(Received for publication, April 15, 1995; and in revised form, August 1, 1995)
From the
Receptor activation of phospholipase D has been implicated in
signal transduction in a variety of cells. Reconstitution of cell-free
guanosine 5`-O-(3-thiotriphosphate)(GTPS)-dependent
phospholipase D activity from human neutrophils requires protein
factors in both the plasma membrane and the cytosol. We previously
proposed that one of the factors is a Ras-family small molecular weight
GTPase of the Rho subtype (Bowman, E. P., Uhlinger, D. J., and Lambeth,
J. D.(1993) J. Biol. Chem. 268, 21509-21512). Herein, we
have used RhoGDI (GDP dissociation inhibitor), an inhibitory
Rho-binding protein, to selectively extract Rho-type GTPases from the
plasma membrane, and have used immunoprecipitation as well as
chromatographic methods to remove cytosolic Rho. Depletion of RhoA from
either the plasma membrane or the cytosol resulted in a partial loss in
GTP
S dependent activity, while removal of RhoA from both fractions
resulted in a nearly complete loss in activity. Activity was nearly
completely restored by adding purified recombinant RhoA, which showed
an EC
of 52 nM, while Rac1 showed little
activity. Cytosol fractionated using DEAE-cellulose chromatography
separated ADP-ribosylation factor and Rho from the major activating
fraction. Gel exclusion chromatography of this fraction revealed an
activating factor of 50 kDa apparent molecular mass. Using
RhoA-depleted membranes, reconstitution of phospholipase D activity
required both RhoA and the 50-kDa factor. Thus, RhoA along
with a non-Rho, non-ADP-ribosylation factor 50-kDa cytosolic factor are
both required to reconstitute GTP
S-dependent phospholipase D
activity by neutrophil plasma membranes.
Phospholipase D (PLD) ()is activated via
receptor-coupled mechanisms and by phorbol esters in a variety of
cells(1, 2, 3) . The enzyme catalyzes the
hydrolysis of phosphatidylcholine to generate free choline and
phosphatidic acid. Phosphatidic acid can be further metabolized via
phosphatidic acid phosphohydrolase to form diacylglycerol, and is the
major source of signaling diacylglycerol in some cell types such as
neutrophil(2, 4) . Phosphatidic acid and
diacylglycerol have been implicated as second messengers involved in
regulation of cell growth, differentiation, inflammation, and in a
variety of cell-specific responses such as the respiratory burst of
granulocytes(4, 5) .
While the occurrence of
receptor-activated PLD has been widely documented, its molecular
mechanism of activation remains poorly understood. GTPS activates
PLD in cell-free systems including liver plasma membranes (6) and in lysates from human neutrophils and HL-60
cells(7, 8) . Unlike the liver system, reconstitution
of GTP
S- and phorbol myristate acetate-stimulated PLD from
neutrophils requires protein factors in both the cytosol and the plasma
membrane(7, 8) . The calcium-dependent (7) activation by either GTP
S or phorbol myristate acetate
has been reported to require the participation of a 50-kDa cytosolic
factor (9) as well as unknown membrane components which include
the PLD catalytic moiety(10) . However, in other studies using
HL-60 membranes, a requirement for a 50-kDa factor was not
seen(10, 11) , or was attributed to a 50-kDa complex
between Rho GTPases and RhoGDI(12) . Thus, there has been
controversy regarding the existence and identity of the putative 50-kDa
factor.
By pre-binding GTPS to the plasma membrane followed by
reisolation to remove free GTP
S, the membrane was shown to contain
the guanine nucleotide-binding site(9) . The GTP-binding
protein did not appear to be a heterotrimeric G protein, based on its
magnesium dependence, lack of activation by fluoride, and lack of
effect of G protein
subunits. Stimulation of GTP-dependent
activation by GDP-dissociation stimulator, an exchange factor that
functions on a variety of small GTPases (but not heterotrimeric G
proteins), identified the activating GTP-binding protein as a member of
the Ras superfamily. Inhibition of GTP
S-activated PLD by RhoGDI
(GDP-dissociation inhibitor) further identified the GTPase as a member
of the Rho subfamily(9) . It was later shown that RhoA can
reconstitute GTP
S-dependent PLD activity in liver plasma membranes (13) .
The identification of the GTP-dependent activating factor has been confounded by recent studies implicating ADP-ribosylation factor (ARF) as the guanine nucleotide-dependent factor and as a cytosolic rather than a membrane factor(10, 11) . These studies utilized permeabilized HL-60 cells or HL-60 plasma membranes (or their extracts) along with cytosol from bovine brain. In these heterologous systems, ARF was identified as a major cytosolic activating factor from brain. Using human neutrophil fractions, we recently showed that the major cytosolic activating factor is approximately 50 kDa by gel exclusion chromatography (9) and separates from fractions containing ARF. The cytosolic factor at a relatively high concentration activated PLD in the absence of added ARF. ARF alone produced a minimal stimulation of PLD when added to plasma membranes. However, in the presence of a low concentration of ARF-depleted cytosol, ARF and the cytosolic factor functioned synergistically to activate PLD(14) . Similar results have been reported recently for HL-60 cells(12) . In the latter study, it was proposed that the membrane GTP-binding factor is membrane-associated ARF rather than a Rho-family GTPase and that the a 50-kDa PLD stimulating cytosolic factor is a complex of RhoA and RhoGDI. Using our earlier separation methods, the 50-kDa activating fraction also probably contained RhoA and RhoGDI, so this possibility could not at that time be ruled out.
In the present study, we have
investigated the role of RhoA and the 50-kDa factor in activation of
phospholipase D in human neutrophils. In neutrophil, approximately 5%
of RhoA is associated with the plasma membrane, while other small
GTP-binding proteins such as Rac2 and CDC42 were detected only in the
cytosol. ()The remainder of RhoA and the majority of the
other small GTPases are cytosolic, largely associated with the
inhibitor protein RhoGDI. The latter is complexed with Rho proteins in
such a way as to mask their isoprenyl group, maintaining solubility and
preventing membrane interactions(15, 16) . Herein, we
have depleted Rho-type proteins from both the plasma membrane and from
the cytosol. We have used this Rho-depleted system in reconstitution
studies to investigate the factors required for PLD activation. We find
that both RhoA and a (non-Rho, non-ARF) 50-kDa cytosolic factor are
required to reconstitute GTP
S-stimulated phospholipase D activity
in neutrophil plasma membranes.
The fusion protein RhoGDI-GST was used to
extract membranes, since it was as effective as RhoGDI in inhibiting
GTPS-stimulated PLD activity. (
)RhoGDI has been
previously shown to be capable of binding to and extracting Rho
proteins from plasma membranes of other cell types(21) . Upon
incubation at 22 °C for 5-10 min, less than half of the RhoA
in neutrophil plasma membranes was released into the soluble fraction.
However, as shown in Fig. 1, greater than 80% of the RhoA was
extracted from membrane by incubation with 20 µM RhoGDI-GST for 20 min at 30 °C and all detectable RhoA was
extracted by incubation for 1 h. A mock incubation which did not
contain RhoGDI-GST failed to extract RhoA.
Figure 1: Extraction of RhoA from neutrophil plasma membranes using recombinant RhoGDI-GST. Plasma membranes (1 mg/ml) were treated with (+RhoGDI-GST) or without (Mock) 20 µM RhoGDI-GST for the indicated times at 30 °C. Membrane (M) and supernatant (S) fractions were reisolated by sucrose density centrifugation as described under ``Experimental Procedures.'' Plasma membranes (18 µg) and supernatant (18 µl) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and visualized using antibody to RhoA, as detailed under ``Experimental Procedures.'' Results are representative of 10 experiments.
Figure 2: Extraction of RhoA and RhoGDI from cytosol. The diagram to the lower outlines the extraction procedure described in the text and is keyed to the results shown on the upper. 10 mg of cytosol (1) was mixed with protein A-bound anti-RhoGDI antibody to obtain RhoGDI-depleted cytosol (3) or mock-treated cytosol (2) in which the specific antibody was omitted. These fractions were characterized by Western blotting using antibodies against either RhoGDI (upper panel) or RhoA (lower panels). The RhoGDI-depleted cytosol (3 and A) was then treated by incubating with RhoGDI-GST/glutathione-agarose to obtain Rho-depleted cytosol (B). For SDS-polyacrylamide gel electrophoresis, the amount of protein run per lane was 8 µg for lanes 1, 2, and 3 and 30 µg for lanes A and B. Transfer to nitrocellulose and immunostaining was carried out as described under ``Experimental Procedures.'' Results are representative of 10 experiments.
Figure 3:
Loss of GTPS-stimulated PLD activity
by extraction of RhoA. Plasma membranes (1 mg/ml) were mixed with or
without 20 µM RhoGDI-GST for 20 min at 30 °C and then
reisolated as described under ``Experimental Procedures.''
RhoA-depleted cytosol was prepared as in the legend to Fig. 2.
Membranes (25 µg) were incubated with 50 µg of cytosol, 1
µM CaCl
, and 1.6% ethanol in the absence or
presence of 10 µM GTP
S as indicated for 20 min at 37
°C. Groups tested were: A, mock-treated cytosol plus
mock-treated membranes; B, mock-treated membranes plus
Rho-depleted cytosol; C, mock-treated cytosol plus
Rho-depleted membranes; and D, Rho-depleted cytosol plus
Rho-depleted membranes. Phospholipase D-catalyzed
transphosphatidylation was monitored by phosphatidylethanol formation (PEth), expressed as a percentage of the total radiolabeled
lipids. The ``mock treated'' activity in this experiment was
60% of the GTP
S stimulated activity seen using naive cytosol and
plasma membrane. The result shown is representative of three
experiments.
Figure 4:
Reconstitution of PLD activity with
purified recombinant Rho proteins in a RhoA-depleted condition.
GTPS-bound forms RhoA-GST, RhoA, and Rac1 were prepared as
described under ``Experimental Procedures.'' RhoA-depleted
membrane and RhoA-depleted cytosol were prepared as described in the
legend to Fig. 2and Fig. 3. Membranes (25 µg) were
incubated with 50 µg of cytosol and incubation buffer containing 1
µM CaCl
, with (slashed bars) or
without (filled bars) 10 µM GTP
S. The
RhoA-depleted fractions were treated with nothing or with 10 µM GTP
S alone (third set of bars), or as indicated with
1 µM RhoA, RhoA-GST, or Rac1 prepared in their
GTP
S-bound forms. The data in panel B are the average of
two experiments, and were normalized to permit comparison with panel A. The results shown are representative of three
experiments.
Figure 5:
Concentration dependence for RhoA
reconstitution of PLD activity. Activation of PLD was assayed with
increasing concentrations of GTPS-bound RhoA. RhoA-extracted
membranes, RhoA-depleted cytosol, and GTP
S-bound RhoA were
prepared as described in the legend to Fig. 4. RhoA-extracted
membranes (25 µg) were incubated with 50 µg of RhoA-depleted
cytosol, 10 µM GTP
S, 1 µM
CaCl
, 1.6% ethanol, and the indicated concentrations of
RhoA. After 20 min, the incubation was terminated and
phosphatidylethanol (PEth) was quantified as described under
``Experimental Procedures.'' Data represent the average of
two to three determinations for each point, obtained in two independent
experiments.
Figure 6:
Chromatographic separation of ARF,
cytosolic factor, and RhoA using DEAE-cellulose. Cytosol was
chromatographed on DEAE-cellulose as described under
``Experimental Procedures.'' Panel A, protein was
eluted using a KCl gradient and step elution as indicated. Panel
B, PLD activity was monitored by phosphatidylethanol (PEth) formation; 20 µl of each fraction was incubated for
25 min at 37 °C in a reaction mixture containing 25 µg of
plasma membrane, 10 µM GTPS, 1 µM CaCl
, and 1.6% ethanol. Panel C, aliquots (20
µl) of column fractions were analyzed for ARF, Rho proteins, and
RhoGDI by Western blotting, as described under ``Experimental
Procedures.'' Fractions 50-66 were pooled for subsequent
chromatography on Superose-12. The experiment shown is representative
of four.
Although Rac2 and CDC42 were also detected in the second half of the major peak of PLD stimulating activity, their presence failed to correlate with PLD stimulating activity (compare Fig. 6, B and C). RhoGDI showed at least three peaks of immunoreactivity, and these corresponded to the presence of Rac2, CDC42, and RhoA. Thus, each of the small GTPases chromatographs as a complex with RhoGDI. The fractions (fractions 50-66) which showed the highest PLD stimulating activity, but which did not contain any detectable ARF, Rho-type proteins, or RhoGDI were pooled, concentrated, and chromatographed on a calibrated Superose-12 column. As shown in Fig. 7, PLD stimulating activity showed a major peak with an apparent molecular mass of 50 kDa. The five most active fractions were pooled and used in subsequent experiments. This fraction is designated the ``50-kDa cytosolic factor.''
Figure 7:
Superose-12 chromatography of cytosolic
factor. Fractions 50-66 from the DEAE column in Fig. 6were pooled, concentrated, and chromatographed on
Superose-12. Aliquots (20 µl) of column fractions (0.5 ml) were
monitored for GTPS-dependent PLD activation (filled
circles) or protein (filled triangles). PLD activity was
measured as described under ``Experimental Procedures.'' The
elution maxima for molecular mass markers bovine serum albumin (66 kDa)
and chymotrypsinogen A (25 kDa) are indicated by arrows. The
experiment shown is representative of four.
Figure 8:
RhoA and the 50-kDa cytosolic factor are
both required for reconstitution of GTPS-dependent PLD activity in
Rho-depleted plasma membranes. RhoA was preloaded with GTP
S as
described under ``Experimental Procedures.'' The 50-kDa
cytosolic factor was obtained from the five highest activity fractions
on Superose-12 chromatography. The final concentrations of GTP
S,
RhoA, and 50-kDa factor were 10 µM, 1 µM, and
5 µg, respectively in a 250-µl reaction mixture containing 25
µg of plasma membrane, 1 µM CaCl
, and 1.6%
ethanol, as detailed under ``Experimental Procedures.'' These
experiments used the Rho-depleted membrane and mock-treated membrane
obtained as described in the legend to Fig. 3. Data represent
the average of two to three determinations. The experiment shown is
representative of two.
We have developed a Rho-depleted cell-free PLD system
consisting Rho-depleted membrane and cytosol. The depleted system has
minimal GTPS-stimulated PLD activity, and provides an optimal
system for investigating the role of small GTP-binding proteins in
activation of the enzyme. It has the advantage that other membrane and
cytosolic components in this complex system are not removed, minimizing
the chance of artifact. It was possible to extract almost all of the
detectable membrane-associated RhoA using RhoGDI-GST at 30 °C for 1
h. Less efficient extraction was seen at room temperature, presumably
due to a less fluid membrane. Partial extraction at the lower
temperature was reported previously(13, 21) . Because
Rho in the cytosol was already largely associated with RhoGDI,
extraction with RhoGDI-GST alone failed to deplete RhoA. (
)We therefore initially used an antibody against RhoGDI to
immunoprecipitate the RhoGDI-RhoA complexes. Following this treatment,
there remained a small quantity of soluble RhoA, which could be
effectively removed by RhoGDI-GST plus glutathione-agarose. We propose
that the small amount of residual RhoA present in the cytosol is due to
free RhoA in equilibrium with RhoGDI-bound RhoA, or possibly RhoA which
is produced in excess of RhoGDI binding sites. The material is not
likely to be nonprenylated RhoA, since only prenylated Rho proteins
bind to RhoGDI (15, 24) . RhoA was the only Rho family
protein found in appreciable quantities in the membrane, so the
efficiency of the extraction method for other Rho family proteins is
not known, although others are reportedly extracted in other
systems(13, 21) . We also monitored the depletion of
the small GTPase Rac (using a general antibody to both Rac1 and Rac2)
from the cytosol, and found that this methodology was also effective in
depleting Rac. Therefore, it appears that the depletion protocol is
generally useful for removing Rho family proteins, and will likely find
a variety of applications in other systems. The methodology allowed us
herein to initially investigate the role of RhoA in GTP
S-dependent
activation of phospholipase D in granulocytic cells.
Using the
Rho-depletion system, depletion of RhoA is associated with loss of
GTPS dependent activity, and recombinant RhoA (but not Rac1)
reconstituted GTP
S-dependent PLD activity. It is usually assumed
that isoprenylation of small GTP-binding proteins is essential for
their effector functions(15, 24) . Recombinant RhoA
and Rac1 were expressed in E. coli, in which isoprenylation
fails to take place. It was recently shown for Rac activation of the
NADPH oxidase that isoprenylation was necessary for recognition by
exchange proteins, but was not needed for the effector
function(18) . We therefore used a GTP
S-preloading
protocol in which chelation with EDTA of magnesium permits release of
bound GDP and subsequent binding of GTP
S. GTP
S-preloaded RhoA
reconstituted a rate of PLD activity similar to that seen in untreated
preparations. The relatively low EC
(50 nM) for
RhoA further indicates that the activation is specific, and that
isoprenylation is not essential, although it is not clear whether an
increase in potency would be seen with isoprenylation. Reconstitution
by RhoA is consistent with our previous studies which showed that
RhoGDI inhibited GTP
S-dependent activation in a neutrophil
cell-free system (plasma membranes plus cytosol)(9) , and with
subsequent studies in liver (13) which showed that RhoA can
activate PLD in isolated plasma membranes. The latter system differs in
several respects from the neutrophil/HL-60 system including its
independence from cytosolic factor and its ATP independence for phorbol ester activation. Thus, it is not clear that the two
systems represent the same PLD isoform or regulation mechanism.
Nevertheless, the fact that both activities are regulated by RhoA
suggests a common link in the regulation of some PLD's.
The
immunodepletion protocol removes Rho-type small GTPases, and
demonstrates the need for non-Rho cytosolic components. However, the
depleted cytosol may contain multiple activating factors (e.g. ARF, others). To further characterize the nature of the cytosolic
activity, we developed chromatographic methods to resolve ARF, Rho-type
GTPases, and RhoGDI from other activities. Using DEAE-cellulose
chromatography, we were able to resolve these factors from a major peak
of PLD stimulating activity. Subsequent gel exclusion chromatography
indicated that the major activity migrates with an apparent size of 50
kDa. Using the RhoA-depleted plasma membranes, it was clear that
reconstitution of GTPS-stimulated PLD activity required both the 50-kDa factor and RhoA.
The mechanism of activation of PLD
by Rho is not yet clear. Signal transducing GTPases (both
heterotrimeric G proteins and small GTPases) frequently function as
molecular switches, binding to their effector enzymes in their GTP (or
GTPS)-associated forms. In GTP
S pre-binding experiments, we
showed that GTP
S binding to a component in the plasma membrane was
sufficient to activate PLD, but that pretreatment of cytosol with
GTP
S failed to activate PLD(9) . Thus, it appears that the
membrane-associated form of RhoA is the form relevant to PLD
activation, although cytosolic RhoA probably serves as a mobile
reservoir to provide additional RhoA to the membrane. Earlier work (10) suggests that the PLD catalytic moiety itself resides in
the plasma membrane. Thus, one model is that GTP association and
translocation of RhoA results in its complexation and activation of a
membrane-associated PLD, as is the case for activation of some forms of
adenylyl cyclase by G
. Ras, which is bound to the
plasma membrane via its isoprenyl and other groups, provides an
attractive alternative model. An effector enzyme for Ras is Raf-1. The
latter is a protein kinase which is located in the cytosol, and which
translocates to the membrane by binding to GTP-Ras where it is active (25) . RhoA and the 50-kDa cytosolic factor are resolved as
different proteins and both are necessary to activate PLD. Thus, we can
speculate that membrane-associated GTP-Rho complexes with the essential
50-kDa cytosolic factor in neutrophil, permitting it to translocate to
the plasma membrane where it participates in PLD activation. Consistent
with this model, in U937 cells, which normally show a similar
requirement for both plasma membrane and cytosol for PLD activity,
pretreatment of permeabilized cells with GTP
S renders PLD activity
in subsequently isolated plasma membranes independent of
cytosol(26) . Thus, GTP
S appeared to induce translocation
from the cytosol to the membrane surface of a required PLD component.
Finally, Rho-GTP may be functioning by inducing the generation of an
important cofactor such as PIP. The latter lipid has been
shown to be essential for PLD activity in brain (27) and U937
cells(28) , and Rho reportedly activates an isoform of
phosphatidylinositol 5-kinase (29) which generates
PIP
. While regulated production of PIP
may also
be important, three findings argue against the possibility that Rho
activation of PIP
generation explains PLD activation in our
system. First, Rho effects on phosphatidylinositol 5-kinase but not PLD
are inhibited by ADP-ribosylation of Rho with botulinus C3 exoenzyme (13, 29) . Second, GTP
S activation in our system
is not further stimulated by ATP (as is seen in U937 cells, presumably
due to the ATP requirement for lipid phosphorylation). Third, addition
of PIP
, while causing a modest stimulation, does not
eliminate the ability of RhoA or cytosolic factor to stimulate high
levels of activity. (
)If Rho were acting by increasing the
synthesis of PIP
, then we would expect the activating lipid
to eliminate the ability of Rho to further activate.
Rho family GTP-binding proteins have been implicated in mediating receptor-coupled regulation of the actin cytoskeleton, and appear to be involved in Ras-induced mitogenic transformation(30) . Microinjected RhoA results in the production of actin stress fibers(31) , while microinjected Rac1 results in membrane ruffling, the latter mediated by submembrane actin cytoskeleton. It is tempting to speculate that PLD may play a role in cytoskeletal changes. However, stress fiber formation is inhibited by C3 exoenzyme ADP-ribosylation of Rho, but PLD activation is not affected. Thus, a more tenable model is that Rho represents a common coordination point for several cellular responses, with one of these being lipid signal transduction and another the regulation of the actin cytoskeleton. A role of Rho-activated PLD in transformation also remains a possibility. Rho may be important in coordinating diverse cellular responses, for example, as occurs during cytokinesis or locomotion.