(Received for publication, May 15, 1995; and in revised form, September 19, 1995)
From the
We previously described the purification of a membrane-bound
diacylglycerol kinase highly selective for sn-1-acyl-2-arachidonoyl diacylglycerols (Walsh, J. P., Suen,
R., Lemaitre, R. N., and Glomset, J. A.(1994) J. Biol. Chem. 269, 21155-21164). This enzyme appears to be responsible for
the rapid clearance of the arachidonate-rich pool of diacylglycerols
generated during stimulus-induced phosphoinositide turnover. We have
now shown phosphatidylinositol 4,5-bisphosphate to be a potent and
specific inhibitor of arachidonoyl-diacylglycerol kinase. Kinetic
analyses indicated a K for
phosphatidylinositol 4,5-bisphosphate of 0.04 mol %. Phosphatidic acid
also was an inhibitor with a K
of 0.7 mol
%. Other phospholipids had only small effects at these concentrations.
A series of multiply phosphorylated lipid analogs also inhibited the
enzyme, indicating that the head group phosphomonoesters are the
primary determinants of the polyphosphoinositide effect. However, these
compounds were not as potent as phosphatidylinositol 4,5-bisphosphate,
indicating some specificity for the polyphosphoinositide additional to
its total charge. Five other diacylglycerol kinases were activated to
varying degrees by phosphatidylinositol 4,5-bisphosphate and
phosphatidic acid, suggesting that inhibition by acidic lipids may be
specific for the arachidonoyl-DAG kinase isoform. Given the presumed
role of arachidonoyl-diacylglycerol kinase in the phosphoinositide
cycle, this inhibition may represent a mechanism for
polyphosphoinositides to regulate their own synthesis.
Diacylglycerol kinases catalyze the ATP-dependent
phosphorylation of sn-1,2-diacylglycerol (DAG) ()to
phosphatidic acid (PA) (1, 2, 3) . As such,
they are widely regarded as attenuators of the DAG signaling and
protein kinase C activation that occur during stimulus-induced PI
turnover(4) . The recent identification of a specific DAG
kinase essential for PI-mediated invertebrate visual transduction is a
striking confirmation of the involvement of DAG kinases in the PI
cycle(5) .
It has recently become evident that DAG kinases are a diverse family of isoenzymes(2) . The first of these to be purified and cloned was an 82.6-kDa isoform expressed predominantly in brain and thymus(6, 7) . Several homologs of this enzyme also have been cloned, each of which has its own highly specific pattern of expression in cells and tissues (8, 9, 10, 11, 12) . Additional DAG kinases, which appear distinct from the cloned isoforms described above, also have been reported(13, 14, 15, 16, 17, 18, 19, 20, 21) . However, detailed enzymologic data on these are not available at this time. Our laboratory has described and purified a membrane-bound DAG kinase highly selective for DAG molecular species containing arachidonate as the sn-2 fatty acyl moiety(21, 22, 23, 24) . This activity can be distinguished from other DAG kinases by a variety of enzymologic properties in addition to its substrate specificity(21, 22, 23, 24) . Arachidonoyl-DAG kinase activity varies widely between different tissues, but it is detectable in all cells and tissues we have examined (21, 22, 23, 24) . Given the marked enrichment of animal cell phosphatidylinositols in arachidonate at the glycerol sn-2 position, the substrate selectivity of arachidonoyl-DAG kinase suggests a special role for this isoform in PI biosynthesis(21, 22, 23, 24) .
This multiplicity of DAG kinases is reminiscent of the diversity
seen in the protein kinase C and PI-specific phospholipase C isoenzyme
families. As with the phospholipases C and protein kinases C, this
diversity most likely reflects multiple mechanisms of enzyme regulation
in the cells expressing the various DAG kinase
isoforms(26, 27, 28) . The detailed
mechanisms of DAG kinase regulation are, however, poorly understood.
The 82.6-kDa DAG kinase and its homologs are thought to translocate to
membranes during cell activation and are activated in vitro by
Ca and phosphatidylserine(29) . Limited
evidence for activation of some DAG kinases by reversible protein
phosphorylation also has been reported (30, 31, 32) .
Acidic phospholipids have
been shown to modulate the in vitro activities of several
membrane enzymes involved in stimulus
transduction(33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) .
Since levels of these lipids fluctuate during cell stimulation, this
modulation is thought to reflect regulation of the enzymes by the in vivo membrane microenvironment. We have now examined the
effects of acidic phospholipids on arachidonoyl-DAG kinase in a well
characterized, mixed micellar assay system. Phosphatidylinositol
4,5-bisphosphate (PIP) was shown to be a potent and
specific inhibitor of arachidonoyl-DAG kinase. Phosphatidylinositol
4-phosphate (PIP) and PA also were inhibitors, but at 18-fold higher
concentrations. Five other DAG kinases were examined and found to be
activated to varying degrees by PIP
and PA, suggesting that
inhibition by acidic lipids may be a specific property of the
arachidonoyl-DAG kinase isoform. Given the likely role of
arachidonoyl-DAG kinase in PI biosynthesis, these data suggest that
inhibition by polyphosphoinositides may represent a mechanism for
feedback regulation of this enzyme by PIP
, its presumed
final product.
To prepare
mono-O-hexadecylpentaerythritol trisphosphate, pentaerythritol
(70 g, 0.51 mol) was partially acetylated with acetic acid (89 ml, 1.55
mol) by refluxing under a Dean-Stark trap in 150 ml of benzene
containing 2 g of toluenesulfonic acid. After 24 h, 28 ml (1.55 mol) of
water had collected in the trap, and the reaction was terminated. The
mixture was deionized with 50 ml of Dowex AG 501-X8. Evaporation of the
benzene yielded 134 g of clear, syrupy product (theoretical 136 g). To
prepare the hexadecyl ether, acetylated pentaerythritol (20 g, 76 mmol)
was stirred with 80 ml of dimethylformamide and 80% NaH (1.1 g, 37
mmol) added. After 1 h, H evolution had ceased, and
1-bromohexadecane (10 g, 33 mmol) was added. The reaction was stirred
for an additional 2 h and then quenched by addition of 150 ml of ice
water. This mixture was extracted 3 times with 50 ml of diethyl ether,
and the pooled extracts passed over a 50-ml bed of silicic acid. To
remove the acetate blocking groups, the ether was evaporated, and the
residue dissolved in 100 ml of methanol. Concentrated HCl (5 ml) was
added, and the mixture refluxed. Methyl acetate (azeotrope with
methanol, b.p. 54 °C) was allowed to distill over. After 30 min,
evolution of methyl acetate had ceased. The mixture was refluxed for an
additional hour, after which, the methanol was evaporated. The product
was precipitated at -20 °C from H
O/ethanol (1:1
(v/v)) and washed at -20 °C with petroleum ether/diethyl
ether (2:1 (v/v)). The residue was dissolved in 80 ml of CHCl
and extracted with 80 ml of H
O/ethanol (4:1 (v/v))
followed by 60 ml of H
O. Ethanol (9 ml) was added to the
CHCl
, and the solution passed over a 50-ml bed of silicic
acid. The solvents were evaporated, and the product was crystallized
from petroleum ether/diethyl ether (1:1 (v/v)). The final yield was 4.7
g. The product was homogeneous by TLC on silicic acid (R
0.08, benzene/ethyl ether/acetic acid, 25:25:1 (v/v). ESI-MS: m/z 361 (100) MH
, m/z 378 (94) MNH
. MS/MS
(MH
): m/z 361 (49)
MH
, m/z 343 (100)
MH
-H
O, m/z 313 (5)
MH
-CH
O-H
O, m/z 255 (5)
C
H
OCH
, m/z 137 (88) pentaerythritol H
, m/z 119 (97) pentaerythritol
H
-H
O, m/z 101 (99)
pentaerythritol H
-2H
O. Some additional
product could be obtained by concentrating the mother liquor, but it
was contaminated with a small amount of faster migrating material
presumed to be the dihexadecyl ether.
To prepare the
trisphosphorylated product, monohexadecylpentaerythritol (2.0 g, 5.6
mmol), dimethylaminopyridine (0.5 g, 4 mmol), and triethylamine (4.0
ml, 28.7 mmol) were dissolved in 20 ml of CHCl and added
slowly to diphenylchlorophosphate (4.0 ml, 25 mmol) in 20 ml of
CHCl
. The reaction was stirred for 1 h and then quenched by
pouring slowly, with stirring, into 100 ml of 0.5 M NaHCO
together with an additional 30 ml of
CHCl
. The CHCl
phase was washed with 125 ml of
0.5 M NaHCO
and passed over a 10-ml bed of silicic
acid. The product migrated as a single spot on TLC (R
0.75, CHCl
/toluene/ethanol, 20:20:5 (v/v)). To remove
the phenyl protecting groups, the CHCl
was evaporated, and
the syrupy product was dissolved in 180 ml of
ethanol/H
O/acetic acid (4:1:1 (v/v)). 0.5 g of PtO
was added, and the mixture was reduced with H
at 25
°C and 60 psi in a Parr hydrogenation apparatus. Hydrogen uptake
was largely complete after 30 min, and the reaction was continued
overnight. A total of 137 mmol of H
was consumed
(theoretical 142 mmol). The catalyst was removed by filtration, and the
solution was concentrated to about 15 ml by evaporation. This material
was dissolved in 100 ml of H
O. The pH was adjusted to 11.0
by addition of 10 N NaOH, and the product was precipitated at
0 °C by the addition of 100 ml of methanol. The precipitate was
collected by filtration and washed successively with 50% ice-cold
aqueous methanol, absolute ethanol 3 times, ethyl acetate 2 times, and
diethyl ether 3 times. Yield was 3.86 g (95%). The final product
migrated as a single spot on oxalate-impregnated TLC plates (R
0.09, CHCl
/acetone/methanol/acetic
acid/H
O, 27:10:9:8:5 (v/v)). ESI-MS: m/z 601 (100) MH
; MS/MS (MH
): m/z 601 (100) MH
, m/z 503 (63) MH
-H
PO
, m/z 405 (17)
MH
-2H
PO
.
H NMR:
0.78-0.87 (3H, t), 1.15-1.32 (26H, m),
1.39-1.53 (2H, m), 3.26-3.34 (4H, s/t), 3.77-3.84
(6H, d).
To prepare 2-hexadecylglycerol-1,3-bisphosphate,
2-hexadecylglycerol was first prepared from 1-bromohexadecane and
1,3-benzylidene glycerol according to the procedure of Serdarevich and
Carroll (50) except that the alkylation reaction was carried
out in NaH/dimethylformamide as described above. The product migrated
as a single spot on TLC (R 0.14, benzene/diethyl
ether/acetic acid, 25:25:1 (v/v)). ESI-MS: m/z 317
(54) MH
, m/z 334 (100)
MNH
. MS/MS
(MNH
): m/z 317 (21)
MH
, m/z 299 (8)
MH
-H
O, m/z 93 (96)
glycerol H
, m/z 75 (100) glycerol
H
-H
O, m/z 57 (67)
glycerol H
-2H
O. MH
fragmented identically to MNH
. The
2-hexadecylglycerol was phosphorylated with three equivalents of
diphenylchlorophosphate in 90% yield using the procedure described
above for monohexadecylpentaerythritol. The product migrated as a
single spot on oxalate-impregnated TLC plates (R
0.21, CHCl
/acetone/methanol/acetic
acid/H
O, 27:10:9:8:5 (v/v)). ESI-MS: m/z 477 (100) MH
; MS/MS (MH
): m/z 477 (81) MH
, m/z 379 (100) MH
-H
PO
, m/z 235 (11)
MH
-C
H
OH, m/z 155 (73)
MH
-C
H
OPO
H
.
H NMR:
0.79-0.87 (3H, t), 1.15-1.32 (26H,
m), 1.38-1.53 (2H, m), 3.41-3.51 (2H, t), 3.55-3.65
(1H, m), 3.77-3.90 (4H, dd).
Hexadecyl phosphate was prepared
by phosphorylation of hexadecyl alcohol with excess
phosphoryltriimidazole as described previously (51) and
crystallized as the monosodium salt from ethanol/water. The final
product migrated as a single spot on TLC with the expected R(51) . Structures of these
polyphosphorylated lipid analogs are shown in Fig. 1.
Figure 1: Structures of multiply phosphorylated lipid analogs used in this work. Structures of phospholipid analogs used in this work are shown together with phosphatidic acid and phosphatidylinositol 4,5-bisphosphate.
Figure 2:
Inhibition of arachidonoyl-DAG kinase by
PIP and PA. Activities were determined in the Triton assay
as described in the ``Experimental Procedures'' section in
the presence of the indicated surface concentrations of phospholipids.
Activity under these conditions in the absence of inhibitor was 17.5
pmol/min/µl.
, 18:0-20:4 PA;
, 16:0-18:1
PA;
, PIP
.
Addition of 20 mol % phosphatidylcholine had no effect on the
PIP inhibition. The addition of up to 50 µM Ca
or 5 mM Mg
also
was without effect. In the presence of 20 mol %
octadecyltrimethylammonium chloride, 50% inhibition occurred at 1.4 mol
% PIP
and 75% inhibition at 2.2 mol %. (
)Presumably, the decreased inhibitory potency of PIP
in OTAC reflects complexation by the quaternary amine. However,
it does demonstrate that PIP
is capable of inhibiting the
enzyme even in micelles bearing a net positive charge. Examination of
surface dilution behavior by varying the micelle concentration while
holding the surface concentrations of PIP
and
18:0-20:4 DAG constant demonstrated that activity in the presence
of 0.04 or 0.10 mol % PIP
was dependent on the PIP
surface concentration and independent of micelle concentration
(data not shown). This is consistent with our previous observation that
several other phospholipids do not alter the surface dilution behavior
of arachidonoyl-DAG kinase(24) . Inositol polyphosphates,
including D-myo-inositol 1,4,5-trisphosphate and
inositol hexaphosphate had no effect on arachidonoyl-DAG kinase
activity at 1.0 mM, nor did they reverse the inhibition by
PIP
(data not shown).
Figure 3:
Inhibition of arachidonoyl-DAG kinase by
multiply phosphorylated amphiphiles. Activities were determined as in Fig. 2, but in the presence of the indicated concentrations of
phospholipid analogs. , dodecyl phosphate;
,
2-hexadecylglycerol bisphosphate;
,
monohexadecylpentaerythritol trisphosphate. Dodecyl phosphate was used
as the monophosphorylated amphiphile instead of hexadecyl phosphate
because the latter precipitated with the Mg
in the
assay buffer.
The bis- and
trisphosphorylated amphiphiles were freely soluble in water, behaving
as detergents. It was important to show under the assay conditions used
that these compounds incorporate completely into Triton micelles, as
failure to do so would render the calculated mol fractions invalid. The
CMC in 100 mM NaCl of monohexadecyl-pentaerythritol
trisphosphate was 34 µM, and for 2-hexadecylglycerol
bisphosphate it was 16 µM. The CMC of PIP under these conditions was less than 2 µM. Assuming
ideal partitioning between the aqueous and micellar
pseudophases(44) , greater than 99% of these amphiphiles must
be in the micelles. (
)Partitioning of these compounds into
Triton micelles was also estimated by cloud point separation as
described under ``Experimental Procedures.'' These results
indicated that greater than 98% of the bis- and trisphosphorylated
analogs and approximately 80% of the dodecyl phosphate were in the
micelles (
)(data not shown). The inability of these
compounds to inhibit arachidonoyl-DAG kinase as effectively as
PIP
cannot, therefore, be due to a failure to partition
into the micelles.
Figure 4:
Dependence of arachidonoyl-DAG kinase on
DAG and ATP. Double-reciprocal plots of the 18:0-20:4 DAG
dependence at four different MgATP concentrations are shown. The inset shows the slopes of the primary plots as a function of
1/[MgATP]. The MgATP concentrations used were, clockwise from
top: , 70 µM;
, 100 µM;
,
150 µM;
, 200
µM.
Figure 5:
Kinetics of arachidonoyl-DAG kinase
inhibition by PIP. Panel A shows double-reciprocal
plots of the 18:0-20:4 DAG dependence at four different PIP
concentrations. Panel B shows effects of the same
concentrations of PIP
on double-reciprocal plots of the
MgATP dependence. The insets show the slopes of the primary
plots as a function of PIP
. To calculate the true K
for PIP
, the horizontal
intercept of the secondary plot for 18:0-20:4 DAG was corrected
for the concentration of MgATP used in the assays (100
µM). No correction of the MgATP intercept is required. The
PIP
concentrations used were as follows:
, 0.06 mol
%;
, 0.04 mol%;
, 0.02 mol %;
, no
PIP
.
Figure 6:
Kinetics of arachidonoyl-DAG kinase
inhibition by PA. Double-reciprocal plots of the MgATP dependence at
four different concentrations of PA are shown. The secondary plot (inset) showed a slight upward curvature. This may indicate a
small degree of cooperativity or simply be a nonspecific effect due to
formation of mixed micelles containing multiple PA molecules, which
would be expected at the higher PA concentrations used. The apparent K for PA, obtained by extrapolation, was
0.7 mol % in two separate experiments. The PA concentrations used were:
, 0.9 mol %;
, 0.6 mol %;
, 0.3 mol %;
, no
PA.
Figure 7:
Effects
of acidic phospholipids on DAG kinase isoenzymes. Panel A shows the effect of PA on the activities of arachidonoyl-DAG
kinase and five other DAG kinase isoforms. Assays were performed in the
octylglucoside assay described under ``Experimental
Procedures'' in the presence of the indicated surface
concentrations of PA. Phosphatidic acid concentrations greater than 20
mol % could not be tested because of insolubility in the assay mixture
above this concentration. Panel B shows the effect of
PIP on these same DAG kinases. In these assays, the
octylglucoside contained 10 mol % phosphatidylserine in addition to the
indicated concentrations of PIP
. The basal activities are
slightly lower with the arachidonoyl-DAG kinase and slightly higher
with the other isoforms because of the phosphatidylserine. Assays at
1.0 and 3.0 mol % PIP
in octylglucoside without
phosphatidylserine showed the same pattern of inhibition and
activation. To facilitate comparison, activities in both panels are
expressed as percentages of the maximal activity observed with that
isoform in the octylglucoside/PA assay. The DAG kinase isoforms used,
maximal activities, and mol fractions of PA at which these activities
were observed are as follows:
, arachidonoyl-DAG kinase, 33
pmol/min/µl, 0 mol %;
, 3T3 DAG kinase A, 1.9 nmol/min/mg, 7.5
mol %;
, 3T3 DAG kinase B, 1.0 nmol/min/mg, 20 mol %;
,
thymus DAG kinase A, 25 nmol/min/mg, 20 mol %;
, thymus DAG
kinase B, 12.3 nmol/min/mg, 20 mol %;
, testis cytosol DAG
kinase, 8.2 nmol/min/mg, 20 mol %.
We have shown arachidonoyl-DAG kinase to be potently and
specifically inhibited by PIP. The K
for PIP
inhibition of the enzyme was 0.04 mol %.
Assuming a Triton aggregation number of 140, this corresponds to one
PIP
molecule/20 micelles in the assay mixture. (
)As arachidonoyl-DAG kinase inhibition was dependent on the
PIP
surface concentration and independent of micelle
concentration, the mechanism must involve intramicellar binding of
PIP
to the enzyme. Rapid equilibration of the solubilized
lipids and enzyme between micelles presumably permits this low level of
PIP
to effectively inhibit the
enzyme(24, 59) . The inability of D-myo-inositol 1,4,5-trisphosphate and inositol
hexaphosphate to inhibit arachidonoyl-DAG kinase is further evidence
that PIP
must incorporate into the micelle before it can
inhibit the enzyme. The observation that PI does not inhibit
arachidonoyl-DAG kinase below 5 mol % points to the phosphomonoesters
as major determinants of the PIP
effect. This conclusion is
also supported by the potent inhibition seen with the multiply
phosphorylated analogs. These analogs were, however, clearly less
potent than PIP
itself, indicating that some structural
feature of PIP
not related to its total charge is also
important. The competitive inhibition kinetics with respect to MgATP
raise the possibility that the PIP
phosphomonoester binding
sites overlap the binding site for trisphosphate moiety of the MgATP,
although an allosteric mechanism cannot be excluded. The observations
that five other DAG kinase isoforms are not inhibited by PIP
or PA are strong additional evidence that this is a specific
property of arachidonoyl-DAG kinase and not a nonspecific effect of the
mixed micellar assay.
Acidic lipids, including polyphosphoinositides
and phosphatidic acid, have been shown to activate several membrane
associated enzymes of stimulus transduction, including PI-specific
phospholipase C-1(33) , several protein kinase C
isoforms(35, 36, 37) , phospholipase
D(38) , ADP-ribosylation factor 1(39) , and an
ADP-ribosylation factor GTPase-activating protein(40) .
Phosphatidylinositol-4-phosphate 5-kinase appears to be inhibited by
PIP
and activated by PA(41, 42) . Casein
kinase I also appears to be inhibited by PIP
(43) .
In some of these cases, as with the DAG kinases, these effects were
shown to occur only with certain isoforms of these
enzymes(37, 42) . The surface concentration of
PIP
in Triton required for half-maximal DAG kinase
inhibition, 0.04 mol %, compares well with the apparent K
of phospholipase C-
1 for PIP
in
Triton, which is 0.03-0.3 mol %, depending on the activation
state of the enzyme and presence or absence of a PA
cofactor(33, 34) . Also, activation of protein kinase
C by PIP
in Triton occurs at 0.08-0.2 mol
%(35, 36) . In other studies, the use of very
different assay methods renders comparisons with the present work
difficult. Taken together, however, these results do suggest that
signaling cascades may be regulated by the lipid microenvironment of
subcellular membranes in which they are located. Inasmuch as PIP
and PA undergo rapid flux during cell
stimulation(60, 61, 62, 63, 64, 65) ,
this hypothesis seems not unreasonable.
The stimulation of some
other DAG kinases by PIP and PA, while raising the
possibility that these enzymes may be activated by PA or
polyphosphoinositides, does not support a role for these isoforms in
regulating the level of PIP
. Stimulation of these isoforms
by phosphatides may reflect a mechanism for their recruitment to
specific intracellular membranes during cell
stimulation(66, 67) . However, detailed studies of
this phenomenon have yet to be performed.
The marked enrichment of
animal cell phosphatidylinositols in arachidonate strongly suggests a
role for arachidonoyl-DAG kinase in PI
synthesis(21, 25) . Given this, the observed
inhibition may represent a mechanism for PIP to regulate
its own synthesis. Work from several groups has shown that the
arachidonate-enriched DAG pool that arises during stimulus-induced PI
turnover is rapidly phosphorylated by a diacylglycerol kinase and that
much of this PA is ultimately converted back to
PI(21, 60, 61, 62, 63, 64, 65) .
Other DAG species are phosphorylated much more
slowly(60, 61, 62, 63, 64, 65) .
As other DAG kinases do not exhibit fatty acyl selectivity, these
results strongly suggest arachidonoyl-DAG kinase is the isoform
responsible for this DAG phosphorylation. Phosphorylation of
arachidonoyl-DAG under these conditions coincides with a transient drop
in the cellular level of
PIP
(63, 68, 69, 70) .
Regulation of arachidonoyl-DAG kinase by PIP
feedback is
thus entirely consistent with available in vivo data.
The
present work demonstrates PIP to be a potent and specific in vitro inhibitor of arachidonoyl-DAG kinase. To what extent
the mixed micellar system reflects the in vivo regulation of
this enzyme remains to be determined. Arachidonoyl-DAG kinase is an
integral membrane protein and is likely to be highly localized in the
cells in which it is found. The intramicellar mechanism of PIP
inhibition in vitro suggests an intramembranous in
vivo mechanism. Any regulation of this enzyme by PIP
would then require the phosphoinositide to be present in the same
membrane compartment as the DAG kinase. The subcellular localization of
the PI cycle, while likely to be of critical importance to its
regulation, is understood only to a limited
extent(71, 72) . Future studies of in vivo DAG kinase regulation will have to address these issues of
compartmentation and multiple enzyme isoforms. Cloning of
arachidonoyl-DAG kinase, which is in progress in our laboratory, will
facilitate new approaches to these questions. The interaction with
PIP
will be amenable to site-directed mutagenesis, and the
subcellular location of the enzyme will be open to immunocytochemical
approaches. Such studies promise a better understanding of PI-mediated
stimulus transduction in animal cells.