From the Division of Pharmaceutical Sciences, College
of Pharmacy and § The Graduate Center for Toxicology,
University of Kentucky, Lexington, Kentucky 40536-0082
Received for publication, November 18, 2002
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ABSTRACT |
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Supernatant protein factor is a 46-kDa
cytosolic protein that stimulates squalene monooxygenase, a downstream
enzyme in the cholesterol biosynthetic pathway. The mechanism of
stimulation is poorly understood, although supernatant protein factor
belongs to a family of lipid-binding proteins that includes Sec14p and The regulation of cholesterol biosynthesis beyond
HMG-CoA1 reductase is
relatively poorly characterized despite its importance in mammalian
physiology. The steps that follow the formation of squalene in
mammalian somatic cells typically yield only cholesterol, and squalene
monooxygenase catalyzes the second and potentially rate-limiting step
in this downstream or committed pathway for cholesterol synthesis. In
addition to being regulated at the transcriptional level by sterols (1,
2), squalene monooxygenase activity is stimulated by supernatant
protein factor (SPF), a cytosolic 46-kDa protein first characterized in
Konrad Bloch's laboratory (3). SPF belongs to a family of
lipid-binding proteins that includes Sec14p, Recent studies from Shibata et al. (4) revealed that the
overexpression of SPF in hepatoma cells increases cholesterol synthesis
by 2-fold. These findings suggest that SPF may have a role in
regulating cholesterol synthesis in vivo. The regulation of
SPF itself has not been described, although two groups (9, 10) have
reported that a cholesterol-supplemented diet did not alter SPF
activity in rat liver cytosol. Unexpectedly, SPF is identical to tocopherol-associated protein (13), a recently identified protein that is thought to be involved in the intracellular processing of The present studies were undertaken to better understand the role SPF
plays in influencing cholesterol synthesis and squalene monooxygenase
activity. A cDNA clone to human SPF was generated by polymerase
chain amplification, and the protein was expressed in Escherichia
coli. Because the purified protein demonstrated unexpectedly weak
activation of microsomal squalene monooxygenase, we explored the
possibility that post-translational modifications to SPF might be
necessary for full activity.
Cloning and Expression of Human SPF and Associated
Proteins--
The SPF cDNA was amplified with Pfu
polymerase (Stratagene) from a human liver cDNA library
(Quick-Clone, Clontech) with primers based on the
reported human SPF sequence (4) and that incorporated an
NcoI restriction site at the translation start codon and an XhoI restriction site immediately following the stop codon.
The amplification product was cloned into the PCR-BLUNT II TOPO vector (Invitrogen), plasmid DNA was isolated, and the sequence of the insert
was determined at the Molecular Structure and Analysis Facility,
University of Kentucky, to confirm identity with SPF. The SPF cDNA
insert was released with NcoI and XhoI, purified by agarose-gel electrophoresis, and cloned into the pTYB4 expression vector (New England Biolabs). SPF protein was purified following the
protocol for expression of intein fusion proteins with the IMPACT T7
system as follows. SPF expression was induced in E. coli
ER2566 cells overnight with 1 mM
isopropyl- SPF/Squalene Monooxygenase Activity Assays--
The microsomal
fraction (100,000 × g pellet, ~15 mg of protein/ml)
and cytosolic fraction (100,000 × g supernatant, ~20
mg of protein/ml) were prepared from the livers of untreated male Harlan Sprague-Dawley rats (~200 g) by standard procedures. Animals were maintained on a normal light-dark cycle with free access to rat
chow and were killed in late morning by decapitation. Squalene monooxygenase activity in rat liver microsomes was determined on the
basis of the procedure described by Wagner et al. (19) with
200 µg of microsomal protein, 30 µM FAD, 40 µM [14C]squalene, 10 µg of
phosphatidylglycerol, and 0.3 mM AMO 1618 (Calbiochem) to
inhibit oxidosqualene cyclase in 200 µl of 20 mM Tris-HCl
buffer, pH 7.4, with 1 mM EDTA. Reactions were started by
the addition of NADPH to 1 mM, incubated in a 37 °C
water bath for 1 h, and were stopped by the addition of 0.5 ml of
10% KOH in methanol after the incubation volume was brought to 1 ml
with water. The tubes were capped, and after saponification at 80 °C for 1 h the neutral lipids were extracted with 3 ml of petroleum ether. The solvent was removed under evaporative centrifugation, and
the lipids were resuspended in 50 µl of the same and spotted onto
silica thin-layer plates. Lipids were fractionated in 5% ethyl acetate
in hexane and visualized and quantified by electronic autoradiography
(Packard Instant Imager). Radiolabeled squalene (14C) was
synthesized by the Chemical Synthesis Facility, Department of
Medicinal Chemistry, University of Utah, at 7 mCi/mmol.
SPF activity (the ability to increase microsomal squalene monooxygenase
activity) was determined by adding either rat liver cytosol to the
above reactions at a ratio of 5 to 1 (cytosol:microsomes) on a per µg
of protein basis unless otherwise indicated or with 0.8 µg of
purified recombinant human SPF (unless otherwise indicated). Maximal
activation of microsomal squalene monooxygenase was determined by
adding Triton X-100 to 0.1% final concentration and was used as a
reference for the cytosolic and recombinant SPF assays. Activity assays
with purified squalene monooxygenase and cytochrome P450 reductase were
carried out as described (18).
Activation of SPF by ATP--
The ability of ATP and other
nucleotides to activate SPF was determined by preincubating cytosol (2 mg of protein) or 5 µg of recombinant SPF with 3 mM ATP
or other nucleotide in the presence of 4.5 mM
MgCl2 for 30 min at 37 °C on the basis of the procedure of Senjo et al. (20). Unbound nucleotide was then removed by centrifugal filtration, and the SPF preparation was resuspended in Tris
buffer. The ability to stimulate squalene monooxygenase was determined
as described above. When included, protein kinase inhibitors were added
before the addition of ATP at the following concentrations:
staurosporine, 50 nM; 4-cyano-3-methylisoquinoline (CMI),
30 nM; protein kinase A inhibitor-(6-22)-amide (PKAI), 2 nM; bisindolylmaleimide I (BIM-I), 20 nM;
rottlerin, 10 µM; H-8, 500 nM; KT5823, 300 nM; AG 213, 5 µM. All inhibitors were obtained from Calbiochem.
Activation of SPF by Protein Kinases--
The ability of various
purified protein kinases to activate SPF was determined with PKA; mouse
recombinant catalytic subunit C Deactivation of SPF by Protein Phosphatases--
The ability of
several protein phosphatases to decrease SPF activity was determined by
adding human protein phosphatase 1, Dietary Manipulation of SPF Activity--
Five male Harlan
Sprague-Dawley rats (250-300 g, final weight) were fed a high fat diet
(15.6% fat, Research Diets Inc., D12266B) containing 0.1% cholesterol
for 10 weeks. A second group of five rats was fed the same diet for 16 weeks, followed by 1 week on standard rodent chow (Teklad Global 2018, containing 5% fat). Livers were removed after decapitation, and
microsomes and cytosol were prepared for the determination of squalene
monooxygenase and SPF activities.
Cloning and Purification of Human SPF--
The published sequence
of human SPF (4) was used to design primers for the amplification of
SPF from a human liver cDNA library. The resulting cDNA
exhibited two differences from the published sequence: at codon 2, where incorporation of an NcoI site into the primer to
facilitate cloning replaced a serine with glycine; and at codon 11, where a lysine was encoded instead of arginine. A lysine is also found
at this latter position in the rat sequence (4). Expression and
purification of SPF from E. coli yielded a largely
homogeneous protein that migrated at 45 kDa, consistent with the
predicted molecular mass of 46 kDa (Fig. 1).
Stimulation of Squalene Monooxygenase--
Triton X-100 (0.1%)
activates microsomal squalene monooxygenase by ~10-fold and is
required for activity with the purified enzyme in a reconstituted
system (8, 18, 21). Consistent with these findings, purified
recombinant SPF was unable to replace Triton X-100 in assays with the
purified recombinant proteins squalene monooxygenase and cytochrome
P450 reductase. Recombinant SPF did stimulate rat liver squalene
monooxygenase in microsomes, although the extent of stimulation
(~2-fold) was significantly less than that obtained with Triton X-100
(Fig. 2). The addition of
phosphatidylglycerol increased the stimulation by recombinant SPF as
has been shown with purified native SPF (22), but the stimulation was
still less than that obtained with rat liver cytosol.
The inability of recombinant SPF to match the stimulation obtained with
cytosol raised the possibility that the bacterial protein was not fully
active and that native SPF contained bound cofactors or was
post-translationally modified. The rat expresses a second protein
closely related to SPF, which has been reported to bind GTP (11), and
both SPF and this related protein contain a putative nucleotide binding
domain at the C terminus (13). Moreover, Senjo et al. (20)
reported that the preincubation of rat liver cytosol or partially
purified SPF with 3 mM ATP for 30 min prevented the
stimulation of microsomal squalene monooxygenase by these preparations.
However, the addition of 3 mM GTP, GDP, or ATP to our
purified recombinant human SPF had no effect on its ability to
stimulate squalene monooxygenase (data not shown). Tocopherol-associated protein is identical to SPF and has been shown to
bind Activation of SPF--
To address the possibility that nucleotide
binding by SPF required additional cytosolic components, we added
various nucleotides to a rat liver cytosol preparation. In contrast to
the inhibition obtained by Senjo et al. (20), the addition
of ATP increased the ability of cytosolic SPF to stimulate squalene
monooxygenase by more than 2-fold (Fig.
3). Other nucleotides, including GTP, were ineffective. The inability of ADP and ATP
To determine whether phosphorylation was occurring, we tested the
ability of a variety of protein kinase inhibitors to block the
activation of SPF by ATP (Fig. 4).
Staurosporine, a broad spectrum inhibitor of protein kinases A, C, and
G, greatly reduced the activation of SPF by ATP. Two inhibitors that
are relatively specific for PKA (CMI and PKAI) and BIM-I (which is
relatively specific for PKC isoforms) also were effective inhibitors of
activation. Inhibitors of PKG (H-8 and KT5823) and AG 213, a broad
range tyrphostin inhibitor of protein-tyrosine kinases, had no effect
on the activation of SPF. These results suggest that phosphorylation by
protein kinases A or C may be responsible for the activation of SPF in rat liver cytosol.
To determine whether recombinant SPF could be activated by
phosphorylation, the purified protein was incubated with various protein kinases in the presence of ATP (Fig.
5). PKA and PKC
To determine whether SPF was phosphorylated in isolated liver
preparations, cytosol was incubated with several protein phosphatases, and the ability of the treated preparations to stimulate squalene monooxygenase was determined. Human protein phosphatase 1 Effect of Dietary Fats on SPF Activity--
To determine whether
SPF activity was regulated by dietary cholesterol, rats were fed a high
fat diet (15.6% fat, 0.1% cholesterol) for 10 weeks, and SPF activity
in liver cytosol was compared with that of rats on a normal chow diet.
The high fat diet reduced SPF activity by half and could be restored by
returning the rats to a chow diet for 1 week (Fig.
7). Similarly, squalene monooxygenase activity was reduced by half with the high fat diet and could be
restored by refeeding a chow diet. SPF activity from the fat-fed animals could not be increased by incubation with ATP or by adding ATP
with PKC Supernatant protein factor has been a puzzle for more than 25 years, and this mystery has deepened with recent observations that
overexpression of SPF in hepatoma cells increases cholesterol synthesis
(4) and that SPF is identical to a vitamin E-binding protein of unknown
function (13, 14). In the present studies, the expression of human
supernatant protein factor in bacteria yielded a purified protein with
weaker than expected ability to stimulate squalene monooxygenase and
raised the possibility that additional cofactors or post-translational
modifications are required for full activity. Although SPF contains a
putative nucleotide-binding domain at its C terminus (4), the addition
of GTP, GDP, or ATP to the purified protein had no effect on activity,
and the inclusion of This finding with cytosolic SPF was supported by the ability of protein
kinases A and C to activate purified recombinant SPF. Moreover, the
addition of both kinases to purified SPF increased the activity of this
protein to a level equal to that of ATP-activated SPF in cytosol (Fig.
8), suggesting that full activation of
SPF requires phosphorylation on two or more sites by multiple kinases. Protein kinases have previously been linked to the regulation of
cholesterol synthesis and homeostasis. Perhaps best characterized is
the down-regulation of HMG-CoA reductase activity by AMP-activated protein kinase (23) and by PKC (24). PKC-tocopherol transfer protein. Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a
relatively weak ability to activate microsomal squalene monooxygenase,
we investigated the possibility that cofactors or post-translational modifications were necessary for full activity. Addition of ATP to rat
liver cytosol increased supernatant protein factor activity by more
than 2-fold and could be prevented by the addition of inhibitors of
protein kinases A and C. Incubation of purified recombinant supernatant
protein factor with ATP and protein kinases A or C
similarly
increased activity by more than 2-fold. Addition of protein phosphatase
1
, a serine/threonine phosphatase, to rat liver cytosol reduced
activity by 50%, suggesting that supernatant protein factor is
partially phosphorylated in vivo. To determine whether
dietary cholesterol influenced the phosphorylation state, cytosols were
prepared from livers of rats fed a high fat diet. Although supernatant
protein factor activity was reduced by more than one-half, it could not
be restored by the addition of ATP or protein kinase C
with ATP,
suggesting that dietary cholesterol reduced the expression of this
protein. Supernatant protein factor thus appears to be regulated both
post-translationally through phosphorylation and at the level of
expression. Phosphorylation may provide a means for the rapid short
term modulation of cholesterol synthesis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tocopherol transfer
protein, and cellular retinol-binding protein (4). The recently
reported crystal structure of human SPF confirms this relationship (5).
The mechanism by which SPF stimulates squalene monooxygenase has not
been established; most evidence suggests that SPF facilitates the
transfer of squalene into and between membrane compartments in the cell
(4, 6, 7), although SPF has not been shown to bind squalene. SPF
in vitro is only effective with membrane-bound (microsomal)
squalene monooxygenase consistent with a lipid transfer function, and
has no effect on the purified enzyme (8).
-tocopherol (14). Tocopherol-associated protein binds
-tocopherol with high affinity and appears to act as a tocopherol-dependent transcription factor (15). The gene
targets of this protein have not been identified, but it is intriguing that
-tocopherol down-regulates the expression of the cholesterol scavenger receptors SR-A (16) and CD36 (17). It should also be noted
that the rat contains a second SPF-like gene of unknown function that
is highly expressed in epithelial tissue (most notably the olfactory
epithelium) and that the encoded protein exhibits high affinity for GTP
(11, 12).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside at 30 °C with slow
shaking. All subsequent steps were carried out at 4 °C. Cells were
broken in a French pressure cell in buffer containing 20 mM
Tris-HCl (pH 7.4), 500 mM NaCl, and 0.1 mM
EDTA, and the lysate was cleared by 12,000 × g
centrifugation for 30 min followed by digestion with 1 µg of DNA
nuclease for 1 h. The cleared lysate was loaded onto a chitin
affinity column (2-ml bed volume), washed with 40 ml of lysis buffer,
and incubated overnight at 4 °C in 4 ml of buffer containing 30 mM
-mercaptoethanol to promote cleavage of the
intein-SPF bond. SPF was eluted with 10-20 ml of the same buffer,
which was then replaced by centrifugal dialysis with 20 mM
Tris-HCl, pH 7.4, and the sample was stored at
80 °C. The purified
protein retains four amino acids (Leu-Glu-Pro-Gly) at the C
terminus that are derived from the intein fusion sequence. Recombinant
human squalene monooxygenase, foreshortened at the N terminus to
facilitate expression, and rat cytochrome P450 reductase were expressed
in E. coli and purified as described (18), excluding Triton
X-100 from the lysis and purification buffers. Protein was quantified
with the Coomassie Plus assay reagent kit (Pierce).
, 5 units (Calbiochem); human
recombinant PKC; isozymes
,
1,
2,
,
,
,
,
,
0.25 unit (Calbiochem); PKG; bovine recombinant isoform I
, 5000 units (Calbiochem); and rat AMP-kinase, 0.1 unit (Upstate USA, Inc.)
according to the supplier's instructions. Each kinase was incubated
with either purified recombinant SPF (5 µg) or cytosol (2 mg of
protein) for 30 min at 30 °C. The ability of each mixture to
stimulate squalene monooxygenase was determined as described above.
isoform (0.1 unit),
protein phosphatase (
PP, 200 units), or Yersinia
enterocolitica protein-tyrosine phosphatase (100 units), to
cytosol (2 mg of protein) and incubating at 37 °C for 30 min in 50 mM Tris, HCl, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, and 2 mM MnCl2
in accord with the instructions for use provided by the supplier
(Calbiochem). The ability of each mixture to stimulate squalene
monooxygenase was then determined as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SDS-polyacrylamide gel electrophoresis of
bacterially expressed squalene monooxygenase, SPF, and cytochrome P450
reductase. Each purified recombinant protein was loaded onto a
10% SDS-polyacrylamide gel and after electrophoresis the gel was
stained with Coomassie Blue. Lane 1, squalene monooxygenase,
2 µg; lane 2, SPF, 2 µg; lane 3, cytochrome
P450 reductase, 3 µg; lane 4, molecular mass markers at
97, 66, 45, and 33 kDa.
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Fig. 2.
Stimulation of microsomal squalene
monooxygenase by cytosolic and recombinant SPF. Squalene
monooxygenase activity in rat liver microsomes was determined as
described under "Experimental Procedures" in the absence ( ) or
presence of rat liver cytosol at a microsomal:cytosolic protein ratio
of 1:2, 1:5, or 1:10 or with 0.8 µg of pure recombinant SPF, 0.8 µg
of pure recombinant SPF with 10 µg of phosphatidylglycerol
(+PG), or 0.1% Triton X-100 (TX100). Each value
represents the mean ± S.E. of from two to eight experiments
carried out in duplicate.
-tocopherol with high affinity (14, 15). The addition of 50 µM
-tocopherol to recombinant SPF similarly had no
effect on its ability to stimulate squalene monooxygenase either in the
presence or absence of GTP, GDP, or ATP. Although these studies do not
rule out the possibility that SPF binds a nucleotide or
-tocopherol,
it appears that they do not influence the ability of the recombinant
protein to stimulate squalene monooxygenase.
S, a nonhydrolyzable form of ATP, to activate SPF suggested that SPF was a substrate for an
ATP-dependent protein kinase and that phosphorylation
activated SPF. This ATP-dependent activation was not
affected by the presence of GTP or
-tocopherol, and the addition of
ATP to microsomes in the absence of cytosol had no effect on squalene
monooxygenase activity (data not shown).
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Fig. 3.
Activation of cytosolic SPF by ATP. The
ability of various nucleotides to activate SPF in rat liver cytosol was
determined by monitoring the ability of the treated preparation to
stimulate squalene monooxygenase activity as described under
"Experimental Procedures." Each value represents the mean ± S.E. of two to three experiments carried out in duplicate.
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Fig. 4.
Inhibition of SPF activation by protein
kinase inhibitors. Rat liver cytosol was incubated with ATP in the
presence of each inhibitor for 30 min before determining the ability of
each preparation to stimulate squalene monooxygenase activity, as
described under "Experimental Procedures." The relative
specificities of the inhibitors are as follows: staurosporine: PKA,
PKC, and PKG; CMI and PKAI: PKA; BIM-I and rottlerin: PKC; H-8 and
KT5823: PKG; AG213: tyrosine kinases. Each value represents the
mean ± S.E. of two or more experiments carried out in
duplicate.
increased SPF activity
by more than 2-fold, similar to the activation obtained by the addition
of ATP to cytosol. PKC isoforms
,
1, and
2 were also able to
activate recombinant SPF, whereas PKG and AMP-activated protein kinase,
which has been shown to down-regulate the activity of HMG-CoA reductase
(23), were ineffective. The addition of GTP, GDP, or
-tocopherol to
incubations with PKC
did not affect the activation of SPF,
indicating that these potential ligands do not impair or augment
phosphorylation by this protein kinase. The addition of protein kinases
with ATP to microsomes alone had no effect on squalene monooxygenase
activity. These results support a role for protein kinase A or C in the
activation of SPF in vitro and possibly in
vivo.
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Fig. 5.
Activation of recombinant SPF by
phosphorylation. Purified recombinant SPF was incubated in the
absence ( ) or presence of the indicated protein kinases and ATP, and
the ability of the preparations to stimulate squalene monooxygenase was
determined as described under "Experimental Procedures." PKC
isoforms are indicated by their Greek letter assignments;
AMPK, AMP-activated protein kinase. Each value represents
the mean ± S.E. of two experiments carried out in
duplicate.
, a serine/threonine phosphatase, and
protein phosphatase, a
serine/threonine/tyrosine phosphatase, both reduced the ability of
cytosolic SPF to stimulate microsomal squalene monooxygenase by ~50%
(Fig. 6). Protein-tyrosine phosphatase,
which is specific for phosphotyrosine, was largely ineffective. These
results indicate that SPF is partially phosphorylated in cytosolic
preparations and that the site of phosphorylation is on one or more
serines or threonines. These results are consistent with the protein
kinase experiments in which the activation of SPF was inhibited by
serine/threonine kinase inhibitors (staurosporine) and catalyzed by
serine/threonine kinases (PKA and PKC).
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Fig. 6.
Deactivation of SPF by protein
phosphatases. Rat liver cytosol was incubated with human protein
phosphatase 1 (PP1),
protein phosphatase
(
PP), or protein-tyrosine phosphatase (PTP)
and the ability of the treated preparations to stimulate squalene
monooxygenase was determined as described under "Experimental
Procedures." Each value represents the mean ± S.E. of three
experiments carried out in duplicate.
to the cytosol, suggesting that the lower activity was
caused by a decrease in the expression of SPF. The expression of
squalene monooxygenase has previously been shown to be regulated by
dietary lipids via a transcriptional mechanism (1, 10).
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Fig. 7.
Down-regulation of SPF and squalene
monooxygenase by a high fat diet. The effect of dietary fat on SPF
and squalene monooxygenase activities was determined by feeding rats a
high fat diet as described under "Experimental Procedures"; refed
animals were returned to a standard chow diet for 1 week before assay.
SPF activity in the cytosol of treated animals was determined with
microsomes from untreated animals (left axis, gray
bars); squalene monooxygenase activity in microsomes from treated
animals was determined with 0.1% Triton X-100 for activation
(right axis, solid bars). Each value represents
the mean ± S.E. of five animals analyzed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tocopherol, a proposed ligand for this protein (14, 15), in the presence or absence of nucleotides similarly had no
effect. However, the addition of ATP but not other nucleotides to rat
liver cytosol had a stimulatory effect that could be blocked by the
prior addition of inhibitors of protein kinases A and C, which argues
that cytosolic SPF was a substrate for one or more of these kinases and
that phosphorylation increased the ability of this protein to stimulate
squalene monooxygenase. SPF contains three consensus recognition sites
for PKA and six sites for PKC.
has recently been shown to
mediate the up-regulation of low density lipoprotein receptor
transcription in response to sterol depletion; notably, this PKC
isoform is directly inhibited by 25-hydroxycholesterol (25). Although
PKC
produced only a modest activation of SPF, the closely related
PKC
isoform potently increased SPF activity. Both isoforms are
expressed in hepatocytes (26) and are members of the novel PKC subclass
that is activated by lipids (including diacylglycerol and
phosphatidylserine) but is independent of calcium (27). PKC isoforms
and
were recently suggested to also be involved in the
regulation of cholesterol levels in the endoplasmic reticulum of
fibroblasts in a complementary fashion, with PKC
stimulation leading
to lower cholesterol levels and PKC
stimulation leading to increased
cholesterol levels (28). These changes in intracellular cholesterol are
believed to modulate cholesterol uptake and biosynthesis (29). Although
in the present studies PKC
was very effective in the activation of
purified recombinant SPF, rottlerin, a relatively specific inhibitor of
this isoform, had little effect on the activation of SPF in cytosol.
Rather, the ability of BIM-I to inhibit SPF activation in cytosol
argues that conventional kinases such as PKC isoforms
,
I, and
II are active in this preparation. Because these PKC isoforms also were effective in activating recombinant SPF the isoform responsible for activation of SPF in vivo remains to be
elucidated.
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Fig. 8.
Comparison of squalene monooxygenase
stimulation by cytosolic and recombinant SPF. The ability of
cytosol and purified recombinant SPF to stimulate microsomal squalene
monooxygenase was determined after treatment of the preparations as
indicated. 2,3-Oxidosqualene formation was measured after incubation
for 1 h as described under "Experimental Procedures." Each
value represents the mean ± S.E. of two or more experiments
carried out in duplicate.
Protein kinase A was also very effective in activating recombinant SPF, and the PKA inhibitors CMI (30) and PKAI, an inhibitory peptide specific to this kinase, were effective in preventing SPF activation in cytosol. PKA has not previously been implicated in the regulation of cholesterol synthesis, although it plays a prominent role in the activation of cholesterol transport into mitochondria for steroidogenesis (31). PKA has also been implicated in the up-regulation of expression of the low density lipoprotein receptor (32). PKA is a cAMP-dependent kinase and thus can be activated by the many signaling pathways that activate adenylate cyclase. The present studies do not allow us to determine which protein kinase (A or C) is active in isolated rat liver cytosol, because the kinase inhibitors CMI and PKAI, which block PKA, and BIM-I, which blocks PKC, all blocked SPF activation despite being used at concentrations reported to allow selective inhibition (30, 33, 34). The limited specificity of protein kinase inhibitors has previously been discussed (35). As noted above, the results with purified recombinant SPF indicate that full activation requires phosphorylation by both kinases (Fig. 8).
Protein phosphatase 1, the principal serine/threonine phosphatase in liver, is activated by a variety of regulatory proteins that respond to glucagon, insulin, and glucocorticoids and has been implicated in cholesterol homeostasis (36) and steroidogenesis (37). Protein phosphatase 2A is also present in liver and is responsible for the dephosphorylation of HMG-CoA reductase (38). In the present study protein phosphatase 1 reduced SPF activity in cytosol by ~50%, indicating that SPF is partially phosphorylated in this preparation. Although this finding suggests that SPF is partially phosphorylated in vivo, predicting that the actual phosphorylation state of SPF in the intact liver by this approach is limited by the possible activation of kinases and phosphatases during isolation of the subcellular fractions. A similar problem was encountered in the analysis of HMG-CoA reductase inactivation by AMP-dependent protein kinase and was solved by rapid cooling of the liver during preparation (39). Although this technique was not employed in the present studies, these results nonetheless strongly suggest that phosphorylation of SPF is physiologically relevant and occurs in the intact liver of normal animals. Partial phosphorylation under normal conditions would allow rapid up-regulation and down-regulation of squalene monooxygenase activity in response to changing sterol levels or other stimuli.
In an effort to identify physiologic conditions that alter the
phosphorylation state of SPF, we obtained livers from rats that had
been maintained on a high fat diet. This diet decreased squalene
monooxygenase activity by approximately 50% as compared with animals
on a standard chow diet, consistent with earlier reports (9, 10). This
diet also decreased SPF activity in contrast with earlier results from
these two groups (9, 10); this may reflect differences in the dietary
treatment regimens. However, the decrease in SPF activity was not
caused by a decrease in the phosphorylation state of SPF; in fact, SPF
appeared to be fully phosphorylated in these preparations because the
addition of ATP with or without PKC did not increase SPF activity.
This increase in SPF phosphorylation combined with the decrease
in overall SPF activity is consistent with a hypothesis in which the
long term regulation of SPF and squalene monooxygenase is mediated
through changes in protein levels, whereas phosphorylation might allow
rapid short term modulation of activity, as has been proposed for
HMG-CoA reductase (23).
SPF was not previously recognized to be a phosphoprotein. As shown
here, phosphorylation amplifies the ability of SPF to stimulate squalene monooxygenase in vitro and may provide a means for
the rapid short term modulation of cholesterol synthesis in
vivo. Although no relationship between phosphorylation and
-tocopherol binding or GTP binding could be found, phosphorylation
may also be important in the other actions of SPF, most notably
its role in
-tocopherol binding and signaling.
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ACKNOWLEDGEMENTS |
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We thank Carine Boustany and Lisa Cassis for providing the livers from the high fat diet-treated animals and Steve Post for sage advice.
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FOOTNOTES |
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* This work was supported by Grant 0150251N from the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported in part by National Institutes of Health Grant T32 ES-07266.
To whom correspondence should be addressed. Tel.:
859-257-1137; Fax.: 859-257-7564; E-mail: tporter@uky.edu.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M211750200
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ABBREVIATIONS |
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The abbreviations used are:
HMG-CoA, hydroxymethylglutaryl-CoA;
SPF, supernatant protein factor;
PKA, protein kinase A;
PKC, protein kinase C;
PKG, protein kinase G;
PKAI, protein kinase A inhibitor-(6-22)-amide;
ATPS, adenosine 5'-O-(thiotriphosphate). BIM, bisindolylmaleimide;
CMI, 4-cyano-3-methylisoquinoline.
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