(Received for publication, January 17, 1996; and in revised form, February 22, 1996)
From the
Rat brain cytosol contains proteins that markedly inhibit the
activity of partially purified brain membrane phospholipase D (PLD)
stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol
4,5-bisphosphate (PIP). Sequential chromatography of the
brain cytosol yielded four inhibitor fractions, which exhibited
different kinetics to heat treatment at 70 °C. Purification of the
most heat-labile inhibitor to homogeneity yielded two preparations,
which displayed apparent molecular masses of 150 kDa and 135 kDa,
respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150-
and 135-kDa proteins yielded similar elution profiles on a C
reverse-phase column, suggesting that the 135-kDa form is a
truncated form of the 150-kDa form. Sequences of two tryptic peptides
were determined. A data base search revealed no proteins with these
sequences.
The purified 150-kDa inhibitor negated the PLD activity
stimulated by Arf, RhoA, or Cdc42. The concentration required for
half-maximal inhibition was 0.4 nM. Concentration dependence
on the 150-kDa inhibitor was not affected by changes in the
concentrations of Arf, PIP, or phosphatidylcholine used in
the assays, suggesting that the inhibition is not due to competition
with the activators or substrate for PLD. The purified inhibitor did
not affect the PIP
-hydrolyzing activity of a phospholipase
C isozyme that was measured with substrate vesicles of lipid
composition identical with that used for the PLD assay. Thus, the
mechanism of inhibition appears to be a specific allosteric
modification of PLD rather than disruption of substrate vesicles.
Phospholipase D (PLD) ()catalyzes the hydrolysis of
phospholipids at their terminal phosphodiester bond, thus producing
phosphatidic acid (PA) and releasing the free polar head group.
Stimulation of PLD occurs in a wide variety of cells treated with
hormones and growth
factors(1, 2, 3, 4, 5, 6) .
PA has been implicated as a biologically active molecule and can be
further metabolized by a PA phosphohydrolase to form diacylglycerol, a
protein kinase C activator (3, 4, 7, 8, 9, 10) .
Most mammalian PLD activity is found in association with membranes and
appears to be specific for phosphatidylcholine (PC) (11, 12, 13, 14, 15) .
There is compelling evidence for the existence of multiple isoforms of
PLD in mammalian cells, and PLD is known to be activated via multiple
pathways involving G proteins, Ca
, unsaturated fatty
acids, protein kinase C, or protein-tyrosine kinases (1, 2, 3, 4, 9, 16, 17, 18, 19, 20, 21, 22) .
However, there is little information on the biochemical and molecular
properties of mammalian PLDs.
Significant progress has been made
recently on the G protein regulation of PLD. Members of the Arf family
of the small G protein (Smg) superfamily were recognized as activators
of PLD, and the presence of PIP in substrate vesicles was
shown to be essential for the detection of Arf-regulated PLD
activity(12, 16, 23, 24, 25) .
Subsequently, a rat brain PLD that is activated by Arf and PIP
but inhibited by oleate was separated from another form of rat
brain that is completely dependent on oleate for activity but
insensitive to Arf and PIP
(17) . Members (RhoA,
Cdc42, and Rac) of another Smg family, Rho, were also found to activate
PLD from several tissues. Membrane PLDs in HL60 cell and porcine brain
were shown to be synergistically activated by Arf and
RhoA(26, 27) , whereas rat liver PLD was activated by
RhoA but not by Arf(28) . Thus, whether the target for Arf and
Rho proteins is identical or not is not clear. Recently, a group led by
Drs. Morris and Frohman (29) cloned a human cDNA corresponding
to the PLD (PLD-1) that is activated by Arf and PIP
but
inhibited by oleate and identified at least two more PLD genes. The
same authors reported that the brain PLD-1 could also be activated by
members of the Rho family.
Rho-activated PLD is also negatively
modulated by Rho GDP dissociation inhibitor (Rho-GDI) which blocks
GDP/GTP exchange on certain Smg
proteins(26, 27, 30, 31) . The
present studies were undertaken to investigate the possibility that
proteins other than Rho-GDI negatively modulate Smg-dependent PLD
activity. We find that rat brain cytosol contains multiple proteins
that can inhibit PLD activity stimulated by Arf proteins. We purified
one of these inhibitors to homogeneity and studied its effects at
various concentrations of Arf, PIP, and PC.
Figure 1: Purification of PLD-inhibiting proteins from rat brain cytosol. The 0-35% ammonium sulfate precipitate from the cytosol of 400 g of rat brains was subjected to sequential chromatography on a DEAE-Sephacel column (A), a preparative HPLC DEAE-5PW column (B), a preparative HPLC Phenyl-5PW column (C), and HPLC Heparin-5PW columns (D and E). Detailed procedures are described under ``Experimental Procedures.'' Bars above the elution profiles indicate those inhibitor fractions that were pooled for subsequent purification or characterization. Fractions thus pooled are hereafter collectively referred to as a ``peak fraction.'' Peak fractions I, II, and III refer respectively to fractions 73-77, 82-88, and 94-98 of A. Peak fraction IA (fractions 27-34) from the HPLC Heparin-5PW column was subjected to SDS-PAGE on an 8% gel, and proteins were visualized by silver staining (F).
Figure 3:
Gel filtration chromatography of two post
Heparin-5PW column fractions of inhibitor IA. A, Fraction 29
containing 170 µg of protein in 1 ml (upper panel) and
fraction 32 containing 230 µg of protein in 1 ml (lower
panel), both from Fig. 1D, were concentrated to 30
µl on a Microcon-30 (Amicon) and applied to a Superose 12 PC 3.2/30
column (3.2 300 mm, Pharmacia). This sizing chromatography was
performed on a Pharmacia-LKB SMART system equipped with a µ
separation unit and a µ precision pump. The column was eluted at a
flow rate of 40 µl/min with 20 mM Hepes (pH 7.5)
containing 150 mM NaCl. Fractions of 40 µl were collected
and assayed for PLD-inhibiting activity as described under
``Experimental Procedures.'' The elution positions of marker
proteins, thyroglobulin (670 kDa), bovine
-globulin (158 kDa), and
chicken ovalbumin (44 kDa) are indicated. B, proteins of each
fraction from the sizing chromatography shown in A were
separated on 8% SDS-polyacrylamide gels and stained with Coomassie
Brilliant Blue. Upper and lower panels represent
proteins originating from the post Heparin-5PW column fractions 29 and
32, respectively. The gel filtration column fraction numbers are
indicated on the top of the gels, and the positions of
molecular size standards (kDa) are shown at right.
The above purification procedure starting with the 0-35% ammonium sulfate precipitate was repeated three times to accumulate enough purified inhibitor IA for characterization.
In an effort to identify any physical property
that distinguishes one inhibitory activity from the others, the
kinetics of heat inactivation of the three fractions was evaluated.
Upon incubation at 70 °C, peak I lost 50% of its inhibiting
activity in 3 min but retained
40% of its activity after 30 min.
However, the activities of peaks II and III increased slightly and
remained at that level during the incubation period (Fig. 2A). At 85 °C, the three inhibitor fractions
lost their activities completely within 30 min but with different
kinetics. Whereas peaks II and III showed slow, monophasic decreases in
activity, peak I displayed a biphasic time course with a rapid
inactivation phase followed by a slow one (data not shown). These
results suggested that the inhibitor in peak I is most likely distinct
from those in peaks II and III.
Figure 2:
Effect of heat treatment on PLD-inhibiting
activity. Peak fractions from various chromatography steps were each
diluted 5-fold with Buffer H and incubated at 70 °C. Aliquots of
the mixture were assayed at the indicated times for PLD-inhibiting
activity as described under ``Experimental Procedures.'' A, peak fractions I (-
), II
(
-
), and III (
-
)
from the DEAE-Sephacel column (Fig. 1A). B,
peak fraction I from the DEAE-5PW column (Fig. 1B)
(
-
), peak fraction IA from the Heparin-5PW
column (Fig. 1D) (
-
), and
peak fraction IB from the Heparin-5PW column (Fig. 1E)
(
-
).
Sequential fractionation of peak I
proteins on a preparative DEAE-5PW HPLC column (Fig. 1B) and a Phenyl-5PW column (Fig. 1C) yielded two inhibitory peaks, IA and IB. Both
peak IA and IB proteins were chromatographed separately under identical
conditions on a Heparin-5PW column. IA activity eluted as a single peak
centered at fraction 30 (Fig. 1D), whereas IB activity
eluted in a main peak centered at fraction 26 and two minor peaks (Fig. 1E). The kinetics of heat inactivation was
followed at 70 °C using peak fraction 30 (IA) and peak fraction 26
(IB) from the Heparin-5PW column steps (Fig. 2B). More
than 95% of IA activity was abolished within 1 min. Under the same
conditions, IB lost rapidly only 30% of its activity and retained
65% of its activity after 30 min. The peak fraction from the
DEAE-5PW step (mixture of IA and IB) lost
50% of its activity in
the rapid phase and retained
35% after 30 min in a fashion similar
to the result shown in Fig. 2A with the
post-DEAE-Sephacel fraction I. These results suggest that the rapid
phase in the inactivation of the post-DEAE Sephacel fraction I was due
largely to the inactivation of inhibitor IA. Whether the biphasic time
course of IB is an intrinsic property of a single inhibitor or reflects
yet another mixture of inhibitors with different heat stabilities
awaits further purification of IB.
Further purification of the heat-stable peak II and III inhibitors from the DEAE-Sephacel column is underway but has not progressed enough to be reported here. Nevertheless, the two peak activities remained heat-stable through several chromatography steps (data not shown).
Two post-Heparin-5PW column fractions, fractions 29 and 32 in Fig. 1D, were subjected to gel filtration chromatography (Fig. 3A). Fraction 29 yielded two protein peaks, one small and one large, centered at fractions with an apparent molecular mass of 300 kDa and 140 kDa, respectively. In contrast, fraction 32 yielded a large peak at the fraction corresponding to 300 kDa followed by a shoulder. For both chromatographies, the PLD-inhibiting activity profiles were parallel to protein elution profiles. SDS-PAGE analysis of the fractions across the gel filtration peaks revealed that the early peak is due to a 150-kDa protein and the late peak (or shoulder) to a mixture of 135- and 120-kDa proteins (Fig. 3B). These results clearly indicated that the inhibitory activity is associated with both the 150-kDa protein and the mixture of 135- and 120-kDa proteins. It was also apparent that the 150-kDa protein exists in a dimeric form, whereas the 135- and 120-kDa proteins exist in monomeric forms. Thus, the segment cleaved by proteolysis might be involved in dimerization.
In another preparation that proceeded swiftly, we resolved an
inhibitor IA preparation into a fraction containing nearly homogeneous
150-kDa protein (>95%) and a fraction containing largely 135-kDa
protein (90%) with a small amount of 120-kDa protein (
10%) (Fig. 4A). These two preparations were digested
separately with trypsin and subjected to a C
column. The
elution profiles were similar as shown in Fig. 4B, in
agreement with the notion that the 135-kDa protein is a truncated
fragment of the 150-kDa protein. At the present time, we do not know
whether the 135-kDa form exists in cells or is an artifact of the
purification procedure. However, the 120 kDa proteins(s) appeared to be
generated during purification.
Figure 4:
SDS-PAGE of the 150- and 135-kDa forms of
inhibitor IA and HPLC of their tryptic peptides. A, the entire
purification procedure for inhibitor IA was carried out rapidly, and
gel filtration chromatography yielded preparations of 150 kDa (left
lane) and a 135-kDa protein (right lane), which were
subjected to SDS-PAGE on 8% gels. The positions of molecular size
standards (kDa) are shown at right. B, HPLC of
tryptic peptides derived from the 150-kDa (upper panel) and
135-kDa (lower panel) inhibitor IA. Proteins (20 µg each)
from the same preparations shown in A were denatured
reductively by treatment with 50 mM Tris (pH 8.0), 6 M guanidine hydrochloride, and 2 mM DTT. The sulfhydryl
groups were labeled with 2-nitro-5-thiobenzoate by adding
Ellman's reagent, 5,5`-dithiobis-(2-nitrobenzoic acid), to a
final concentration of 10 mM. The
2-nitro-5-thiobenzoate-conjugated proteins were precipitated with 10%
trichloroacetic acid, and the pellet was washed with acetone, suspended
in 50 mM Tris (pH 8.0), and digested with trypsin overnight at
37 °C. The trypsin-digested peptides were applied to a C column (4.6
250 mm) that had been equilibrated with 0.1%
trifluoroacetic acid. The elution was performed at a flow rate of 1
ml/min with a 60-ml linear gradient of 0 to 60% acetonitrile in 0.1%
trifluoroacetic acid. Peptides were detected by measuring absorbance at
215 nm. The sequences of the two indicated peptides were demonstrated
to be EANAPAFD (
) and GSVPLFWE
(
).
The sequences of two peptides through the first eight residues were determined to be EANAPAFD and GSVPLFWE. A search of the data base did not reveal any proteins with these sequences. Upon incubation at 70 °C, both the 150- and 135-kDa proteins rapidly lost their inhibitory activity as did the mixture of both proteins as shown in Fig. 2B.
Figure 5: Effect of 150-kDa inhibitor IA on PLD activity stimulated by Arf, Cdc42, or RhoA. PLD activity stimulated by 10 nM Arf, 10 nM Cdc42, or 150 nM RhoA was measured in the absence (solid bars) or the presence (open bars) of 0.5 nM inhibitor IA. PLD activity was measured as described under ``Experimental Procedures'' for Arf-stimulated PLD.
The concentration-dependent inhibition of Arf-stimulated PLD
activity by the 150-kDa inhibitor IA was studied at 3 different
concentrations of Arf (Fig. 6A). Half-maximal
inhibition was observed at 0.4 nM inhibitor IA (calculated
based on monomeric molecular mass) in the presence of 100 nM Arf, and the inhibition profile was nearly unchanged when the Arf
concentration was reduced by 20-fold. The inhibitor concentration
dependence was also measured with respect to PIP and PC (Fig. 6, B and C). Neither PIP
nor
PC concentration variance affected the profiles of inhibitor IA
concentration dependence.
Figure 6:
Effect of changes in the concentration of
Arf, PIP, or PC on PLD inhibition by the 150-kDa inhibitor
IA. Concentration dependence of PLD inhibition by the 150-kDa inhibitor
IA was measured in the presence of three different concentrations of
Arf (A) and four different concentrations of PIP
(B) or PC (C). PLD activity profiles were
determined as described under ``Experimental Procedures'' for
the case of fixed concentrations of Arf, PIP
, or
PC.
Frequently, inhibitors identified for the enzymes acting on
lipid substrates are hydrophobic molecules that interact
nonspecifically with lipid vesicles. However, the inhibitory action of
inhibitor IA is unlikely to be due to a hydrophobic interaction because
the inhibitor is a cytosolic protein that elutes in very early
fractions from a hydrophobic column (Fig. 1C). Another
possible interaction target for the inhibitor is PIP, which
is a cofactor for the PLD reaction. A number of cytosolic proteins are
known to bind PIP
tightly and inhibit the hydrolysis of
PIP
by phospholipase C (PLC)(33) . However, the
concentrations (up to 5 nM) of inhibitor IA used in this study
are too low to cause inhibition directly by binding PIP
and
do not interfere with the presumed binding of PIP
to PLD (Fig. 6B). Furthermore, when the effect of inhibitor IA
was studied with a phospholipase C (PLC) isoform, PLC-
1, using
substrate vesicles of lipid composition identical to that used for the
PLD assay, PLC activity was not affected by inhibitor IA (data not
shown).
A growing body of evidence suggests the existence of
multiple PLD isoforms, and the PLD preparation used in this study is
likely to contain more than one isoform of
PLD(25, 26, 29) . The effect of Arf and RhoA
on rat brain PLD was shown to be additive. However, it is not clear
whether the additivity is due to the existence of separate PLD enzymes
or separate interaction sites for Arf and RhoA on the same PLD. In the
former case, inhibitor IA inhibits both Arf- and RhoA-dependent
enzymes. In the latter case, inhibitor IA reduces the activity of a
specific PLD independently of the activator. Neither mechanism
eliminates the possibility that inhibitor IA inhibits basal activity in
the absence of activators like PIP and Smg proteins.
However, we could not evaluate the effect of inhibitor 1A on the basal
activity, because the basal activity of our partially purified PLD
preparation is too low to be measured quantitatively.
The
concentration of inhibitor IA required for half-maximal inhibition is
0.4 nM, while the cellular concentration of inhibitor IA
appears to be much higher than 0.4 nM, as homogeneous
preparation of inhibitor IA from cytosol required only several
thousandfold purification according to rough estimates. Furthermore,
the interaction of inhibitor IA with PLD is not affected by the
presence of activated (GTPS-bound) Arf. Therefore, an increase in
the concentration of a GTP-bound Smg protein in response to
extracellular signals is not likely to be sufficient to relieve the
inhibition. A specific mechanism by which receptor signaling can
modulate the interaction between the inhibitor and PLD might be
involved. Modulation by phosphorylation is one possibility. Considering
that inhibitor 1A is extremely sensitive to proteolysis, degradation of
the inhibitor in response to a PLD-activating signal is also a
possibility.
While our work was in progress, Geny et al. (34) reported the identification of a bovine brain cytosolic protein that inhibits Arf-dependent PLD activity associated with HL-60 cell membrane. This inhibitory protein was eluted from a Superose 12 column in the void volume which corresponds to an apparent molecular mass larger than 300 kDa. The partially purified bovine brain inhibitor was heat-stable. Thus, inhibitor IA is unlikely to be a rat homolog of the heat-stable bovine brain inhibitor. Preliminary results from gel filtration chromatography of the peak II inhibitor from the DEAE-Sephacel column (Fig. 1A) indicate that the apparent size of inhibitor II is much larger than that (300 kDa) of untruncated IA. The peak II inhibitor, which was also heat-stable, is a better candidate for a rat homolog of the bovine brain inhibitor.