fractions containing the 120-kDa protein; lanes 3 and 6, 140 µg of protein from a Mono Q column fraction
containing the 120-kDa protein. The blots were washed and incubated
with either 200 nM [P]NDPK or 200
nM [
-
P]GTP for 1 h at room
temperature. The blots were washed and analyzed by autoradiography. The
positions of NDPK and 120-kDa proteins are indicated.
[
P]NDPK used in this experiment was made by
phosphorylating NDPK with [
-
P]GTP and
separating [
P]NDPK from
[
-
P]GTP by gel
filtration.
[P]NDPK electrophoresed on a nondenaturing
gel and transferred to nitrocellulose gave a doublet with the same
mobilities as shown in Fig. 6for NDPK phosphorylated after
electrophoresis and blotting, and
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. 6for partially purified 120-kDa protein phosphorylated
after electrophoresis and blotting.
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
µmolmin
mg
. Values
for rat liver ATP-citrate lyase vary from 4 to 12
µmol
min
mg
.
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 ATP-citrate 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 P-labeled 120-kDa protein should result the loss of bound
P. As shown in Fig. 7, the addition of ADP or
citrate to the 120-kDa protein phosphorylated with either
[
-
P]ATP or [
P]NDPK
resulted in the removal of
P from the 120-kDa protein.
Figure 7:
Citrate and ADP remove P from
the 120-kDa protein. Partially purified 120-kDa protein (50 µg/ml)
was incubated in Buffer A containing 5 mM MgCl
and
either 1 µM [
-
P]ATP (lanes
1-4) or 1 µM [
P]NDPK (lanes 5-8). After 10 min at room temperature, the
samples in lanes 1 and 5 were frozen. The following
additions were made to the other samples: none (lanes 2 and 6), 10 mM citrate (lanes 3 and 7),
25 µM ADP (lanes 4 and 8). After an
additional 15 min at room temperature, the samples were analyzed by
basic SDS-PAGE and autoradiography.
The rate of transfer of phosphate from NDPK to ATP-citrate lyase was
determined using 0.1-0.5 µM
[P]NDPK and 5 µM ATP-citrate lyase
120-kDa subunit. After 2 min, about half of the
P was
transferred to ATP-citrate lyase. When [
-
P]
ATP was used, ATP-citrate lyase was maximally phosphorylated in less
than 10 s.
Rat liver NDPK incorporated 0.5-0.6 mol of 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
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
(data not shown).
Incubation of PC12 cell cytosol with
[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
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
and higher concentrations of ATP were
required for phosphorylation in EDTA (data not shown).
[
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 ATP-citrate 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 ATP-citrate 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 [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 t for
transfer of phosphate from NDPK to ATP-citrate 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) .