(Received for publication, September 27, 1995; and in revised form, January 19, 1996)
From the
Reversible phosphorylation of CTP:phosphocholine
cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine
biosynthesis, is thought to play a role in regulating its activity. In
the present study, the hypothesis that proline-directed kinases play a
major role in phosphorylating cytidylyltransferase is substantiated
using a c-Ha-ras-transfected clone of the human keratinocyte
cell line HaCaT. Cellular extracts from epidermal growth
factor-stimulated HaCaT cells and from ras-transfected HaCaT
cells phosphorylated cytidylyltransferase much stronger as compared
with extracts from quiescent HaCaT cells. The tryptic phosphopeptide
pattern of cytidylyltransferase phosphorylated by cell-free extracts
from ras-transfected HaCaT cells was similar compared with the
patterns of cytidylyltransferase phosphorylated by p44mitogen-activated protein kinase and p34
kinase in vitro, whereas in the case of casein
kinase II the pattern was different. Furthermore, in
c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as
compared with untransfected HaCaT cells. This higher phosphorylation of
cytidylyltransferase in the ras-transfected clone was reduced
to a level below the phosphorylation of cytidylyltransferase in
untransfected cells, using olomoucine, a specific inhibitor of
proline-directed kinases. The reduced phosphorylation of
cytidylyltransferase in olomoucine-treated cells correlated with an
enhanced stimulation of enzyme activity by oleic acid.
In mammalian cells, the main pathway for the biosynthesis of
phosphatidylcholine (PC) ()is via CDP-choline and
CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) (CT) is the
rate-limiting enzyme of this pathway(1) . In addition to the
structural function as a component of cellular membranes, PC has been
identified to be involved into signal transduction via the PC
cycle(2) . In a recent study, the coordination of PC metabolism
with the cell cycle was investigated, and evidence was provided that
the net biosynthesis of PC is restricted to the S-phase of the cell
cycle (3) .
CT exists as a soluble, inactive form that can be activated by translocation to membranes. Many mechanisms that modulate this translocation process have been firmly established(4, 5, 6, 7) , and reversible phosphorylation of CT has been shown to influence translocation of CT between cytosol and membranes(7, 8, 9, 10) . In many systems cytosolic CT is highly phosphorylated, whereas the membrane-bound, active form of CT is dephosphorylated. However, in a recent study it was shown that dephosphorylation of CT is not required for membrane binding(11) .
The physiological role of
phosphorylation and the kinases involved in this process are still
discussed. Whereas CT is a substrate for cAMP-dependent kinase in
vitro(12) , neither cAMP-dependent protein kinase (13, 14, 15) nor protein kinase C (16) seem to phosphorylate CT in vivo. In rat
hepatocytes and HeLa cells only serine residues of CT are
phosphorylated in vivo(14, 17) , and the
phosphorylation sites of CT from rat liver were
identified(18) . This study revealed that phosphorylation of CT
is confined to the carboxyl-terminal region of CT and that many serine
residues reside in potential sites for proline-directed kinases. We
have shown recently that growth factors can stimulate phosphorylation
of CT in HeLa cells and that CT is a substrate for p44 MAP kinase in vitro(19) , suggesting that
the ras/Raf/MAP kinase-signaling pathway is involved in this process.
In the present study, we investigated the phosphorylation of CT in vivo and in vitro. In ras-transfected
HaCaT cells, the phosphorylation of CT was increased by 2-fold as
compared with the phosphorylation of CT in untransfected cells. The
enhanced phosphorylation of CT in ras-transfected cells was
reduced in the presence of olomoucine, a specific inhibitor of
proline-directed kinases, such as p34 and
p44
MAP kinase(20) . Using this
experimental approach we could show that phosphorylation of CT
interferes with the activation of the enzyme by oleic acid and protects
the enzyme against proteolytic digestion. Furthermore, cell-free
extracts from quiescent and EGF-stimulated HaCaT cells as well as ras-transfected HaCaT cells were used to phosphorylate CT in vitro. The tryptic phosphopeptide patterns of CT
phosphorylated with cell-free extracts from HaCaT and ras-transfected HaCaT cells were compared with the patterns of
CT phosphorylated with purified MAP kinase, cyclin-dependent kinase,
and casein kinase II in vitro. The results presented here
substantiate the hypothesis that p44
MAP kinase
is involved in the phosphorylation of CT, but other proline-directed
kinases, such as p34
kinase, represent probable
candidates as well.
Prior to EGF stimulation, confluent cells were starved for 24 h in keratinocyte basal medium (Clonetics, San Diego, CA) containing no supplements. Under these conditions, proliferation of HaCaT cells and ras-transfected HaCaT cells was totally abolished.
Figure 1:
Activation of MAP kinases in HaCaT
cells and ras-transfected HaCaT cells. A, in
vitro phosphorylation of MBP by cell lysates from quiescent cells
without stimulation (0 min) and from cells that had been stimulated for
2 and 10 min with 100 ng/ml EGF. Equal amounts of protein (8 µg)
were loaded into each lane of the gel. Bands corresponding to MBP were
excised from the gel, and incorporated radioactivity was determined by
scintillation counting. B, the same cell lysates (25 µg
protein/lane) were analyzed by Western blot using a rabbit anti-MAP
kinase antibody that recognizes both isoforms, p42 and
p44
. The phosphorylated high molecular mass forms are
indicated by asterisks. The experiments were repeated, and
similar results were obtained.
MAP kinases are activated by phosphorylation(30) .
It is known that the phosphorylation of both isoforms, p42 and p44
, leads to a mobility shift in the SDS gel.
As shown in the Western blot of Fig. 1B, p42
and p44
are expressed in HaCaT cells and become
phosphorylated time-dependently after EGF stimulation. In ras-transfected HaCaT cells, both isoforms of MAP kinase
appear as phosphorylated, high molecular weight forms that were not
influenced by EGF stimulation.
Figure 2:
Phosphorylation of cytidylyltransferase in vivo. 2 10
ras-transfected
HaCaT cells were incubated in phosphate-free medium containing 150
µCi/ml [
P]P
(carrier-free) and
different concentrations of olomoucine or iso-olomoucine for 2 h. As an
additional control, 2
10
untransfected HaCaT cells
were labeled with phosphate-free medium containing 150 µCi/ml
[
P]P
(carrier-free) in the absence
of kinase inhibitors. Subsequently, the cells were washed twice with
cold phosphate-buffered saline and homogenized by sonication. CT was
immunoprecipitated with the antibody SA 2 and immunoprecipitated
proteins were separated on SDS-PAGE (10%) as described under
``Experimental Procedures.'' The dried gel was exposed to
Kodak XAR-5 film for 24 h. Bands corresponding to CT were analyzed by
video densitometry. The optical density of immunoprecipitated CT from ras-transfected cells in the absence of kinase inhibitors was
defined as 100%. The experiment was repeated, and similar results were
obtained.
To address
the question of which kinase(s) phosphorylate CT in the cells, the
purine analogue olomoucine was used in cell culture experiments. This
compound was shown to specifically inhibit the activity of cell
cycle-regulating kinases and p44 MAP kinase (20) . Iso-olomoucine, on the contrary, is confirmed as a
general kinase inhibitor with much lesser specificity for these
kinases. Fig. 2shows that the phosphorylation of CT observed in ras-transfected HaCaT cells is inhibited by olomoucine in a
concentration-dependent manner. In the presence of 100 µM olomoucine, the phosphorylation of CT in ras-transfected
HaCaT cells is reduced by 72%, whereas the phosphorylation of CT is
reduced by only 39% in the presence of the same concentration of
iso-olomoucine (see Fig. 2).
Figure 3:
Effect of reduced CT phosphorylation on
oleic acid-mediated enzyme activation. 6 10
ras-transfected HaCaT cells were incubated with 100
µM olomoucine or 0.25% Me
SO as controls for 4
h. Subsequently, the cells were homogenized in 1 ml of buffer
containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl,
0.06% Triton X-100, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride by use of a tight-fitting Dounce
homogenizer, and CT activity was measured as described. The values are
given in percentages of basal activity determined in the presence of
liposomes containing no oleic acid. In extracts of cells treated with
Me
SO, basal activity was 0.85 ± 0.22 nmol
CDP-choline formed per ml, and in extracts of cells treated with 100
µM olomoucine basal activity was 0.72 ± 0.2 nmol of
CDP-choline formed per ml ± S.D. (n = 3). *,
significantly different from controls at p < 0.02.**,
significantly different from controls at p <
0.01.
We also tested the
possibility that phosphorylation of CT affects enzyme stability to
endogenous proteases. For this, cellular extracts from olomoucine- and
MeSO-treated cells were incubated for different time
periods at 37 °C in the absence of protease inhibitors. After the
incubation, the reaction was stopped by the addition of SDS-sample
buffer and CT, and digestion products of CT were visualized by Western
blot analysis using the CT-specific antibody SA 2. As shown in Fig. 4, degradation products of CT with an apparent molecular
mass below 30 kDa were observed after 20 min in extracts from
olomoucine-treated cells. On the other hand, no degradation products of
this size were observed in extracts from control cells.
Figure 4:
Effect
of reduced CT phosphorylation on enzyme stability. 6 10
ras-transfected HaCaT cells were incubated with 100
µM olomoucine or 0.25% Me
SO as controls for 4
h. Subsequently, the cells were homogenized on ice in 1 ml of buffer
containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1%
Triton X-100, 1 mM dithiothreitol, 100 µM olomoucine, 100 nM ocadaic acid, and 10 mM NaF
by use of a tight-fitting Dounce homogenizer. Control extraction buffer
contained 0.25% Me
SO instead of 100 µM olomoucine. Cellular extracts were then incubated at 37 °C for
the indicated time periods, and CT and degradation products were
analyzed by Western blot using CT antibody SA 2
(1:3000).
Figure 5:
Phosphorylation of cytidylyltransferase by
cellular extracts from HaCaT cells and ras-transfected HaCaT
cells. 2 10
serum-starved HaCaT cells and ras-transfected HaCaT cells were incubated with EGF (100
ng/ml) or with medium without supplements for 5 min. Cells were
homogenized in 200 µl of buffer A as described under
``Experimental Procedures.'' 20 µl of the extracts were
used to phosphorylate rat liver CT that was previously dephosphorylated
with 5 units of alkaline phosphatase. CT was immunoprecipitated with
protein A-Sepharose using antibody SA 2 and separated by SDS-PAGE. A
representative autoradiogram from two independent experiments is
shown.
Figure 6:
Phosphorylation of cytidylyltransferase by
different kinases in vitro. Rat liver CT expressed in Sf21
insect cells was immunoprecipitated using protein A-Sepharose and
antibody SA 2. The washed immune complex was dephosphorylated with
alkaline phosphatase and incubated with 2 10
µmol/min p44 MAP kinase, p34
kinase,
and casein kinase II for 30 min at 30 °C. Phosphorylated CT was
separated by SDS-PAGE. A representative autoradiogram from two
independent experiments is shown.
Additionally, the effect of olomoucine on the
phosphorylation of CT by cellular extracts from ras-transfected cells in vitro was investigated. The
strong phosphorylation of CT catalyzed by cellular extracts from ras-transfected HaCaT cells was reduced in the presence of
olomoucine far more potently than in the presence of the unspecific
kinase inhibitor iso-olomoucine. To verify that olomoucine inhibits
p44 MAP kinase and p34
kinase and not
casein kinase II in our system, we investigated the effect of
olomoucine on the phosphorylation of CT by the purified kinases in
vitro. Although the phosphorylation of CT is reduced in the case
of p44
MAP kinase and p34
kinase,
olomoucine has no inhibitory effect on casein kinase II activity (see Table 1).
In
another set of experiments, immunoprecipitated CT was phosphorylated by
MAP kinase, cyclin-dependent kinase, casein kinase II, and cell-free
extracts from quiescent HaCaT cells and ras-transfected HaCaT
cells, followed by digestion with L-1-tosylamido-2-phenylethyl
chloromethyl ketone-treated trypsin and two-dimensional separation of
phosphorylated peptides. In order to obtain a sufficiently high P label in the phosphorylated peptides, the purified
kinases were used in this assay at the highest specific activity
available. The peptide maps obtained in the presence of purified
kinases were compared with the patterns of digested CT phosphorylated
by cell-free extracts from quiescent HaCaT cells and ras-transfected HaCaT cells (Fig. 7). Confirming the
results in the previous section, phosphorylation of the different
peptides by extracts from ras-transfected cells was higher as
compared with phosphorylation by extracts from normal HaCaT cells.
However, with the exception of spots 10 and 12, the patterns of HaCaT
cells and ras-transfected HaCaT cells were identical.
Furthermore, the patterns obtained by phosphorylation with MAP kinase
and cdc2 kinase (especially the spots numbered 2, 3, 4, 5, 6, 7, 10,
and 12) resembled the pattern of ras-transfected HaCaT cells.
In contrast, when casein kinase II was used in the assay two
predominant phosphopeptides were obtained (spots 4 and 7), resulting in
a different phosphopeptide pattern as compared with the other patterns.
Figure 7:
Phosphopeptide mapping of P-labeled cytidylyltransferase. Rat liver CT was
phosphorylated by cellular extracts from quiescent HaCaT cells, ras-transfected HaCaT cells, p44 MAP kinase,
p34
kinase, and casein kinase II as described
under ``Experimental Procedures.'' CT was separated by
SDS-PAGE and blotted onto nitrocellulose membrane. The enzyme was
localized on the blot by autoradiography, excised, and digested with
trypsin. Phosphopeptides were separated two-dimensionally by thin layer
electrophoresis and chromatography as described under
``Experimental Procedures.'' The numerical designation of
each spot was arbitrary, and sample origins are marked with
.
Reversible phosphorylation is a universal mechanism regulating enzymatic processes in eukaryotic cells. It has been known for some time that CT is regulated by reversible phosphorylation(1, 7, 9, 31) . However, the kinases involved in this process remain to be identified. Many approaches have used different protein kinase activators (13, 14, 16) and kinase inhibitors (15) as well as phosphatase inhibitors (10) to investigate the phosphorylation of CT. In the present paper, a different approach is presented using the human keratinocyte cell line HaCaT and a c-Ha-ras-transfected clone of this cell line. In untransfected HaCaT cells, MAP kinases became activated rapidly after EGF stimulation of the cells. On the other hand, MAP kinases were already fully activated in ras-transfected HaCaT cells without EGF stimulation, confirming the well known effect that EGF treatment and ras transfection stimulate MAP kinase activity(30) . Cell culture experiments revealed that ras-transfected HaCaT cells contained a highly phosphorylated form of endogenous CT, suggesting that the activation of MAP kinases might also be important for the phosphorylation of CT in vivo. The increased phosphorylation of CT in ras-transfected cells did not influence CT activity when determined in the absence of stimulating liposomes. However, we could clearly demonstrate that activation of CT by oleic acid was enhanced when phosphorylation of the enzyme was reduced by olomoucine treatment. This is in accordance to previous findings by Yang and Jackowski(32) , who showed that a mutant of CT lacking the COOH-terminal phosphorylation domain is much more sensitive to stimulation by lipids when compared with the wild type enzyme. Furthermore, we tested the hypothesis that phosphorylation of CT influences the stability of the enzyme. Using extracts from olomoucine-treated cells that mainly contained dephosphorylated CT, we could demonstrate that CT was more susceptible to digestion by endogenous proteases in vitro. In this context it has been shown that down-regulation of CT by cholecystokinin treatment of pancreatic acinar cells correlates with a decrease in CT phosphate levels, indicating that phosphorylation of CT protects the enzyme from degradation(33) .
Phosphorylation of CT by extracts from
quiescent HaCaT cells was very low, and EGF stimulation as well as ras transfection of HaCaT cells obviously activated kinases or
inactivated phosphatases. As a consequence, the phosphorylation of CT
by extracts from EGF-stimulated or ras-transfected HaCaT cells
was much stronger. Additionally, olomoucine, a purine analogue that was
shown to specifically inhibit cyclin-dependent kinases and p44 MAP kinase (20) reduced phosphorylation of CT in vivo and in vitro, suggesting an involvement of
proline-directed kinases in this process.
Phosphorylation of CT by
cAMP-dependent protein kinase or PKC has finally been ruled out.
Because the cDNA of rat liver CT contains many consensus
phosphorylation sequences for proline-directed kinases (like
p34 kinase and MAP kinase) and one consensus sequence
for casein kinase II(34) , we tested these kinases in their
ability to phosphorylate rat liver CT in vitro. All three
kinases were able to phosphorylate CT, and p34
kinase
catalyzed the strongest incorporation of
P into CT. The
advantage of the approach presented here, using rat liver CT as a
substrate for phosphorylation by cellular extracts and purified
kinases, is that the tryptic phosphopeptide patterns of CT can be
compared with each other. Using this system, difficulties arising from
differences in the amino acid sequence of CT from different species are
ruled out. The number of different spots obtained was similar to
patterns presented by other groups, using rat liver CT as
well(18) . Interestingly, the pattern obtained after
phosphorylation of CT by cellular extracts from ras-transfected HaCaT cells was similar to the patterns
obtained after phosphorylation by purified p44
MAP kinase
and p34
kinase. On the other hand, phosphorylation of CT
by casein kinase II revealed a different tryptic phosphopeptide
pattern, suggesting that this kinase plays a minor role in
phosphorylating CT. In this context, it is interesting to note that a
mutant of CT with an alanine residue in the potential phosphorylation
site for casein kinase II showed the same properties as compared with
the wild type enzyme(35) .
The findings presented here, together with results showing that growth factors stimulate phosphorylation of CT in HeLa cells(19) , substantiate the hypothesis that proline-directed kinases, such as MAP kinases or cyclin-dependent kinases, are involved in the phosphorylation of CT.