(Received for publication, July 21, 1994; and in revised form, December 19, 1994)
From the
Phospholipase D (PLD) activation by guanine nucleotides requires
protein cofactors in both the plasma membrane and the cytosol. HL-60
cytosol was fractionated by ammonium sulfate and gel-permeation
chromatography. Two cytosolic protein fractions were found to
reconstitute the GTPS (guanosine
5`-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution
assay consisting of
H-labeled HL-60 membranes and eluted
column fractions. The major peak of reconstituting activity was in the
region of 50 kDa, and a second discrete peak of PLD reconstitution
activity was observed in the region of 18 kDa. Rho GDP/GTP exchange
inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2
were entirely complexed with Rho GDI and eluted with an apparent
molecular mass of 43 kDa by gel filtration chromatography. The partial
overlap between cytosolic Rac2 and RhoA with the
50-kDa peak of reconstituting activity was not consistent with the
participation of cytosolic Rho-related GTPases in the
activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family
of small GTP-binding proteins, reduced GTP
S-stimulated PLD
activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac,
could be partially separated from the PLD-stimulating factor(s) by
gel-permeation chromatography. Moreover, recombinant Smg GDS
failed to stimulate GTP-dependent PLD activity. Cytosolic
ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa
peak of reconstitution activity. Faint amounts of membrane-bound ARF
were also detected using the monoclonal antibody 1D9. The effects of
the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD
activity were synergistic. The weak stimulatory effect of ARF alone
suggested that the GTP
S-stimulated PLD activity is dependent on
the presence of another protein(s), presumably ARF-regulatory proteins.
We propose that a membrane-bound GTP-binding protein, possibly ARF, may
be involved in the activation of PLD when combined with the
component(s) of the 50-kDa fraction.
Phosphatidylcholine hydrolysis by a type D phospholipase (PLD) ()generates two proximal second messengers, phosphatidic
acid and diacylglycerol, which in turn control the functional responses
of various cell types(1) . Phosphatidic acid may have a
messenger role in regulating stimulus-secretion coupling and in
activating the respiratory burst in
granulocytes(2, 3, 4, 5, 6) .
So far, PLD activation has been demonstrated in neutrophils and HL-60
cells upon stimulation with N-formylated peptides, C5a,
platelet-activating factor, and leukotriene
B
(7, 8, 9, 10) . The
recent cloning of chemotactic and chemokine receptors revealed that
these proteins belong to the family of seven transmembrane G
protein-coupled receptors(11) . These receptors appear to be
coupled to a pertussis toxin-sensitive G protein in granulocytes.
ADP-ribosylation of the G
class of G protein by pertussis
toxin abrogates a variety of biochemical responses, including PLD
activity in fMLP-stimulated neutrophils(12, 13) .
Additional support for the existence of GTP-binding proteins in the
regulation of PLD activity comes from the observation that, in
permeabilized cells as well as in cell-free systems, PLD can be
stimulated by GTPS(14, 15, 16) . In
streptolysin O-permeabilized cells, GTP
S stimulates PLD
independently of phospholipase C(14) . Activation of PLD
involves the interactions of several neutrophil components, some
located in the plasma membrane and other(s) in the
cytosol(17) . A PLD-associated stimulatory GTP-binding protein
has been reported to reside in the plasma membrane(18) . More
recently, several laboratories reported the activation of PLD by a
small GTP-binding protein isolated from brain
cytosols(19, 20, 21) . Evidence for the
involvement of a small GTP-binding protein regulating PLD activity
includes the following observations: (i) in permeabilized cells or
cell-free systems, PLD was not stimulated by fluoroaluminate, an
activator of heterotrimeric but not of small, G proteins; (ii) Bowman et al.(18) reported that PLD activity is stimulated
by Smg GDS, a GDP/GTP dissociation stimulator, and inhibited
by Rho GDI, a GDP/GTP dissociation inhibitor; (iii)
ARF proteins can reconstitute the GTP
S-dependent stimulation when
combined with an enriched preparation of PLD (20) or with
permeabilized HL-60 cells previously depleted of their cytosolic
content(21) .
The GDP/GTP exchange proteins Rho GDI
and Smg GDS are not totally specific and appear to be capable
of regulating multiple small GTP-binding proteins from different
families. Smg GDS has been found to be active on
p21,
p21
/Rap1/Krev1,
p21
, and p21
. Rho GDI regulates members of the Rho family of GTP-binding
proteins, including p21
,
p21
, and CDC42Hs(22, 23) . In
light of the foregoing observations, we investigated the possibility
that several small GTPases might control PLD activity in
Me
SO-differentiated HL-60. This report provides evidence
that cytosolic ARF, but not cytosolic Rho-related proteins,
regulates PLD in HL-60 granulocytes. The results also indicate that
activation of PLD by GTP
S is dependent on the presence of a 50-kDa
cytosolic factor.
The lipid samples were dried and spotted on Silica gel 60 plates. Plates were developed using a solvent system consisting of chloroform/methanol/acetic acid (65:15:2, by volume) for separation of PEt. Lipids were located by staining with Coomassie Blue(28) , and areas of the silica plate containing appropriate lipids were scraped off and quantitated by liquid scintillation counting. The results were corrected for quenching and recovery. Unless otherwise specified, data are presented in the text as the mean ± S.E. of a minimum of three separate experiments.
The
reconstituting PLD activity was recovered in the proteins precipitated
from 35 to 75% ammonium sulfate saturation. Then 350 µl (3-5
mg of protein) of the 35-75% ammonium sulfate fraction was loaded
onto a TSK 2000 column (7.5 mm, inner diameter, 60 cm, Beckman)
equilibrated in Pipes buffer. Proteins were eluted at a flow rate of
0.5 ml/min. The molecular size of the eluted proteins was determined by
reference to standard proteins of known molecular weight and blue
dextran. Fractions of 0.5 ml were collected, and the PLD-reconstituting
activity of each fraction (50-100 µl) was determined in the
presence of
H-labeled membranes as described above.
Figure 1:
Inhibition of
GTPS-stimulated PLD activity by Rho GDI. HL-60 cell
postnuclear fractions (500 µl) in incubation buffer containing 8
mM MgCl
and 1 µM CaCl
were incubated in the absence or presence of 0.6 µM,
1.2 µM, 3.0 µM, and 6 µMrho GDI for 30 min at 4 °C. Samples were then incubated with (opencircles) or without (filledcircles) 20 µM GTP
S in the presence of
1.4% ethanol for 20 min at 37 °C. Reactions were stopped and
samples processed for [
H]alkyl-PEt measurement as
described under ``Experimental Procedures.'' Data are the
mean ± S.E. of three independent experiments. Where absent,
error bars are smaller than the symbol.
PLD was marginally stimulated by physiological concentrations of GTP (Fig. 2). Smg GDS, which stimulates GDP/GTP exchange on
multiple small GTPases, would be expected to enhance GTP-stimulated PLD
activation if nucleotide exchange is rate-limiting. Fig. 2shows
that the addition of Smg GDS to HL-60 postnuclear homogenates
failed to enhance significantly the levels of PEt accumulation elicited
by GTP. It is noteworthy that GTPS-stimulated PLD activity was
similarly unaffected by the stimulatory exchange factor (not
illustrated), indicating that nucleotide exchange was not limiting
under these conditions. The effect of maximally stimulatory
concentrations of GTP
S was considerably greater than that elicited
by 200 µM GTP (Fig. 3). As expected, the
stimulatory effects of maximal doses of GTP
S (10 µM)
was reversed by GTP, which, unlike GTP
S, is not resistant to
hydrolysis by GTP-binding proteins. At 200 µM, GTP was
found to reduce the rate of PEt synthesis elicited by GTP
S by 87.5
± 2.6% (Fig. 3). Because HL-60 cell membranes have been
reported to possess high affinity GTPase activities, it was conceivable
that introduction of GTP to postnuclear homogenates resulted in the
rapid hydrolysis of GTP to GDP. GDP may then be responsible for the
observed inhibitory effect. If GTP hydrolysis to GDP by GTPases
precedes and is causally related to inhibition of PLD, the response to
GTP
S should be less (or at best equally) sensitive to inhibition
by GTP than by GDP. A detailed concentration dependence of the effects
of GTP and GDP is illustrated in Fig. 4. The response is
expressed as a percentage of the maximal response obtained with 10
µM GTP
S for comparison. PEt accumulation in response
to GTP
S became progressively smaller as the concentration of GDP
or GTP was increased. GTP was more efficient than GDP at inhibiting
GTP
S-stimulated PLD activity, with half-maximal effects obtained
at 30 and 60 µM, respectively. The difference between the
effects of the two guanine nucleotides was maximal at 30 and 100
µM. Although statistically insignificant, a small
accumulation of PEt was, however, observed in response to 200
µM GTP (Fig. 2). Furthermore, the order of potency
GTP > GDP implies that rapid hydrolysis of GTP to GDP by
miscellaneous GTPases is unlikely to be the dominant process in the
inhibition of the response to GTP
S, suggesting that the
interaction of GTP and GTP
S is competitive.
Figure 2:
Effects of Smg GDS on
GTP-stimulated PLD activity. PLD was assayed in incubation buffer
containing 8 mM MgCl, 1 µM CaCl
, and 1.4% ethanol. Reactions were initiated by
the addition of 200 µM GTP (hatched bars) or
vehicle alone (filled bars) with the indicated concentrations
of Smg GDS. Following incubation at 37 °C for 20 min,
reactions were stopped, and [
H]alkyl-PEt was
quantitated as described under ``Experimental Procedures.''
Data are the mean ± S.E. of three independent
experiments.
Figure 3:
Inhibition of GTPS-stimulated PLD
activity by GTP. Aliquots (500 µl) of HL-60 postnuclear homogenates
were incubated for 20 min at 37 °C with GTP
S (10
µM) or GTP (200 µM) alone or in combination.
PLD activity was quantitated as described under ``Experimental
Procedures'' and [
H]alkyl-PEt formation
expressed as a percentage of total lipid-associated radioactivity. Data
are the mean ± S.E. of three independent
experiments.
Figure 4:
Concentration dependence of the effects of
GTP and GDP on GTPS-stimulated PLD activity. HL-60 cell
postnuclear fractions (500 µl) were stimulated with 10 µM GTP
S in the absence or the presence of the indicated
concentrations of GTP or GDP. Following incubation at 37 °C for 20
min in the presence of 1.4% ethanol, samples were assayed for
[
H]alkyl-PEt formation as described under
``Experimental Procedures.'' Data are the mean ± S.E.
of six independent experiments. *, p < 0.05, for values
compared to the adequate controls using a Student's paired t-test.
Figure 5:
Restoration of GTPS-stimulated PLD
activity by cytosolic proteins precipitated from 35 to 75% ammonium
sulfate saturation. Membranes, cytosols, 0-35% and 35-75%
ammonium sulfate fractions were prepared as described under
``Experimental Procedures.'' Freshly isolated
[
H]alkyl-phosphatidylcholine-labeled membranes (8
10
cell eq) alone or in combination with cytosol
(200 µg); 0-35% ammonium sulfate fraction (200 µg) and
35-75% ammonium sulfate fraction (200 µg) in 0.5 ml of
incubation buffer containing 8 mM MgCl
, 1
µM CaCl
, and 1.4% ethanol were stimulated with
20 µM GTP
S. After 20 min at 37 °C, the reactions
were stopped, and [
H]alkyl-PEt formation was
quantitated as described under ``Experimental Procedures.''
Data are the mean ± S.E. of six independent
experiments.
Figure 6:
Gel-permeation chromatography of
35-75% ammonium sulfate protein fraction. Proteins precipitated
from 35-75% ammonium sulfate saturation were loaded on a TSK 2000
SW column. Aliquots (50-100 µl) of eluted fractions were
combined with H-labeled membranes, and
[
H]alkyl-PEt formation was monitored as described
in Fig. 6. Results are representative of data obtained in four
separate experiments under identical
conditions.
Figure 7: Immunoblot analysis of nucleotide exchange factor and small GTP-binding proteins. Aliquots (80 µl) of column fractions were processed for SDS-PAGE/Western blotting and transferred to Immobilon PVDF membranes as described under ``Experimental Procedures.'' Membranes were exposed to anti-Smg GDS, anti-Rho GDI, anti-ARF (1D9), anti-H/N-Ras (142-24E5), and anti-Rap1 antibodies for immunoblot analysis. Data presented are representative of results obtained in four separate experiments with similar results.
Figure 8: Identification of nucleotide exchange factors and ARF proteins in eluted column fractions: comparison to PLD activity. Data presented in Fig. 7were plotted to enable comparison between the presence of Smg GDS, Rho GDI, ARF, and PLD activity. The amounts of Smg GDS, Rho GDI, and ARF in column fractions were determined by Western blot analysis in experiments like those in Fig. 7and scanning densitometry as described under ``Experimental Procedures.'' Data were normalized to the fraction with the highest integrated optical density value. One of four similar experiments is shown.
To characterize further the component(s) in the 50-kDa
fractions derived from granulocyte cytosols, the distribution of
GTP-binding proteins, Rho GDI, and Smg GDS were
examined in the eluted column fractions by immunoblot analysis. The
localization of Rho GDI was assessed using a GDI-specific
antibody. The same strips were also probed with mAb 142-24E5, which
recognizes a neutrophil Ras-related protein, Rap1,
and a subtrate for botulinum toxin D (31, 32) . As
shown in Fig. 7, column fractions 31-34 contain a 27-kDa
band recognized by the antibody against Rho GDI on Western
blots. Monoclonal antibody 142-42E5 revealed an intense band at about
24 kDa in fraction 32. When films were overexposed, small amounts of
antigenic material were also detected in fractions 31 and 33,
respectively. This 24-kDa protein was not recognized by a monoclonal
antibody against p21, Y13-259 (not illustrated), or
a rabbit antiserum raised against human Rap1 (Fig. 7).
The first peak of PLD reconstitution (fractions 29-34) was found
to overlap the fractions containing Rho GDI and the antigenic
material recognized by mAb 142-24E5.
Since a correlation between the stimulation of PLD and the activation of a Rho-related small GTP-binding protein has been drawn(18) , the presence of the these small GTPases was investigated by immunoblotting. As illustrated in Fig. 9, Rac2 copurified in parallel with RhoA and Rho GDI with an apparent molecular mass of 45-43 kDa. As estimated by Western blotting, Rac2 and RhoA were entirely complexed to Rho GDI. In contrast, Rac1 eluted by gel filtration between Rac2/RhoA complexes and ARF proteins in the 24-kDa region. Additionally, Rac1 was predominantly recovered in a region devoid of PLD reconstitution activity ( Fig. 7and Fig. 8). Although the presence of RhoA and Rac2 was clearly detected in the first peak of reconstituting activity, PLD activation is unlikely to be due to RhoA/Rac2 GDI, inasmuch as these complexes were not detectable in the reconstitutively active fractions 29 and 30 ( Fig. 7and Fig. 8).
Figure 9: Distribution of Rho-related small GTP-binding proteins. Aliquots (80 µl) of column fractions were processed for SDS-PAGE/Western blotting and transferred to Immobilon PVDF membranes as described under ``Experimental Procedures.'' Membranes were exposed to anti-Rho GDI, anti-ARF (1D9), anti-RhoA, anti-Rac2, and anti-Rac1 antibodies for immunoblot analysis. These results are from a single experiment representative of three others performed with identical results. Data presented in Fig. 7and Fig. 9are from two independent experiments.
The presence of Smg GDS
in the reconstitutively active PLD fraction was examined using a Smg GDS-specific antibody. As shown in Fig. 7, the
resolved Smg GDS was essentially found in column fractions
28-30 but was not recovered in column fractions 32-34,
which contained most of the PLD-stimulating activity. Conversely,
fractions 28 and 29 contained substantial amounts of Smg GDS
but no or little PLD-stimulating activity ( Fig. 7and Fig. 8), excluding Smg GDS as the reconstituting factor
for GTPS-stimulated PLD activity.
Figure 10:
Effects of PLD-inducing factors on a
solubilized PLD activity. PLD was extracted from HL-60 membranes as
described under ``Experimental Procedures.'' The solubilized
PLD activity (75 µg) was mixed in 0.5 ml of incubation buffer with
phospholipid micelles composed of a lipid extract from
[H]alkyl-phosphatidylcholine-labeled cells
(200,000 cpm/assay). The samples were stimulated with 20 µM GTP
S for 60 min at 37 °C in the presence of 1.4% ethanol.
Where indicated aliquots (100 µl) of the 50-kDa or the 18-kDa peak
of PLD-inducing activity were added alone or in combination. Data are
the mean ± S.E. of four independent experiments. *, p < 0.05, for values compared to the adequate controls using a
Student's paired t-test.
The present study demonstrates the presence in HL-60 cytosols
of protein factors that will reconstitute GTPS-stimulated PLD
activity in a reconstitution assay consisting of previously labeled
HL-60 membranes. The major peak of PLD-reconstituting activity was
recovered in fractions with an apparent molecular mass of 50 kDa. The
50-kDa cytosolic component is antigenically distinct from the 18-kDa
ADP-ribosylation factor, a small GTP-dependent regulatory protein
previously found to be an activator of PLD. The component of this
fraction is able to support a strong accumulation of PEt and is
suggested to be another cytosolic regulatory element of PLD activity or
perhaps other proteins with which ARF interacts.
In recent years,
efforts to elucidate the biochemical and molecular mechanisms of PLD
activation have focused on Ras-related small GTPases with molecular
masses between 18 and 30 kDa and on larger, heterotrimeric, G proteins
that have been linked to receptor-mediated signal transduction. The
most definitive evidence for a functional role of G proteins comes from
studies in permeabilized HL-60 cells or cell-free systems prepared from
human granulocytes. A GTPS-dependent PLD activity can be measured
in postnuclear fractions obtained from HL-60 cells (33) or
human neutrophils(18) . Cytosols and membranes isolated from
HL-60 postnuclear fractions do not support PLD activation when assayed
separately. However, a GTP
S-dependent PLD activity is observed in
combined fractions of membranes and cytosols of HL-60 cells. The
observation that the cytosolic reconstituting activity precipitated
between 35 and 75% saturation of ammonium sulfate further emphasizes
the central and critical role of cytosolic cofactor(s) in PLD
activation.
Cytosols from bovine and rat brains were found to
provide an essential factor for the GTPS-dependent stimulation of
an enriched preparation of PLD and of PLD activity in HL-60 cells
depleted of their cytosol by permeabilization, respectively. This
factor was purified to homogeneity and identified as a member of the
ARF subfamily of small GTPases(20, 21) . It is
noteworthy that the presence of the 50-kDa peak of reconstituting
activity was not detected after fractionation of rat brain cytosol by
amonium sulfate precipitation, followed by heparin-agarose and gel
filtration chromatography with Pipes buffer. However, using conditions
comparable to those reported by Geny et al.(19) , we
observed that the marked instability of the 50-kDa cytosolic
protein(s), especially in Pipes buffer, was an obstacle to further
purification. Moreover, the absence of this PLD reconstitution activity
in brain tissues cannot be totally excluded. Using an autologous
reconstitution assay, Bowman et al.(18) reported the
presence in neutrophil cytosols of a protein factor essential for the
GTP
S-stimulated PLD activity. However, in contrast to other
studies(20, 21) , this group reported the
participation of a membrane-associated low molecular weight GTP-binding
protein, presumably a member of the Rho subfamily of G proteins, in PLD
activation.
The results obtained in this study demonstrate that the
inhibitory GDP/GTP exchange factor Rho GDI prevents the
activation of PLD by GTPS in HL-60 postnuclear fractions.
Immunoblots probed with a rabbit anti-human Rho GDI
demonstrate that GDI is detectable and is specifically localized in the
50-kDa peak of PLD reconstituting activity. The presence of Rho GDI in fractions that contained most of the
PLD-reconstituting activity suggests that PLD activity may be
underestimated in these fractions. GDI eluted by gel filtration with an
apparent molecular mass of 45-30 kDa, although the calculated
molecular mass of Rho GDI is 26.5 kDa. Copurification of Rho GDI with the Rho-related GTP-binding proteins has been
reported in phagocytic granulocytes(34, 35) . Indeed,
our experiments revealed the presence of Rac2 and RhoA in column fractions containing Rho GDI. Both
GDP- and GTP-bound forms of small GTPases can form stable complexes
with Rho GDI (24, 34) . The association of Rho-related GTP-binding proteins with GDI appears to account
for the maintenance of these proteins in the cytosolic compartment,
thus preventing interactions with their effector
proteins(34, 36) . Rho GDI has been reported
to inhibit several cell functional responses, including activation of
the NADPH oxidase in neutrophils (37) and organization of
polymerized actin in Swiss 3T3 cells(38, 39) .
Among the small GTP-binding proteins, p21 and
p21
are both common substrates of Rho GDI and
the stimulatory GDP/GTP exchange factor, Smg
GDS(22, 23) . It is noteworthy that Smg GDS
is not able to enhance significantly the GTP(
S)-dependent PLD
activity in HL-60 postnuclear fractions. The results are not consistent
with the recent report concerning the ability of Smg GDS to
stimulate the activity of PLD in neutrophils(18) . The reason
for this discrepancy remains unclear, but a possible explanation would
be that GTP hydrolysis rather than GDP/GTP exchange is the
rate-limiting step in HL-60 cells. Consistent with this idea, the rate
of PEt accumulation can be significantly stimulated by GTP
S but
not by GTP. Moreover, this study demonstrates a marked decrease in
GTP
S-stimulated PLD activity when cells are incubated in the
presence of GTP. Such inhibition is expected, provided GTP and
GTP
S compete at the GTP-binding site of small GTPases for the
formation of activated GTP(
S)-bound GTP-binding proteins. Due to
its nonhydrolyzable nature, the binding of GTP
S to a GTP-binding
protein would serve to maintain it in its activated state. Cytosolic RhoA/Rac2 GDI complexes are unable to account for the
activation of PLD, inasmuch as neither GTP-binding proteins nor GDI
could be detected in several reconstitutively active fractions.
However, our study does not address the possible role of
membrane-associated Rho-related proteins in PLD activation. RhoA, Rac1, and ARF were all present in HL-60
membranes (data not shown). It is therefore possible that
membrane-associated Rho-related proteins and membrane-bound
ARF synergize with cytosol derived-regulatory proteins. In a recent
report by Malcolm et al.(40) , only RhoA was
found to reconstitute a full PLD response in Rho GDI-washed
liver membranes. Thus, extraction of Rho A from HL-60 membrane
would explain the inhibition of the GTP
S-stimulated PLD activity
by Rho GDI.
Several investigators have presented evidence implicating ARF proteins in the activation of PLD(20, 21) . Taking into account the fact that this reconstitution factor has been purified from brain cytosols, it is highly possible that ARF proteins present in HL-60 cytosol were involved in the activation of a membrane-bound PLD. HL-60 cells express genes for ARF proteins(41) . ARF is present in HL-60 cytosol, and cytosolic ARF was found exclusively in the 18-kDa peak of PLD reconstitution. The weak stimulatory effect of cytosolic ARF itself does not preclude a role for ARF in the regulation of PLD activity, since we observed that preparation of HL-60 membranes contains antigenic material recognized by the monoclonal antibody 1D9.
Membrane-bound ARFs are likely to be the active (GTP-bound)
proteins. Based on preincubation with guanine nucleotides, the plasma
membrane has been shown to contain GTP-binding proteins that support
PLD activation(18) . It seems likely that the strong dependence
on the 50-kDa fraction reflects the presence of ARF regulatory
proteins, presumably a nucleotide-exchange factor specific for a
particular ARF protein. Accelerated nucleotide exchange would promote
both GTPS binding to ARF and subsequent coupling to PLD. Several
studies have documented the presence of an ARF-specific guanine
nucleotide-exchange protein in Golgi membranes that is inhibited by
brefeldin A(42, 43, 44) . Attempts to observe
an inhibition of GTP
S-stimulated PLD activity by concentrations of
brefeldin A as high as 20 µg/ml proved unsuccessful. This result
does not exclude the involvement of a nucleotide-exchange factor in the
activation of PLD. The brefeldin A-sensitive component is not yet
known. Indeed, a nucleotide-exchange protein for ARF has been
documented in bovine brain and shown to become insensitive to
inhibition by brefeldin A with purification(45) . Interaction
of Rho GDI with ARF is unlikely inasmuch as GDI had no
significant effect on either GTP
S binding to or GDP dissociation
from human ARF. (
)Thus, it appears that the
GTP
S-activated PLD activity requires both a small GTP-binding
protein, presumably membrane-bound ARF, and a cytosolic cofactor of
approximately 50 kDa.
In conclusion, we have further characterized a 50-kDa factor present in HL-60 cytosol that is essential to PLD activation. This factor is antigenically distinct from the ARF proteins. The molecular mass of this protein and the discrete peak of PLD stimulation by ARF proteins alone strongly suggest that the 50-kDa cytosolic component is another regulator of PLD or an ARF-regulatory protein. Further purification and antibody production are required to define the nature and the function of this 50-kDa protein. We are currently involved in these studies.