From the
Phospholipase D, which has been extracted from porcine brain
membranes and chromatographically enriched 100-fold, was activated
better by impure preparations of Arf than by purified or recombinant
Arf. Examination of brain cytosol with this enriched preparation of PLD
activity revealed at least three stimulatory components. One of these
is Arf or the first cytoplasmic factor. A second peak of
PLD-stimulating activity (cytoplasmic factor II, CFII) was resolved
from Arf by anion exchange and gel filtration. This CFII can be further
separated into multiple activities by chromatography with
heparin-agarose. The activities were differentiated by their
stimulatory properties as measured in the absence or presence of
guanosine 5`-O-(3-thiotriphosphate) (GTP
While all of the CFII pools
stimulated PLD activity to some degree and showed synergistic
activation when administered in conjunction with Arf, they could be
classified into two groups with distinct behavior. When used together,
pools from the two respective groups showed synergistic activation of
PLD. The first set of pools contained the RhoA monomeric G protein.
Recombinant RhoA was used to show that it could indeed activate this
enriched PLD activity and act synergistically with Arf proteins. A
related monomeric G protein, Cdc42, was also effective. The second set
of CFII pools were devoid of RhoA and, in contrast to the first group,
demonstrated significant stimulating activity in the absence of guanine
nucleotides.
These data indicate that the PLD activity from brain
can be modulated by several cytosolic factors and that Arf-sensitive
PLD may represent a complex activity that can be regulated in an
interactive fashion by a variety of cellular signaling events.
Phospholipase D (PLD)
Characterization
of mammalian PLD (4, 5, 6) has lagged behind
that of the plant enzyme (for reviews, see Refs. 4-7, and see
Ref. 8 for the first cloned enzyme from plants). Furthermore, the
regulation of PLD activity is not well understood. Several lines of
evidence indicate that GTP-binding regulatory proteins (G proteins) are
involved (for review, see Ref. 6). For example, stimulation of PLD
activity by a variety of receptors that are known to act through
heterotrimeric G proteins indicate their involvement. Recently, two
groups have demonstrated, either through in vitro assays with
exogenous substrate (9) or through use of permeabilized
cells(10) , that PLD activity can be stimulated by the monomeric
G protein, Arf. Further evidence of involvement of the monomeric G
proteins comes from the demonstration that Rho GDI, a GDP dissociation
inhibitor of the Rho family of monomeric G proteins, could inhibit
activation by GTP
The previous accompanying manuscript (13) describes the partial purification and characterization of
Arf-sensitive PLD activity from porcine brain. During this
purification, it became apparent that there were other factors that
could stimulate the same PLD activity. This report describes the
initial resolution of these other cytosolic factors from Arf and
initial attempts to identify the components and characterize their
activities.
Porcine brain cytosol (5-6 g of protein) was diluted
with an equal volume of Solution A (20 mM Tris-Cl, pH 8, 1
mM EDTA, 1 mM dithiothreitol, and 1 µM GDP) containing phenylmethylsulfonyl fluoride/TLCK/TPCK and loaded
onto a 1-liter column of DEAE-Sephacel (Pharmacia Biotech Inc.), which
had been equilibrated with the same solution. Bound protein was eluted
with a 2-liter linear gradient of NaCl (0-300 mM) in
Solution A, followed by a 1.2-liter wash with 1 M NaCl in
Solution A. Fractions of 27 ml were collected, and samples were assayed
for Arf activity and the capacity to stimulate PLD activity. CFII
activity eluted over approximately 25 fractions centered around 110
mM NaCl (see ``Results'' for basis of CFII peak
selection). Fractions with activity were combined with protease
inhibitors and concentrated to 30 ml by pressure filtration through an
Amicon PM 10 membrane.
The concentrated pool of activity from DEAE
was loaded onto a 1.2-liter column of Ultrogel AcA 44 (Sepracor), which
was both equilibrated and eluted with 1.2 liters of Solution B (20
mM NaHepes, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, and 1 µM GDP).
Fractions of 20 ml were collected.
A portion (60 ml) of the pool of
CFII activity from the AcA 44 step was loaded onto a 50-ml column of
heparin-Sepharose CL-6B (Pharmacia), which had been equilibrated with
Solution B containing protease inhibitors. Bound protein was eluted
with a 200-ml linear gradient of NaCl (50-500 mM) in
Solution B, followed by a 75-ml wash with 1.5 M NaCl in the
same solution. The flow-through of applied material was collected as
fractions 1-8; subsequent fractions contained 4.5 ml. Three
regions of PLD stimulatory activity were observed (see Fig. 3);
these were designated A, B, and C, and representative pools were
collected as indicated.
Arf activity was determined by its ability to promote
ADP-ribosylation of recombinant G
Recombinant human RhoA(18) , Rac1(19) , and Cdc42
containing posttranslational modifications were purified from Spodoptera frugiperda (Sf9) cells after expression using a
baculovirus system. Human Rho GDI (20) and nonmodified RhoA
containing the Val-14 activation mutation (18, 21) were
purified following overexpression in E. coli. Nonprenylated
recombinant human Rab1a, Rab3a, and Rab5 (all containing a
hexahistidine tag at their amino terminus), were also purified
following expression in E. coli(22) . These Rab
proteins, and the prenylated recombinant Rab1a without a hexahistidine
tag, were provided by Dr. Miguel Seabra. Recombinant
G
The original isolation of Arf as a cytosolic factor for
stimulation of PLD activity was accomplished with either extracts of
membranes (9) or permeabilized cells (10) as the source
of the enzyme. The accompanying report (13) described the use of
Arf to partially purify and characterize PLD activity from porcine
brain. During the course of these studies, it became apparent that
cruder preparations of Arf were more effective than highly purified and
recombinant Arf in stimulating more enriched preparations of PLD. This
prompted a further evaluation of cytosol in which enriched porcine PLD
(purified about 100-fold from membranes) was used as the source of
activity.
Separation via anion
exchange (Fig. 1) revealed clear overlapping peaks of activity
with both conditions. Binding of GTP
The three pools were subjected to further resolution on
hydroxylapatite. The profiles of activities eluted by identical
chromatography of the three pools through this matrix are shown in Fig. 4. Whereas Pools A and C eluted as apparent singular peaks
with uniform behavior, Pool B from the heparin-Sepharose appeared to
separate into two activities. One pool (labeled B) enhanced stimulation
of PLD activity in the presence of Arf but had little effect in the
presence of GTP
Only additive or less than
additive effects are observed when CFII A is combined with CFII B or
when CFII B` is combined with CFII C ( Fig. 6and 7). The
combination of this result and similarities in synergistic action
suggest that CFII A and CFII B may represent the same or similar
factors. Such a relationship may also exist between CFII B` and CFII C.
Fig. 8demonstrates a further difference between the factors
designated CFII B and CFII B`. Whereas factor B required guanine
nucleotide for activity, factor B` had substantial activity in the
absence of nucleotide. The addition of Arf in the absence of GTP
While RhoA may account for at
least some of the activity of CFII pools A and B, it does not explain
the additional activity of the CFII A pool or the activities of the
more robust CFII B` pool and that of the CFII C pool. Other selected
monomeric G proteins have been tested for potential action on PLD.
Cdc42, a member of the Rho family of monomeric G proteins, proved an
effective activator of PLD activity and showed similar synergistic
activity with added Arf (Fig. 10). The action of this protein
could also be attenuated by Rho GDI. Other proteins that were tried but
proved ineffective are shown in . Nonprenylated Rab1a,
Rab3a, or Rab5 had essentially no activity by themselves or in the
presence of native Arf or the CFII peak from gel filtration. The
prenylated Rab1a was similarly unresponsive in comparison with RhoA.
Interestingly, RhoA showed synergism with both Arf and the CFII
fractions in which it resides, albeit at lower concentrations. The
latter result resembles the interactive effects of the CFII pools (A
and B) that contain RhoA with the pools (B` and C), which are devoid of
this G protein (Fig. 6).
A phospholipase D activity has been described that can be
activated with the monomeric G protein, Arf(9, 10) . The
development of an in vitro assay, which uses exogenous
substrate (9) to assay this activity, has allowed extensive
purification of the enzyme as described in the companion
report(13) . Use of enriched preparations of porcine PLD and
reexamination of the stimulation provided by cytoplasmic Arf revealed
the presence of other stimulatory factors in cytosol. Resolution of
these activities yielded at least three unique components; Arf, RhoA,
and a third factor that has not yet been identified. The identification
of the Rho protein was anticipated by two previous reports that
implicated Rho in the function of PLD. Thus, Bowman et al.(11) showed that the addition of Rho GDI inhibited a PLD
activity observed in human neutrophils. More recently, addition of RhoA
to plasma membrane preparations derived from rat liver caused a 2-fold
enhancement of basal PLD activity(12) . The inability of the
latter group to remove Arf or Cdc42 from their membrane preparation
presumably precluded observation of stimulatory effects by these two
proteins. The efficacy of recombinant RhoA, or the cytoplasmic factor
pools that contained the native protein on the enriched PLD activity
used in this study suggests the likelihood that this action of RhoA is
a direct interaction with the PLD enzyme. The efficacy of Cdc42, a
protein related to RhoA, suggests that stimulation of PLD activity may
be a general functional property of the Rho family. Since the
regulation by Arf suggests a potential role for PLD in protein traffic,
other speculative candidates for regulation of this enzyme would
include the Rab family of small G proteins. Exploration of various
members of this and other monomeric G protein families has just begun.
The effect of RhoA by itself was relatively modest. Its efficacy is
most apparent in its ability to facilitate activation of PLD by Arf.
This effect could be achieved by enhancing activation of the Arf
protein itself. However, significant stimulation induced by Rho alone
indicates a more direct action on the PLD; it is more likely that Rho
acts through PLD to increase interaction of the enzyme with Arf. One
caveat to the action of RhoA is that a complete titration of the
protein could not be achieved with current preparations of recombinant
protein. It is plausible that higher concentrations of the monomeric G
protein will yield more robust stimulation of PLD in the absence of
Arf.
This pattern of regulation was repeated in the interaction
between Arf and the cytosolic factor pools that did not contain RhoA.
In this case, the CFII B` pool showed substantial activity on its own.
It also had synergistic effects when combined with the factor pools
containing RhoA. The identity of this third factor is unknown. Since a
significant proportion of its activity could be obtained in the absence
of guanine nucleotides, it is likely to represent more than another G
protein, and this factor(s) is likely to facilitate PLD activity by
another mechanism. One explanation for the synergism between Arf and
CFII B` would be that the latter acts as a catalyst for nucleotide
exchange on Arf. While this is possible, the effect of the factor on
PLD activity in the absence of nucleotide suggests a more direct
mechanism. A second possibility for synergism is that these factors
would prevent degradation of the substrate by other contaminating
activities such as a phospholipase A
The
molecular mechanism behind the requirement for PIP
What is
the significance of the regulation of this enzyme by Arf, the Rho
family, and potentially other GTP-dependent or independent proteins?
The role of Arf in intracellular protein traffic suggests that PLD
plays an important role in these processes, perhaps by modulating
membrane lipids to facilitate formation or fusion of transport
vesicles. RhoA and Cdc42 have been shown to mediate different
cytoskeletal-associated assembly processes; thus, RhoA can regulate the
formation of actin stress fibers and focal adhesions(21) , and
Cdc42 is essential for bud site assembly in Saccharomyces
cerevisiae (30, 31). RhoA has also been implicated in the
regulation of phosphatidylinositol 3-kinase (32) and
phosphatidylinositol 4-phosphate 5-kinase (18) activities.
Evidence indicates that Cdc42 activates a phosphatidylinositol 3-kinase (33) as well as a serine/threonine-protein kinase(34) .
It would seem more than coincidence that Rho and/or Cdc42 would
regulate synthesis of PIP
Overall, the results delineated in this study indicate that
PLD activity can be regulated by a variety of cytoplasmic factors. It
is likely that membrane factors yet to be identified also impinge on
this enzyme. These data then predict that activation of the enzyme and
its sequelae mediate effects of multiple cellular pathways. It is
possible that regulation of PLD by several factors is indicative of the
use of this enzyme by a variety of signal transduction systems to
achieve different end points in remote locations in the cell.
Alternatively, the potential synergistic effects of these regulatory
molecules suggest a dynamic capability of the enzyme to provide a
robust response in the presence of multiple intracellular stimuli or
the need for several interacting components to be stimulated before
marked activity is initiated. This would allow the system to perform in
a highly cooperative fashion. Such a response might be most desirable
if this enzyme is involved in regulated secretory events as proposed by
Cockcroft (35) or in controlled budding or fusion of vesicles
during protein transport. The synergism between Arf and Rho raises the
intriguing possibility that the functions of these proteins are
interrelated in a fashion that remains to be discerned.
We thank John Long and Steve Gutowski for excellent
technical assistance and Kim Edwards for help in the preparation of
this manuscript. We also thank Miguel Seabra for providing recombinant
Rab proteins and Tsung Chuang for preparing recombinant RhoA and Rho
GDI.
Addendum-During the review of this manuscript, a communication
by Lambeth et al.(36) was published that provided
evidence for an unidentified activity from cytosol, which could
stimulate PLD activity in neutrophil membranes and potentiate the
effects of Arf.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S) alone and in
the presence of added Arf and GTP
S.
(
)hydrolyzes
phospholipids to produce phosphatidic acid and their respective head
groups. This activity can be regulated by a variety of hormones and
growth factors and one of the products, phosphatidic acid, has been
touted as a putative second messenger (1, 2) and has
also been suggested to have fusigenic properties(3) .
Phosphatidic acid is also a precursor of the second messenger,
diacylglycerol, an activator of protein kinase C.
S of PLD in neutrophil membranes (11) and
that recombinant RhoA could stimulate a PLD activity in rat liver
plasma membranes(12) .
Preparation of PLD and Arf
Porcine brain
membranes and cytosol were prepared as described in the accompanying
report(13) . Briefly, PLD was extracted from the membranes with
sodium cholate, exchanged into n-octyl--D-glucopyranoside, and partially
purified through three steps of chromatography, which concluded with
passage through a Mono Q anion-exchange column. The PLD in this
preparation is enriched about 100-fold. A highly enriched preparation
of Arf (approximately 25% pure) was obtained from porcine brain cytosol
after four steps of chromatography, which terminated with gel
filtration through Sephadex G-75(9) . Myristoylated recombinant
human Arf5 (myrArf5) was produced by co-expression with a yeast
myristoyl transferase in Escherichia coli(14) .
Preparation of CFII Factors
CFII activity is
defined as the ability to stimulate PLD activity in the absence of Arf
or an ability to enhance PLD activity in the presence of Arf (see
``Results'' for further description). All procedures were
conducted at 4 °C. Protease inhibitors were included when indicated
at the following concentrations: 21 µg/ml N-p-tosyl-L-lysine
chloromethyl ketone (TLCK), 21 µg/ml tosylphenylalanyl chloromethyl
ketone (TPCK), 21 µg/ml phenylmethylsulfonyl fluoride, 2.5
µg/ml leupeptin, 0.1 unit of trypsin inhibitor/ml of aprotinin, 10
µg/ml soybean trypsin inhibitor, 1 µg/ml pepstatin A, and 21
µg/ml N
-p-tosyl-L-arginine methyl
ester.
Figure 3:
Chromatography of CFII factors through
heparin-Sepharose CL-6B. A portion (60 ml) of the pool of activity from
the Ultrogel AcA 44 step was fractionated through heparin-Sepharose and
assayed as described under ``Experimental Procedures.''
Assays to measure activation of PLD were the same as indicated in Fig.
1, except for the use of 0.5 µg of PLD, 1 µg of porcine Arf,
and the inclusion of 2.8 mMn-octyl--D-glucopyranoside. Fractions selected
for pools of activity are indicated by the linesbetweenpanels: PoolA, fractions 8-10; PoolB, fractions 25-28; and PoolC, fractions 36-42.
Each pool of activity from heparin-Sepharose
was loaded onto a fast protein liquid chromatography hydroxylapatite
column (100 7.8 mm Bio-Gel high performance hydroxylapatite
column, Bio-Rad) equilibrated with Solution C (20 mM NaHepes,
pH 7.5, 1 mM dithiothreitol, 100 mM NaCl, 10
µM CaCl
, 5 mM potassium phosphate, pH
7.5, and protease inhibitors). The column was eluted at 0.5 ml/min with
a 50-ml linear gradient of 5-350 mM potassium phosphate,
pH 7.5, in Solution C. Fractions of 2 ml were collected in tubes
containing a 10-µl aliquot of 200 µM GDP to achieve a
concentration of 1 µM GDP in the fractions. Pools of CFII
activities were collected as indicated. Stimulatory fractions from B
were separated into two pools, designated CFII B and CFII B`. The pools
were concentrated 5-10-fold by ultrafiltration using Centricon 10
microconcentrators (Amicon) and stored in aliquots at -80 °C.
Measurement of PLD and Arf Activity
PLD activity
was assessed by its ability to hydrolyze dipalmitoyl-PC using an assay
described previously (9, 13) with minor modifications.
Briefly, 5 µl of mixed lipid vesicles containing 3 nmol of
phosphatidylethanolamine, 0.3 nmol of phosphatidylinositol
4,5-bisphosphate (PIP), 0.3 nmol of dipalmitoyl- PC, and
[choline-methyl-
H]dipalmitoyl-PC to
yield approximately 30,000 cpm/assay were added to a mixture of PLD,
cytosolic factors, and regulatory ligands (such as GTP
S) in a
total volume of 30 µl. The assays also contained 1.7 mMn-octyl-
-D-glucopyranoside (which inhibits the
assay(15) ), due to the presence of the detergent in the PLD
preparation. Reactions were allowed to proceed for 30 min at 37 °C.
Variations from this protocol are indicated in the figure legends.
in the presence of
activated cholera toxin. Aliquots (10 µl) of column fractions were
assayed essentially as described previously(9) , except 20 pmol
of bovine brain heterotrimeric G protein
subunits was
employed, the concentration of activated cholera toxin added to the
assay was increased to 500 µg/ml, and the reaction was terminated
after 60 min at 30 °C.
Immunoblot Analysis
Proteins in column fractions
were resolved by electrophoresis through 12% SDS-polyacrylamide gels (16) and transferred to nitrocellulose. Following incubations
with primary antibody (1 µg/ml) and horseradish
peroxidase-conjugated secondary antibody (Amersham Corp.),
immunoreactive proteins were visualized by enhanced chemiluminescence
(Amersham Corp.). Affinity-purified antibodies raised against peptides
representing portions of RhoA and Rho GDI were obtained from Santa Cruz
Biotechnology.
Miscellaneous Methods
Guanine nucleotide binding
activity was quantified by the filter binding method(17) .
Aliquots (5 µl) of column fractions were assayed for 60 min at 30
°C in a 60-µl volume of solution containing 50 mM NaHepes, pH 8, 1 mM EDTA, 1 mM dithiothreitol,
800 mM NaCl, dimyristoyl-PC (3 mM) vesicles, 0.1%
sodium cholate, 5 mM MgCl, and 1 µM [
S]GTP
S (4000 cpm/pmol).
, obtained by expression in E. coli and
purified according to published procedures(23) , was a gift of
the laboratory of Dr. Alfred Gilman. Heterotrimeric G protein
subunits were purified from bovine brain as described
previously(24) . Protein concentrations were determined by the
method of Bradford(25) , using bovine serum albumin as a
standard.
Resolution of Stimulatory Cytosolic Factors from Arf
The
profiles of activities obtained from the first two steps of
chromatographic resolution of porcine brain cytosol are shown in Figs.
1 and 2. Activities obtained after mixing enriched PLD with aliquots of
each fraction were measured under two conditions; in the presence of
either GTPS or added Arf and GTP
S.
S and the presence of Arf in
the fractions was also assessed for comparison. Two observations are
worthy of note. First, GTP
S stimulation of PLD activity (
)
did not correlate well with the presence of Arf (
); that is,
fractions that contain little or no Arf can stimulate PLD activity.
Second, even in the presence of near saturating levels of Arf (
),
additional stimulating activity was observed in
fractions(60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85) following
the peak of Arf. These results indicated that cytosol contains
stimulatory factors for PLD other than Arf.
Figure 1:
Resolution of CFII activity and Arf
activity by chromatography through DEAE-Sephacel. Porcine brain cytosol
was fractionated and assayed as described under ``Experimental
Procedures.'' Aliquots (5 µl) of fractions were examined for
their capacity to stimulate PLD activity by assays that contained 0.2
µg of PLD, 10 µM GTPS, and 8.2 µg of porcine
brain Arf as indicated. Fractions 61-85 were collected as the
CFII pool.
To pursue these other
factors, a pool (CFII) of fractions that contained stimulating activity
but which were mostly devoid of Arf activity was concentrated and
subjected to gel filtration (Fig. 2). Again, two assay conditions
used to look at PLD-stimulating activity resulted in definable peaks of
activity. GTPS alone (
) revealed two peaks of activity; the
peak with slower migration corresponded to fractions that contained
Arf. A peak (CFII) with faster migration lacked Arf but stimulated PLD
activity in the presence of GTP
S alone or enhanced activity in the
presence of added Arf and GTP
S. It is of interest to note that the
stimulating peak devoid of Arf also contained GTP
S binding
activity.
Figure 2:
Resolution of CFII factors and Arf by
chromatography through Ultrogel AcA 44. The pool of activity from the
DEAE-Sephacel column was fractionated and assayed as described under
``Experimental Procedures.'' Assays for PLD-stimulating
activity were the same as indicated in Fig. 1. Fractions 30-38
were collected as the CFII pool (180 ml, 450 mg of
protein).
Resolution of PLD-stimulating Activities on
Heparin-Sepharose and Hydroxylapatite
The CFII peak of PLD
stimulating activity with faster migration through gel filtration was
further resolved on heparin-Sepharose (Fig. 3). Multiple
activities appeared to emerge. Whereas a well defined peak of activity
with the capacity to stimulate PLD in the presence of GTPS alone
was observed, the capability to enhance PLD activity in the presence of
Arf and GTP
S was broadly distributed. ADP-ribosylation activity
and Arf immunoreactivity were not detectable in the heparin fractions
(data not shown). Interestingly, fractions in the latter part of the
elution, which could enhance stimulation of PLD in the presence of Arf,
were devoid of GTP
S binding activity. Fractions were selected to
represent potential resolved activities (Fig. 3). Pool A (early
in the elution) contained GTP
S binding activity but required Arf
to be effective. Pool B could activate PLD in the absence of Arf. Pool
C (later in the elution) represented a capacity to activate PLD in the
presence of Arf but had little intrinsic GTP
S binding activity.
S alone; a second pool (labeled B`) stimulated PLD
activity in the presence of GTP
S alone as well as showing
enhancement of stimulation by Arf. Another criterion for the separation
of the B and B` pools was either the presence or absence, respectively,
of the RhoA protein (see below). Selected fractions were pooled as
indicated and concentrated for further characterization.
Figure 4:
Chromatography of CFII factors through
hydroxylapatite. Pools A (top panel), B (middle
panel), and C (bottom panel) from the heparin-Sepharose
CL-6B column were fractionated through a Bio-Gel high performance
hydroxylapatite column and assayed as described under
``Experimental Procedures.'' Stimulation of PLD activity was
measured as described for Fig. 3. Fractions containing activity were
pooled as indicated in each panel.
Interactions among Arf and the CFII Pools
The
stimulatory affect of Arf is compared with activation obtained with the
CFII peaks in Fig. 5. These were all measured in the presence of
GTPS. Within the ranges of concentrations that could be tested,
both Arf and the CFII B` pool gave robust stimulation. The other three
factors were either less efficacious or less potent. For example, the
effect of CFII B was still increasing at the highest level of factor
tested, and the exact nature of the activation by Peak CFII C is hard
to evaluate due to an inhibitory action when high concentrations of the
pool were used.
Figure 5:
Activation of PLD with resolved cytosolic
factors. PLD activity was determined as described under
``Experimental Procedures.'' Assays contained 10 µM GTPS, 1 µg of PLD, and the indicated amount of cytosolic
factor.
The observations are of particular interest when the
action of Arf was examined in the presence of the cytosolic pools (Fig. 6). Besides their own component of stimulatory action, the
cytosolic factors increased the potency of Arf. This is most pronounced
for the CFII B` pool, which increased the potency of Arf by
approximately 10-fold. The other three factors induce a more modest
effect, about a 3-fold increase in the potency of Arf. At maximal
concentrations of Arf, the cytosolic factors gave no more than additive
effects. Therefore, the primary result of combining the factors with
Arf was a synergistic action at suboptimal concentrations of Arf. This
synergism with Arf was also observed when the factors were titrated
against fixed concentrations of Arf ( Fig. 6and Fig. 7).
Figure 6:
Concentration dependence of Arf, CFII A,
and CFII B for the activation of PLD in the presence of other cytosolic
factors. Enzyme activity was determined as described under
``Experimental Procedures.'' Assays contained 10 µM GTPS, 1 µg of PLD, and either 1.2 µg of porcine brain
Arf, 1 µg of CFII A, 5 µg of CFII B, 0.6 µg of CFII B`, or
1.5 µg of CFII C as indicated for titration curves. In addition,
assays contained the indicated variable amounts of Arf, CFII A, or CFII
B. Activities shown for the lowest concentration in the titrations were
essentially the same as no addition of the titrated
factor.
Figure 7:
Concentration dependence of CFII B` and
CFII C for the activation of PLD in the presence of other cytosolic
factors. Assays were performed with the indicated amounts of factors as
described in Fig. 6 and the indicated variable amounts of CFII B` or
CFII C.
When the factors were mixed with each other, synergistic action
could also be observed between factors CFII A and either CFII B` or
CFII C and between factors CFII B and either CFII B` or CFII C (Fig. 6). Since the cytosolic factors were not sufficiently
concentrated to saturate their effects on PLD, it is not clear whether
the synergism is due to a shift in the potency of the factors or to an
actual synergism in total efficacy.
S
produces no effect and a clear synergism between Arf and the two
factors was observed in the presence of the activating nucleotide.
Figure 8:
Differential requirement of GTPS by
CFII B and CFII B` for activation of PLD activity. PLD activity was
determined as described under ``Experimental Procedures.''
Assays contained 0.9 µg of PLD and as indicated, 10 µM GTP
S, 0.5 µg of Arf, and various amounts of the cytosolic
factors.
Evaluation of Potential CFII Factors
The ability
of the non-Arf cytosolic factors to stimulate PLD activity in the
presence of GTPS but the absence of Arf suggested the involvement
of other GTP-binding proteins. At least one of these is the RhoA
protein. When aliquots from the fractions eluted from hydroxylapatite (Fig. 4) were separated by SDS-polyacrylamide gel electrophoresis
and immunoblotted with antibodies for RhoA (Fig. 9), the
monomeric G protein was detected in fractions used for factor pools
CFII A and CFII B. The other two pools, CFII B` and CFII C, were devoid
of immunoreactivity for this protein. Actually, the presence or absence
of RhoA was part of the basis for defining the two pools from column B.
The fractions were also tested for the presence of Rho GDI (Fig. 9), a protein that inhibits GDP dissociation from
Rho(26, 27) . Interestingly, this protein was only
observed in the CFII B peak. The presence of Rho GDI in the B pool
could explain its apparent lower activity relative to pool A in spite
of a higher abundance of RhoA. Alternatively, pool A may contain
another stimulatory component. The latter possibility is emphasized by
the detection of RhoA in only the early fractions of the activity peak
derived from high performance hydroxylapatite chromatography of pool A;
stimulation by the latter fractions is indicative of a separate but,
perhaps, similar factor.
Figure 9:
Immunological detection of RhoA and Rho
GDI in the CFII pools. Aliquots (5 µl) of indicated fractions from
hydroxylapatite chromatography of pools A, B, and C shown in Fig. 4 were resolved by SDS-polyacrylamide gel
electrophoresis and subjected to immunoblot analysis with the indicated
antibodies as described under ``Experimental Procedures.''
The left lanes contained 60 ng of recombinant RhoA (rRhoA) or Rho GDI as indicated. Load refers to the
pool from heparin-Sepharose, which was applied to the respective column
being examined.
Fractions from the gel filtration procedure (Fig. 2) and the isolation on heparin-Sepharose (Fig. 3)
were also blotted for the presence of RhoA (data not shown). As
anticipated, the protein migrated with the higher mobility activity
during gel filtration. Both peaks A and B from the heparin column also
contained RhoA as anticipated. RhoA was not detected in the
preparations of either Arf or the enriched PLD used for these
experiments.
Effect of Recombinant RhoA, Cdc42, and Other Selected
Monomeric G Proteins on PLD Activity
While RhoA was present in
stimulatory fractions, the effect could be due to the presence of other
GTP-binding proteins. Therefore, recombinant RhoA, produced in Sf9
cells, was tested for its ability to stimulate enriched PLD activity (Fig. 10). Activation of PLD activity was observed with
concentrations of the protein in the low nanomolar range.
Unfortunately, an unidentified inhibitory activity in the preparation
prevented observation of the protein at higher concentrations, which
might provide greater activation. Of equal interest was the ability of
RhoA to show synergistic activation of PLD when combined with Arf in
the form of myrArf5. The effects of RhoA, but not Arf, were attenuated
by the addition of Rho GDI, presumably by inhibiting nucleotide
exchange on RhoA. The relatively modest activity of RhoA alone and the
enhancement of Arf activity resembles the action of CFII A and CFII B,
the cytosolic factor pools that contain this small G protein. However,
Rho GDI was only partially effective in blocking the action of CFII A
(data not shown), consistent with the likelihood that there is another
stimulatory factor in this pool.
Figure 10:
Activation of PLD with recombinant RhoA
and Cdc42. Enzyme activity was measured as described under
``Experimental Procedures,'' except that the reactions were
performed in a 60-µl volume. Assays contained 1 µM
GTPS, 2.5 µg of PLD, and the indicated concentrations of
recombinant RhoA or Cdc42 either in the presence or absence of 35
nM myrArf5. As indicated, assays also contained 380 nM Rho GDI.
The recombinant RhoA from Sf9 cells
is modified, at least in part, by isoprenylation of its C
terminus(18) . In order to assess the requirement for this
modification, a mutant RhoA, which contained the Val-14 activation
mutation, was expressed in E. coli(18, 21) ,
purified, and mixed with PLD. Only very low activity (2-5 pmol of
PC hydrolyzed) was observed, even at high concentrations (0.5-2
µM) of the nonmodified protein. A small synergism was
observed when Arf was added in conjunction with increasing
concentrations of the rRhoA(Val-14) (57-71 pmol of PC hydrolyzed versus 50 pmol with Arf alone). This indicates that the
nonmodified protein had activity but that it was poor relative to the
RhoA expressed in Sf9 cells, which is posttranslationally modified,
minimally with an isoprenyl group.
. This seems unlikely
in view of the linearity of the assay (see Fig. 6in the
companion paper(13) ), and no evidence of nonspecific substrate
degradation or preservation was observed when PC labeled in the acyl
moieties was utilized. Finally, the enhancement of stimulation by CFII
B` by GTP
S may be due to the coincident presence of a stimulating
G protein in the CFII B` pool or to the potential presence of a
GTP-binding protein associated with the PLD preparation itself but
which is not effective in the absence of other factors.
(9, 13) in the expression of Arf-dependent PLD
activity is unknown. The discernment of RhoA as a regulatory factor for
this activity may offer part of the explanation. The dissociation of
Rac from Rho GDI was stimulated by acidic phospholipids; PIP
was particularly effective(28) . If this is also the case
with RhoA, PIP
could facilitate its activation in the CFII
B preparation. Interestingly, acidic lipids, especially
PIP
, had marked effects on activation of Arf(29) .
In contrast to these results, acidic lipids other than PIP
and phosphatidylinositol 3,4,5-trisphosphate (PIP
)
are not effective in the PLD assay. It is possible, if not probable,
that PIP
has multiple effects; facilitation of G protein
activation is one of these that contributes to the overall mechanism of
regulation observed in these in vitro experiments.
and PIP
and that both
these regulatory proteins and PIP
/PIP
facilitate the activity of PLD. This may indicate that this
lipase is one of the key mediators of the action of the Rho family in
cells.
Table: Effect of monomeric G proteins on PLD activity
S, guanosine 5`-O-(3-thiotriphosphate); GDI, GDP
dissociation inhibitor; CFII, cytoplasmic factor II, cytosolic
activators of phospholipase D other than Arf; G protein, GTP-binding
regulatory protein; myrArf5, myristoylated recombinant Arf5; TPCK,
tosylphenylalanyl chloromethyl ketone; TLCK, N
-p-tosyl-L-lysine
chloromethyl ketone; PIP
, phosphatidylinositol
4,5-bisphosphate; PIP
, phosphatidylinositol
3,4,5-trisphos-phate; PC, phosphatidylcholine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.