(Received for publication, November 9, 1995)
From the
Phospholipase D (PLD) which was partially purified from
membranes of porcine brain could be stimulated by multiple cytosolic
components; these included ADP-ribosylation factor (Arf) and RhoA,
which required guanine nucleotides for activity, and an unidentified
factor which activated the enzyme in a nucleotide-independent manner
(Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C.(1995) J. Biol. Chem. 270, 14944-14950). Here, we report
purification of the latter factor, its identification as the
isoform of protein kinase C (PKC
), and characterization of its
regulation of PLD activity.
Stimulation of PLD by purified PKC
or recombinant PKC
(rPKC
) occurred in the absence of any
nucleotide and required activators such as Ca
or
phorbol ester. This action was synergistic with stimulation of PLD
evoked by either Arf or RhoA. Dephosphorylation of rPKC
with
protein phosphatase 1 or 2A resulted in a loss of its kinase activity,
but had little effect on its ability to stimulate PLD either alone or
in conjunction with Arf. Staurosporine inhibited the kinase activity of
PKC
without affecting activation of PLD. Finally, gel filtration
of PKC
that had been cleaved with trypsin demonstrated that
stimulatory activity for PLD coeluted with the regulatory domain of the
enzyme. These data indicate that PKC may regulate signaling events
through direct molecular interaction with downstream effectors as well
as through its well characterized catalytic modification of proteins by
phosphorylation.
Hydrolysis of phospholipids by phospholipase D (PLD) ()yields phosphatidic acid and the respective head groups.
Phosphatidic acid is an apparent second messenger for multiple
signaling events, and its production can be stimulated by a variety of
stimuli, including hormones that activate G protein-mediated pathways
and growth factors that function through tyrosine kinases. The
phosphatidic acid produced may act directly in downstream functions or
serve as a precursor for the production of diacylglycerol or
lysophosphatidic acid and their subsequent sequelae. (For more recent
reviews, see (1, 2, 3) .)
The stimulation
of PLD in intact cells can be readily observed through measurement of
the production of phosphatidic acid or phosphatidyl alcohols. However,
molecular details of the mechanisms that effect this regulation are
just beginning to emerge. Experiments which used permeabilized and
broken cell preparations and assays which utilized exogenous lipid for
substrate have elucidated at least three unique regulators of PLD
activity. The first of these was revealed from the development of an
assay which could use an exogenous lipid substrate to measure
regulation of a PLD activity by guanine nucleotides. The activity
showed a marked dependence on the inclusion of phosphatidylinositol
4,5-bisphosphate (PIP) in the phospholipid vesicles which
contained the labeled substrate, phosphatidylcholine(4) . This
unique requirement was confirmed with another system in which the
utility of employing phosphatidylinositol 3,4,5-trisphosphate to
measure PLD activity was also demonstrated(5) . While the
molecular action of PIP
has not yet been determined, its
requirement for the measurement of PLD that has been substantially
purified (6) suggests direct action as a cofactor or regulator.
A second PLD activity that can be detected with an assay that uses a
mixture of phosphatidylcholine and oleate (7) does not appear
to be regulated by guanine nucleotides and can be separated from the
PIP
and G protein-dependent
activity(6, 8) .
Phospholipase D activity, measured
either in membranes or in a partially purified form can be stimulated
by cytosolic factors. Indeed, an enriched form of PLD activity is
essentially dependent on these factors(6) . The first of these
factors to be purified and identified was Arf. It was purified from
brain cytosol as a factor which either conferred guanine nucleotide
sensitivity on a partially isolated PLD activity (4) or could
reconstitute guanine nucleotide stimulated PLD activity in
permeabilized cells(9) . The Arf proteins are GTP-binding
proteins that occupy a unique niche in the Ras superfamily of small G
proteins(10) . All of the Arf proteins tested to date are
effective activators of PLD activity(6, 8) . The first
Arf protein was identified as a factor which facilitated ADP-ribosylation (hence Arf) of G proteins by cholera toxin(11) . More recently, Arf has
been identified as a functional component of pathways for protein
traffic in cells; specifically, it is a cytosolic protein required for
binding of coatomer and formation of coated vesicles from Golgi
membranes (see (12) for review). The speculation that
regulation of PLD activity may also be important in protein transport
is supported by the observations that PLD activity is higher in
membranes enriched in Golgi (13) . Further, basal PLD activity
appeared to be constitutively active and no longer sensitive to Arf in
Golgi membranes obtained from cells resistant to the fungal metabolite,
brefeldin A, an inhibitor of protein transport through Golgi.
A
second group of cytosolic proteins, which have been identified as
regulators of PLD activity, are members of the Rho family of monomeric
G proteins. Experiments with membranes derived from neutrophils showed
that GTPS stimulation of PLD activity could be attenuated by
treatment with a Rho guanine nucleotide-dissociation inhibitor (GDI)
protein(14) . Exton and colleagues (15) demonstrated
that treatment of plasma membranes from rat liver with Rho GDI reduced
guanine nucleotide-stimulated PLD activity; this activity could be
restored with a purified RhoA protein. Singer et al.(16) demonstrated more directly that two purified recombinant
proteins from the Rho subfamily, RhoA and Cdc42, could stimulate an
enriched preparation of PLD and that this stimulation was synergistic
with activation induced by Arf. The synergism observed in vitro among the three regulatory mechanisms (PIP
, Arf, and
Rho) may suggest some cooperative regulation of PLD activity by
multiple pathways in vivo.
Another identified pathway for
regulation of PLD activity is through protein kinase C (PKC). The PKC
family has numerous members that have been studied extensively (for
reviews, see (17) and (18) ). These enzymes are
currently divided into three subgroups. The classical proteins (cPKC),
consisting of the ,
, and
isoforms, are stimulated by
Ca
, diacylglycerol, and phosphatidylserine. The new
isoforms (nPKC;
,
,
,
, µ) are not regulated
by Ca
and the atypical members of the family (aPKC;
and
) appear to be regulated by second messengers other than
diacylglycerol. Phorbol esters, which mimic diacylglycerol and activate
PKC, have been used in numerous experimental paradigms which utilize
intact cells to show stimulation of PLD activity. Similarly,
down-regulation of PKC in various cells has led to attenuation of
hormone-regulated PLD activity and thus suggested a functional role for
these enzymes (see Refs. 1, 2, and 19 for reviews of earlier work).
Recent experiments by Lambeth and colleagues (20) have
demonstrated that the addition of classical forms of PKC, but not the
new or atypical forms, can stimulate PLD activity in membranes from
neutrophils in an ATP-dependent fashion. The addition of the classical
forms of PKC to Chinese hamster lung fibroblast membranes could also
evoke a stimulation of PLD activity; in this case, the stimulation
could be obtained in the apparent absence of ATP(21) . We have
previously reported resolution of a cytosolic factor that could
stimulate the activity of partially purified PLD in the absence of any
nucleotide(16) . We have purified this factor and now identify
it as PKC. Like the resolved factor, purified PKC
and the
recombinant protein (rPKC
) stimulate PLD activity in the absence
of nucleotides. This action of PKC is synergistic with stimulation
evoked by either Arf or RhoA, requires the presence of an activator
such as Ca
or phorbol 12-myristate 13-acetate (PMA),
and most interestingly, is totally independent of protein kinase
activity.
The concentrated pool of activity was diluted 2-fold with Solution B containing 4000 mM NaCl and loaded onto a fast protein liquid chromatography phenyl-Superose HR10/10 column (Pharmacia Biotech Inc.) which had been equilibrated with Solution B (2000 mM NaCl). The column was washed with 10 ml of equilibration buffer, and bound protein was eluted at 0.5 ml/min with a 90-ml linear descending gradient of NaCl (2000-0 mM) in Solution B followed by a 30-ml isocratic step without added NaCl. Stimulatory fractions (36 ml centered around 400 mM NaCl) were combined with protease inhibitors and concentrated to 12 ml. The concentrate was diluted with 8 ml of Solution B containing 2000 mM NaCl and subjected to a second step of phenyl-Superose chromatography, with elution facilitated by a 60-ml descending gradient of NaCl (1000-0 mM) in Solution B followed by a 20-ml isocratic step without added NaCl. Activity eluted at the same salt concentration as before; these factions were combined with protease inhibitors and concentrated to 0.5 ml.
The concentrated protein was
diluted to 5 ml with Solution B and loaded onto a 5-ml Hi-Trap heparin
column (Pharmacia). Bound protein was eluted at 1 ml/min with a
two-step gradient of 0-750 mM NaCl (15 ml), then
750-2000 mM NaCl (10 ml) in Solution B. Fractions which
contained the purified 80-kDa stimulatory factor (PKC) were
identified by SDS-PAGE and silver staining and stored at -80
°C.
A DNA construct encoding glutathione S-transferase (GST) fused through a thrombin cleavage site to
human RhoA with four additional amino acids at its amino terminus
(Ile-Leu-Glu-Ser) was assembled in a baculovirus transfer vector
derived from the pGEX-KG plasmid(26) . The construct was
inserted into baculovirus by recombination and used to direct
expression of protein in Sf9 cells as described elsewhere(24) .
The cells were lysed by N cavitation in Solution C (50
mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM DTT, and
10 µM GDP) containing PMSF/TPCK/TLCK, and the lysate was
cleared of particulate material by centrifugation at 100,000
g for 60 min. Recombinant GST-RhoA was purified from the
supernatant by chromatography through glutathione-Sepharose (Pharmacia)
following standard procedures. The protein was cleaved with thrombin
(1:600 thrombin:GST-RhoA by weight) for 24 h at 4 °C in Solution C
containing 1% n-octyl-
-D-glucopyranoside and 2.5
mM CaCl
; free GST and uncleaved GST-RhoA were
removed by a second passage through glutathione resin.
Separation of a
micelle-bound regulatory domain from the catalytic domain was performed
by a modification of a procedure described by Lee and
Bell(29) . Triton X-100 (1.5% w/v) mixed micelles containing 15
mol % PS and 5 mol % DOG were diluted 10-fold into the quenched digest,
and the resultant solution was incubated at 20 °C for 10 min. A
100-µl aliquot of the solution was applied to a column (9
0.7 cm) of Ultrogel AcA 44 resin and eluted with digestion solution
containing 100 mM NaCl and 0.02% (w/v) Triton X-100. Fractions
of 120 µl were collected.
The activity of protein kinase C was
measured by the phosphorylation of a synthetic peptide substrate (amino
acids 4-14 of myelin basic protein (Calbiochem)) using a
modification of a Whatman P-81 ion exchange paper binding assay
described previously(31) . Reactions were performed in a
30-µl volume at 37 °C in 20 mM Tris-Cl, pH 7.5, 5
mM MgCl, 10 µM [
-
P]ATP (2-5
10
cpm), 25 µM substrate peptide, 0.2 µM CaCl
, and 0.1% (w/v) Triton X-100 mixed micelles
containing 15 mol % PS and 5 mol % DOG, and subsequently terminated by
pipetting a 15-µl aliquot onto P-81 paper. Where indicated,
CaCl
, PS, and DOG were omitted from the assay mixture. In
some instances, the phospholipid vesicles described for the PLD assay
were substituted for Triton X-100 mixed micelles.
Figure 1:
Purified cytosolic
factor and rPKC. The cytosolic factor which stimulated PLD in a
nucleotide-independent fashion and rPKC
were purified as described
under ``Experimental Procedures.'' Samples (50 ng) of each
were resolved by SDS-PAGE on a 12% acrylamide gel. Proteins were
visualized by staining with silver.
The yield of purified
PKC from porcine brain (approximately 150 µg from 30 g of
cytosolic protein) was insufficient for extensive characterization of
its interaction with PLD and other cytosolic factors. Therefore,
recombinant protein was produced with a baculovirus expression
system(25) . As shown in Fig. 1, a homogenous
preparation of rPKC
was obtained from the cytosol of infected
cells after five steps of chromatography (see ``Experimental
Procedures'') and a yield of 0.5 mg/liter of culture. The
stimulation of PLD activity using the recombinant protein was similar
to that of native porcine PKC
.
Figure 2:
Activation of PLD by rPKC. Assays of
PLD activity were conducted for 30 min as described under
``Experimental Procedures.'' Assays contained 0.9 µg of
PLD, the indicated nucleotide (10 µM), and variable
amounts of rPKC
. Activities shown for the lowest concentration of
rPKC
in the figure were essentially the same as obtained in the
absence of rPKC
.
Figure 3:
Dependence of the activation of PLD on the
concentration of rPKC and Arf. Assays for enzyme activity were
conducted for 7.5 min as described under ``Experimental
Procedures.'' Assays contained 10 µM GTP
S, 10
µM ATP, 0.9 µg of PLD, and the indicated fixed or
variable concentrations of rPKC
and Arf. Activities shown for the
lowest concentration in the titrations were essentially the same as for
no addition of the titrated factor.
Routine assays of PLD activity measure the release of water-soluble choline from phosphatidylcholine. The lipid products of phosphatidylcholine hydrolysis in the presence of PKC and Arf were also analyzed to ensure that the synergism elicited by these two proteins reflects only the stimulation of PLD activity, not a stimulation of PLD in conjunction with another lipase. Phosphatidic acid was the only detectable product of phosphatidylcholine hydrolysis stimulated by PKC and Arf either alone or in combination (summarized in Table 1). Production of phosphatidyl alcohol and concomitant reduction in phosphatidic acid formation occurred in the presence of ethanol, as expected for the transphosphatidylation reaction characteristic of PLD enzymes (for review, see (32) ).
Synergism was also obtained
when PLD was stimulated with either rPKC or Arf in the presence of
recombinant RhoA (rRhoA) (Fig. 4). The rRhoA required
preactivation with GTP
S to be effective in this assay. While the
RhoA produced little stimulation by itself, it enhanced the activity of
the other two factors 3-4-fold. A similar increase occurred for
the combination of PKC
and Arf. Interestingly, the synergistic
action was higher (6.3-fold) when all three factors were employed
together.
Figure 4:
Activation of PLD by rRhoA, Arf, and
rPKC. Phospholipase D activity was determined as described under
``Experimental Procedures.'' Assays were conducted for 7.5
min and contained 0.9 µg of PLD, 10 µM GTP
S, 10
µM ATP, and combinations of 1 µM rRhoA, 20
nM Arf, and 10 nM rPKC
as
indicated.
The modulators described above
activate PKC through interactions with its regulatory (lipid-binding)
domain. Staurosporine inhibits the kinase activity of the
proteolytically generated catalytic fragment of PKC while having no
effect on the binding of phorbol esters to the regulatory
domain(33) . Concentrations of staurosporine which inhibited
77% of the kinase activity of PKC had little effect on activation
of PLD by the enzyme or its synergism with Arf (Table 2, part B).
Figure 5:
Effect of dephosphorylation of rPKC
by protein phosphatases 1 (PP1) and 2A (PP2A) on its
kinase activity and ability to activate PLD. Dephosphorylation
reactions were performed with the indicated concentrations of protein
phosphatase catalytic subunits as described under ``Experimental
Procedures.'' Top, samples of phosphatase-treated
rPKC
(75 ng) were resolved by SDS-PAGE on 9% acrylamide gels and
visualized by staining with silver. Bottom panels, assays for
PLD and kinase activity were performed for 30 min and 15 min,
respectively, as described under ``Experimental Procedures.''
Data are expressed as percent activity of phosphatase-treated rPKC
relative to rPKC
treated with the same buffer but without the
phosphatase. The PLD assays contained 0.9 µg of PLD, 30 nM rPKC
, 1.25 µM okadaic acid, and 1 µM GTP
S in the presence of 100 nM Arf or no added
nucleotides in the absence of Arf. The amount of phosphatidylcholine (PC) hydrolyzed by untreated rPKC was 8 pmol without Arf and
83 pmol with Arf. The phosphorylation of substrate peptide was
monitored in the presence of PS, DOG, Ca
, 1.25
µM okadaic acid, and 30 nM rPKC
. Untreated
rPKC
phosphorylated 85 pmol of
peptide.
Figure 6:
Separation of the regulatory and catalytic
domains of trypsin-treated rPKC by gel filtration. Recombinant
PKC
was treated with trypsin and subjected to chromatography
through Ultrogel AcA 44 as described under ``Experimental
Procedures.'' Top panel, aliquots (19 µl) of the
fractions were resolved by SDS-PAGE on 12% acrylamide gels and
polypeptides were visualized by silver staining. Lanes designated rPKC and Digest contained 125 ng of the protein
before and after proteolysis, respectively. Bottom panel,
aliquots (2.5 µl) of the fractions were analyzed for their ability
to activate PLD or phosphorylate substrate peptide as described under
``Experimental Procedures.'' Assays for PLD were conducted
for 30 min and contained 0.9 µg of PLD, 10 µM GTP
S, and 10 µM ATP in the presence or absence
of 100 nM Arf. Phosphorylation of substrate peptide was
assessed after a 15 min incubation in the presence or absence of PS,
DOG, and Ca
.
Analysis by SDS-PAGE (top panel) indicated that digestion
of the 80-kDa rPKC (rPKC lane) resulted in almost
complete conversion (Digest lane) into fragments which
included those of 50-52 kDa, the reported size (29) of
the kinase domain, and 34 kDa, the size of the regulatory domain. An
antibody raised against a peptide corresponding to the 12
carboxyl-terminal amino acids of PKC
recognized only the
50-52-kDa fragments (data not shown). Additional fragments of
40-43 kDa were also produced which probably represent further
degradation of the catalytic domain; they did not contain the
lipid-binding domain, based on their inability to bind to mixed
micelles of Triton X-100 and lipid (see below). The 21-kDa protein in
the rPKC and Digest lanes is soybean trypsin
inhibitor; staining in the 66-kDa region is an artifact. Proteolysis
had only a minor effect on the ability of PKC to activate PLD;
undigested PKC (30 nM) in the presence of Ca
stimulated 86 and 26 pmol of phosphatidylcholine hydrolysis with
and without 100 nM Arf, respectively. This compares with
stimulated hydrolysis of 102 and 21 pmol of phosphatidylcholine after
digestion.
When mixed micelles of Triton X-100, PS, and DOG were
added to trypsinized PKC and the resulting solution was applied to
an AcA 44 size exclusion column, the elution pattern shown in the top panel of Fig. 6was obtained. The majority of the
34-kDa fragment and a detectable residue of undigested PKC eluted well
before the 50-52- and 40-43-kDa pieces. The larger size of
the regulatory domain is expected from its association with the mixed
lipid/detergent micelles. Stimulatory activity for phospholipase D (bottom panel) coeluted with the 34-kDa fragment; this
indicates that the regulatory domain alone may be sufficient to provide
stimulation of PLD by PKC. A peak of protein kinase activity eluted
well after the major portion of PLD stimulatory activity, and
overlapped almost exactly with the 50-52-kDa fragments. This
kinase activity is not dependent on Ca
or
phospholipids, as expected for removal of the regulatory domain. It is
apparent that the isolated kinase domain is not an effective activator
of PLD, even when assayed in the presence of 10 µM ATP.
PKC was identified as a cytosolic factor from porcine
brain capable of stimulating PLD activity in the absence of added
nucleotides. This action of the enzyme through a molecular mechanism
which did not involve its kinase activity was supported by three other
experimental findings: 1) staurosporine significantly inhibited kinase
activity at a concentration which did not inhibit activation of PLD, 2)
dephosphorylated (kinase-deficient) PKC stimulated PLD, and 3) the
isolated active kinase domain produced by trypsin treatment of the
enzyme did not stimulate activity, whereas the regulatory domain was an
effective activator. This robust phosphorylation-independent
stimulation by PKC
was observed with a PLD activity that had been
extracted from porcine brain membranes, was purified away from other
known regulators such as Arf and RhoA, and which displayed essentially
no basal activity. This nucleotide independence of PKC
action
concurs with the report of a 2-3-fold stimulation of PLD activity
in the absence of added ATP following addition of the classical
isotypes of PKC to a crude membrane preparation from Chinese hamster
lung fibroblasts(21) . In contrast, stimulation of PLD activity
in human neutrophil membranes was reported to require phosphorylation
of a target protein by added cPKCs(20) . The latter
observations may reflect the regulation of a different isotype of PLD
or an alternative pathway in neutrophil membranes for activation of the
same PLD.
The phosphorylation-independent mechanism for activation
of PLD by PKC remains unidentified. Here, as in other
studies(20, 34) , stimulation of PLD required
activators of PKC (e.g. Ca
or PMA). Low
micromolar concentrations of PIP
were also needed for
activation (data not shown), although the general requirement of this
lipid for PLD activity makes it unclear whether this effect involves a
direct interaction between PIP
and PKC. It should be noted
that the PIP
-containing vesicles used in the PLD assay
promoted activation of PKC as assessed by stimulation of its kinase
activity (data not shown). Mixed micelles of detergent and PIP
have also been reported to stimulate the kinase activity of
PKC(35) , presumably reflecting the interaction of anionic
lipids with its lipid-binding domain. Current models predict that
activators of PKC promote a molecular transformation that disinhibits
the kinase active site of the enzyme and exposes membrane association
sites in its regulatory domain. Presumably, this same molecular
transformation induces an interaction site for stimulation of PLD.
Binding of PKC to an anionic phospholipid surface could facilitate this
activation of PLD through increased protein-protein interaction. In the
absence of pure PLD it is not clear whether this regulation involves a
direct complex of PKC with PLD, or an interaction between PKC and a
protein which in turn affects PLD.
Protein kinase C not only
stimulates PLD activity, but also enhances the interaction of PLD with
other activators. Clear synergism of PKC with Arf or RhoA was observed
with the partially purified PLD. This corresponds with earlier reports
of the potentiation of GTPS-stimulated PLD by phorbol ester in
permeabilized cells and cell-free systems (reviewed in(3) ).
Cooperative stimulation of PLD activity was also observed when RhoA and
PKC were added to HL60 membranes which had been pretreated with Rho GDI
to remove endogenous Rho proteins(36) . Arf has been shown to
act synergistically with members of the Rho
family(16, 37, 38) , and also with a
``50-kDa'' fraction prepared by gel filtration of cytosol
from HL-60 cells (39) and human neutrophils (40) which
contains members of the Rho family and at least one other unidentified
activator. It should be noted that RhoA and the nucleotide-independent
factor identified here as PKC
coeluted during gel filtration of a
cytosolic fraction from porcine brain(16) .
Inactivation of
the kinase activity of PKC by dephosphorylation had no effect on
its synergistic activation of PLD with Arf. Therefore, this action also
arises through a phosphorylation-independent mechanism. Since both PKC
and Arf alone activate PLD, their synergism probably involves a
cooperative interaction through different sites on the enzyme.
Interestingly, treatment of rat basophils with phorbol esters is
reported to promote Arf binding to the Golgi membrane(41) .
Treatment of HL-60 cells with PMA or fMet-Leu-Phe promotes
translocation of Arf to membranes and correlates with increased
stimulation of PLD by GTP
S(42) . It is unclear how factors
that promote PKC translocation to membranes in turn promote Arf
translocation, but it is possible that membrane-associated PKC serves
as a nucleating agent for the formation of an active PLD complex that
includes other activators such as Arf and RhoA. It seems probable that
the most efficacious regulation of PLD may occur through the formation
of highly cooperative complexes which are facilitated by the merger of
a variety of signal transduction pathways.
The coelution of PLD
stimulatory activity with the regulatory domain, and not the kinase
domain, during gel filtration of PKC treated with trypsin, indicates
that this lipid-binding portion of PKC contains a site for
complexation with PLD and/or related proteins. While it is possible
that a small amount of uncleaved PKC detected with the regulatory
domain contributed to this activity, it cannot account for the total
activity observed; fractions of the regulatory domain were estimated to
contribute less than 0.5 nM uncut PKC to the PLD assays, and
activation or synergism with Arf has never been detected at
subnanomolar concentrations of PKC (see Fig. 2and Fig. 3).
Overall, these studies have important implications
for the function of PKC in the cell. Following an agonist-induced
increase in cytosolic free Ca and diacylglycerol (e.g. through activation of phospholipase C), PKC
translocates to the cell membrane where it is tethered in an active
form by lipids (diacylglycerol, PS, and PIP
) and receptor
proteins, such as receptors for activated protein kinase C
(RACKs)(43) . Here, it may effect signal transduction in a
bimodal manner, through phosphorylation of substrates and, as
demonstrated here for PLD, by direct protein-protein interaction. It
will be of interest to determine whether this latter action of PKC is
operative on other downstream effectors of the kinase and whether this
dual mode of regulation may be a functional mechanism used by other
protein kinases.