Specificity and Structural Requirements of Phospholipase C-
Stimulation by Rho GTPases Versus G Protein 
Dimers*
Daria
Illenberger
,
Claudia
Walliser,
Bernd
Nürnberg,
Maria
Diaz
Lorente, and
Peter
Gierschik
From the Department of Pharmacology and Toxicology, University of
Ulm, Albert-Einstein-Allee 11, Ulm D-89081, Germany
Received for publication, August 13, 2002, and in revised form, October 25, 2002
 |
ABSTRACT |
Phospholipase C-
2
(PLC
2) is activated both by heterotrimeric G protein
- and 
- subunits and by Rho GTPases. In this study, activated
Rho GTPases are shown to stimulate PLC
isozymes with the rank order
of PLC
2 > PLC
3
PLC
1.
The sensitivity of PLC
isozymes to Rho GTPases was clearly different
from that observed for G protein 
dimers, which decreased in the
following order: PLC
3 > PLC
2 > PLC
1 for
1
1/2 and
PLC
2 > PLC
1 >>>
PLC
3 for
5
2. Rac1 and Rac2
were found to be more potent and efficacious activators of
PLC
2 than was Cdc42Hs. The stimulation of
PLC
2 by Rho GTPases and G protein 
dimers was
additive, suggesting that PLC
2 activation can be
augmented by independent regulation of the enzyme by the two stimuli.
Using chimeric PLC
1-PLC
2 enzymes, 
dimers, and Rho GTPases are shown to require different regions of
PLC
2 to mediate efficient stimulation of the enzyme.
Although the catalytic subdomains X and Y of PLC
2 were
sufficient for efficient stimulation by 
, the presence of the
putative pleckstrin homology domain of PLC
2 was
absolutely required for the stimulation of the enzyme by Rho GTPases.
Taken together, these results identify Rho GTPases as novel PLC
regulators, which mediate PLC
isozyme-specific stimulation and are
potentially involved in coordinating the activation of
PLC
2 by extracellular mediators in intact cells.
 |
INTRODUCTION |
Many extracellular signaling molecules elicit intracellular
responses by activating inositol phospholipid-specific phospholipases C
(PLCs),1 which hydrolyze
phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) to
produce the second messengers inositol 1,4,5-trisphosphate and
diacylglycerol. These two second messengers modulate intracellular events through the regulation of intracellular free Ca2+
and protein kinase C isozymes, respectively. The mammalian PLC isozymes
can be divided into four major families: PLC
, PLC
, PLC
, and
PLC
(1). The PLC
and PLC
subclasses have been shown to be
regulated through G protein-coupled and protein-tyrosine kinase-linked receptors, respectively. The mechanisms by which PLC
isozymes and PLC
are coupled to membrane receptors are less well understood (for recent reviews, see Refs. 1-4). Stimulation of
PLC
, of which four isozymes
(PLC
1-PLC
4) are known, is mediated by
members of the
q subfamily of G protein
subunits
and, excepting PLC
4, by G protein 
dimers (1-4).
Activated
q subunits stimulate PLC
in the rank order
of efficacy of PLC
1
PLC
3 > PLC
2. PLC
4 is also activated by
q subunits. The sensitivity of PLC
isozymes to 
dimers decreases in the order: PLC
3 > PLC
2 > PLC
1 (3, 4).
Mammalian PLC
isoforms are differentially expressed in various
tissues and cell types (5, 6). At the protein level, PLC
1 is highly expressed in the central nervous system
but is also present in several other tissues, e.g. adrenal
gland, parotid gland, lung, and kidney (7-9). The PLC
2
polypeptide is present at high levels in neutrophils and cultured
myeloid cells but has also been detected in other cells types and
tissues, including platelets (10), T lymphocytes (11), osteoblasts
(12), vascular and tracheal smooth muscle cells (13, 14), cerebellum
(12), spleen, and thymus (15). Myeloid cells have been found to contain both PLC
2 and PLC
3 (9, 16). However,
PLC
2 appears to be predominantly important in these
cells, because inactivation of the PLC
2 gene caused an
almost complete loss of formyl peptide receptor-stimulated inositol
phosphate formation in mouse neutrophils (15). Furthermore,
PLC
2, but not PLC
3, was activated by
complement C5a and formyl peptide receptors in transfected cells (17). The latter findings are intriguing in light of the fact that
PLC
3 has been shown to be stimulated to a remarkable
extent by G protein 
dimers in cell-free assays (9). In addition
to myeloid cells, various other cell types and tissues contain the
PLC
3 isoform (8, 9). In contrast, the
PLC
4 protein shows a more limited tissue distribution
and is primarily found in the retina and in specific regions of the
brain (18, 19).
We have previously reported the identification of a
PLC
2-stimulating GTP-binding protein present in
cytosolic fractions of bovine neutrophils (20). This cytosolic protein
was shown to be a member of the Rho subfamily of GTPases, Cdc42Hs
and/or Rac, associated with the Rho GDP dissociation inhibitor LyGDI
(21). Rho GTPases form a subgroup of the Ras superfamily of GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions, including gene expression, cell cycle progression, and
reorganization of the actin cytoskeleton (22-25). The activity of the
Rho GTPases is determined by the ratio of their GTP/GDP-bound forms,
which is regulated by at least three regulatory proteins: guanine
nucleotide dissociation inhibitors, guanine nucleotide exchange
factors, and GTPase-activating proteins (25).
Using purified proteins, we have previously demonstrated that
PLC
2 is activated by GTP
S-liganded Cdc42Hs and Rac1,
but not by RhoA, through direct protein-protein interaction (21). This stimulation has been shown to be independent of LyGDI but to require both C-terminal processing of the Rho GTPases and the integrity of
their effector regulating domain (21). Like G protein 
dimers,
activated Rho GTPases stimulated a deletion mutant of PLC
2, PLC
2
, lacking a C-terminal
region necessary for stimulation by G protein
q subunits
(21). The specificity of PLC
stimulation by Rho GTPases and the
mechanisms of enzyme activation by Rho GTPases remained unknown. The
goal of the study was to elucidate the sensitivity of PLC
isozymes
to stimulation by Rho GTPases versus G protein 
dimers, known activators of these enzymes, and to identify and to
compare the structural requirements of PLC
stimulation by these two
regulatory proteins. The results show that the three PLC
isozymes
tested, PLC
1, PLC
2, and
PLC
3, are differentially sensitive to stimulation by
activated Rho GTPases and 
dimers. The specificity of Rho
GTPase-mediated PLC
stimulation also differs from that reported for
q-mediated PLC
stimulation, with PLC
2
being considerably less sensitive than PLC
1 and
PLC
3 (1-4). This study identifies PLC
2
as the PLC
isozyme most sensitive to stimulation by Rho GTPases,
especially Rac1 and Rac2. Using PLC
1-PLC
2
chimeras constructed on the basis of structural domains predicted by
the crystal structure of PLC
1, we demonstrate that the
presence of the catalytic subdomains X and Y of PLC
2 is
sufficient for 
-dimer stimulation. In contrast, the presence of
the putative pleckstrin homology (PH) domain of PLC
2 is
absolutely required for stimulation of the enzyme by activated Rho
GTPases. Taken together, the results demonstrate, for the first time,
the unique regulation of the activity of PLC
2 by
monomeric GTPases and G protein 
dimers requiring different
structural elements of this enzyme.
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EXPERIMENTAL PROCEDURES |
Recombinant PLC
Isoforms, C-terminally Deleted PLC
s
Mutants, and PLC
1-PLC
2
Chimeras--
Construction of recombinant baculoviruses for expression
of the bovine PLC
1 and human PLC
2 have
been described (26, 20). The cDNA of human PLC
3 (27)
was cloned into the EcoRI site of the baculovirus transfer
vector pVL1393 (Invitrogen, Carlsbad, CA).
The cDNAs of C-terminally deleted PLC
constructs were generated
such that the proteins retained their C-terminal-most portions (GENPGKEFDTPL, PLC
1; QDPLIAKADAQESRL,
PLC
2; and GADSESQEENTQL, PLC
3), which had
been used as synthetic peptides to generate PLC
subtype-specific
antisera in rabbits (28). PLC
2
, a deletion mutant of
human PLC
2, lacking a C-terminal region
(Phe819-Glu1166) necessary for stimulation by
q, has been shown previously to be indistinguishable
from wild-type PLC
2 in terms of its interaction with
PI-4,5-P2, Ca2+, and 
dimers (29). The
deletion mutants PLC
1
and PLC
3
, lacking the corresponding C-terminal regions,
Val817-Leu1203 and
Ala873-Ser1216, respectively, were generated by
the PCR overlap extension method (30). Two PCR amplifications were
performed using bovine PLC
1 cDNA
(GenBankTM accession number J03137) cloned into
pVL1392 as template and the following two pairs of oligonucleotides as
primers: 5'-CGTGGATTCATCTAACTATATGCC-3' (upstream, sense),
5'-GTTTTCTCCTTCATATCGGATTGGATTTGACAAAGC-3' (internal, antisense),
5'-TCCAATCCGATATGAAGGAGAAAACCCAGGAAAAGAG-3' (internal, sense), and
5'-CGCATGTTAACCCAAAGATATGG-3' (downstream, antisense). The two
amplified fragments, flanking the sequence to be deleted, were
re-annealed and re-amplified using the upstream and downstream primers.
The final PCR product and the pVL1392-PLC
1 were digested
with NheI and BamHI, and the wild-type fragment was replaced by the corresponding mutant fragment. In the case of
PLC
3
, the two fragments flanking the upstream and
downstream regions of the deletion were amplified using
pVL1393-PLC
3 as template and the following two pairs of
oligonucleotides as primers: 5'-TGAGCGAGAGCTCCGCG-3' (upstream,
sense), 5'-ACAGGTGCCCGCTCCTCTGGTCCATCAGGC-3' (internal, antisense),
5'-GATGGACCAGAGGAGCGGGCACCTGTCGG-3' (internal, sense), and
5'-GGTTCTTGCCGGGTCCCAGG-3' (downstream, antisense). The final PCR
product and the vector pVL1393-PLC
3 were digested with
BlpI, and the wild-type fragment was replaced by the
corresponding mutant fragment.
The PLC
1-PLC
2 chimeras were generated by
the PCR overlap extension method. In chimera A, the N-terminal amino
acids of PLC
2 (residues 1-138) were replaced by the
corresponding residues of PLC
1 (residues 1-142), so
that the putative PH domain was substituted. Substitution of the PH
domain and the putative four EF-hand motives of PLC
2
(residues 1-303) for the corresponding residues of PLC
1 (residues 1-307) resulted in chimera B. Chimera A was constructed from
the vectors pVL1392-PLC
1 and
pVL1393-PLC
2
by PCR amplification using the following
two pairs of primers: 5'-GAATTCATGGCCGGGGCACAGC-3' (upstream,
sense), 5'-GGGAGGCGTTGGCCGTCAGCAGGTTTGTTGCCAAA-3' (internal, antisense), 5'-TTTGGCAACAAACCTGCTGACGGCCAACGCCTCCC-3' (internal, sense), 5'-GGATCCTCAGAGGCGGCTCTCCT-3' (downstream, antisense). The two amplified fragments were re-annealed and re-amplified using the
upstream and the downstream primers, which introduced an
EcoRI and a BamHI recognition site, respectively.
Chimera B was constructed using the same upstream and downstream
primers and the following two internal primers:
5'-GAAGAAAATGGAGTCGTTGCCCAGGACAAGCTGCT-3' (internal, antisense)
and 5'-CAGCTTGTCCTGGGCAACGACTCCATTTTCTTCTC-3' (internal, sense).
The final fragments were then ligated at their EcoRI and
BamHI sites, and the resulting constructs were inserted into
the baculovirus transfer vector pVL1392.
The entire PCR-amplified regions were sequenced and found to be
identical to the expected sequences. Production of recombinant baculoviruses, expression, and isolation of PLC
isoforms were carried out according to published protocols (26).
Recombinant Rho GTPases--
The production of recombinant
baculoviruses using BaculoGoldTM DNA (BD Pharmingen, San
Diego, CA) and pVL1393 transfer vectors containing the respective Rho
GTPase cDNA ligated into the BamHI/EcoRI site
has been described previously (21). Isoprenylated membrane-bound Rho
GTPases were solubilized by extracting the membranes with buffer
containing 23 mM sodium cholate as described previously (21). Rac2-LyGDI heterodimers were purified from cytosolic fractions of
baculovirus-infected insect cells (31).
[35S]GTP
S Binding--
Binding of
[35S]GTP
S to Rho GTPases was assayed as described
previously (21) with minor modifications. Briefly, samples (0.05-0.5 µg of protein extracted from membranes of baculovirus-infected insect
cells with buffer containing sodium cholate) were incubated at 30 °C
in an incubation mixture (40 µl) containing 25 mM
Hepes-NaOH, pH 8.0, 1 mM EDTA, 1 mM
dithiothreitol, 20 mM MgCl2, 100 mM
NaCl, 0.1% (v/v) GENAPOL® C-100 (Calbiochem, La Jolla, CA, HPLC
grade), and 100 nM [35S]GTP
S (296 GBq/mmol). The incubation was terminated after 6 h as described
earlier (31). We also used [35S]GTP
S binding to
estimate the concentrations of activated GTP
S-bound Rho GTPases
under conditions of the PLC assay (see below) in the presence of 100 µM [35S]GTP
S (115 GBq/mmol).
Phospholipase C Assay--
Phospholipase C activity was
determined as described (21) with minor modifications. In brief, 5 µl
of detergent-solubilized Rho GTPase and/or 5 µl of purified 
were supplemented with 10 µl of soluble fraction of
PLC
-baculovirus-infected insect cells and incubated at 25 °C for
time periods as indicated in the figure legends in a volume of 60 µl
containing 50 mM HEPES-NaOH, pH 7.2, 70 mM KCl,
3 mM EGTA, 2 mM dithiothreitol, 33 µM [3H]PI-4,5-P2 (185 GBq/mol),
536 µM phosphatidylethanolamine, and 150 nm free
Ca2+. In all experiments comparing the effects of Rac2 and

, the final concentration of sodium cholate was 2 mM.
Only 
-mediated stimulation of wild-type versus
C-terminally deleted PLC
isozymes (Fig. 6) was measured in the
absence of sodium cholate. Results obtained in control experiments
using purified PLC
2
(31) were indistinguishable from
that obtained with soluble fractions of PLC
2
-baculovirus-infected insect cells.
Miscellaneous--
Purification of
1
1 isolated from bovine retinal rod outer
segment membranes has been described elsewhere (32). The method used to
purify membrane-associated recombinant
1,5
2-His dimers is described in a
previous study (33). SDS-PAGE, immunoblotting, and determination of
protein concentrations were performed as described previously (21).
Polyclonal antibodies against Rho GTPases were from Santa Cruz
Biotechnology (Santa Cruz, CA). Specific antisera against
PLC
1, PLC
2, and PLC
3 were
a kind gift from Dr. P. J. Parker (28). The data are presented as
means ± S.D. of triplicate determinations.
 |
RESULTS |
Stimulation of Recombinant PLC
Isozymes by 
Dimers--
To determine the specificity of PLC
stimulation by G
protein 
dimers, the PLC
isozymes PLC
1,
PLC
2, and PLC
3 were produced as
recombinant proteins in Sf9 insect cells. Fig.
1A shows that all three
isozymes were expressed as soluble proteins in infected insect cells
and migrated at molecular masses of ~150, 140, and 160 kDa,
respectively, corresponding to the masses described for the native
PLC
isozymes (34, 35, 28). When the amounts of the three PLC
isozymes were normalized according to their maximal activity at 1 mM free Ca2+ and 3.3 mM sodium
deoxycholate (not shown) and then assayed at a more physiological
Ca2+ concentration of 150 nM, recombinant
PLC
2 displayed an ~3-fold higher basal activity than
recombinant PLC
1 and PLC
3 (Fig.
1B). In all experiments shown in this study, the amounts of
the three recombinant PLC
preparations were adjusted to equal basal
phospholipase C activity at 150 nM free Ca2+ in
the presence of 2 mM sodium cholate. Under these
conditions, purified 
dimers of bovine retinal transducin
(
1
1, 300 nM) activated
PLC
1 and PLC
2 only slightly (1.6-fold and
3.0-fold, respectively), but caused a marked (~20-fold) stimulation
of PLC
3 (Fig. 2).

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Fig. 1.
Expression of
PLC 1,
PLC 2, and
PLC 3 in baculovirus-infected
insect cells. A, soluble fractions of Sf9 insect
cells that had been infected with baculovirus-encoding bovine
PLC 1 (10 µg of protein) (lane 1), human
PLC 2 (30 µg of protein) (lane 2), and human
PLC 3 (60 µg of protein) (lane 3) were
subjected to SDS-PAGE and immunoblotting using PLC subtype-specific
antisera. The apparent molecular weights of the marker proteins
are indicated. The PLC 1, PLC 2, and
PLC 3 migrated at ~150, 140, and 160 kDa, respectively.
Additional immunoreactive proteins of lower molecular masses were
detected in all three preparations. Because soluble fractions of
non-infected insect cells did not contain proteins reactive with the
antisera used in this experiment (not shown), these proteins most
likely correspond to proteolytic fragments of the full-length PLC
isozymes. B, aliquots of the three soluble fractions were
incubated in the presence of 150 nM free Ca2+
for the times indicated at the abscissa with phospholipid
vesicles containing PI-4,5-P2. The reaction was terminated
by the addition of chloroform/methanol/concentrated HCl, and the
mixture was analyzed for inositol phosphates. See "Experimental
Procedures" for details. Prior to this experiment, the amounts of the
samples containing soluble PLC 1, PLC 2,
and PLC 3 were adjusted to give equal maximal PLC
activities in the presence of 1 mM free Ca2+
and 3.3 mM sodium cholate (42) (not shown)
(PLC 1, 0.4 µg of protein/sample; PLC 2,
1.5 µg of protein/sample; PLC 3, 5.6 µg of
protein/sample).
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Fig. 2.
Stimulation of
PLC 1,
PLC 2, and
PLC 3 by
1 1.
Aliquots of soluble fractions of insect cells expressing
PLC 1 (0.3 µg of protein/sample),
PLC 2 (0.5 µg of protein/sample), or
PLC 3 (4.0 µg/sample) were incubated for the
times indicated at the abscissa in the absence (open
symbols) or presence (closed symbols) of 300 nM 1 1 purified from bovine
retinal rod outer segments with phospholipid vesicles containing
PI-4,5-P2. The incubation was performed at 150 nM free Ca2+.
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Stimulation of Recombinant PLC
Isozymes by Rho
GTPases--
To investigate the specificity of PLC
2
stimulation by Rho family members, the recombinant Rho GTPases Rac1,
Rac2, and Cdc42Hs were produced in baculovirus-infected insect cells,
extracted from the membrane of infected cells with detergent-containing buffer, and reconstituted with a recombinant C-terminal deletion mutant
of PLC
2, PLC
2
, in the presence of 100 µM GTP
S. Fig. 3 shows
that both Rac1 and Rac2 caused a marked (~13-fold) stimulation of
PLC
2
. Rac2 was slightly more potent than Rac1. Thus,
half-maximal stimulation was observed at ~40 nM Rac2 and
100 nM Rac1. Cdc42Hs was a less potent (EC50:
400 nM) and less efficacious (~8-fold) stimulator of
PLC
2
than Rac1 and Rac2. In additional experiments, we observed a similar rank order of potency of Rho GTPases (Rac2
Rac1 > Cdc42Hs) to stimulate PLC
2 when
full-length enzyme rather than PLC
2
was used (not
shown). RhoA had no effect on full-length PLC
2 or
PLC
2
when tested under the same conditions (not
shown).

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Fig. 3.
Stimulation of
PLC 2 by
the human Rho GTPases Cdc42Hs, Rac1, and Rac2. The recombinant Rho
GTPases were extracted with buffer containing sodium cholate from
membranes of baculovirus-infected insect cells and incubated at
increasing concentrations with soluble proteins of insect cells
expressing PLC 2 (0.2 µg of protein/sample) and
phospholipid vesicles containing PI-4,5-P2. PLC activity
was measured for 1 h in the presence of 100 µM
GTP S. The concentrations of Rac1 (filled triangles), Rac2
(filled circles), and Cdc42Hs (filled squares)
were estimated by determining the binding of [35S]GTP S
under the same buffer conditions. The Rho GTPases did not affect the
activity of PLC 2 in the presence of 100 µM GDP (not shown).
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Next, the most potent Rho GTPase, Rac2, was incubated at increasing
concentrations in the presence of either GDP or GTP
S with soluble
preparations of recombinant wild-type PLC
1,
PLC
2, and PLC
3. Fig.
4 shows that PLC
2 was
clearly the PLC
isoform most sensitive to stimulation by
GTP
S-activated Rac2. Thus, half-maximal and maximal (~4-fold)
stimulation was observed at approximately 80 and 500 nM
Rac2, respectively. Rac2 also appeared to stimulate PLC
2
in the presence of GDP, albeit to a much lesser (~1.6-fold) extent.
At high concentrations of Rac2, a reduction of PLC
2
stimulation was observed both in the presence of GDP and GTP
S,
suggesting an inhibitory effect of the membrane extracts on PLC
activity. PLC
1 and PLC
3 were also
activated by Rac2, but to a much lower extent and only at much higher
concentrations of GTP
S-activated Rac2 (
1 µM).
Additional measurements (not shown) of PLC
stimulation by
C-terminally modified Cdc42Hs and Rac1 revealed an at least 10-fold
higher potency of these GTPases toward PLC
2 than toward PLC
1 or PLC
3. RhoA, a Rho GTPase
incapable of stimulating PLC
2 (21), affected neither
PLC
1 nor PLC
3 activity under the same conditions. These data show that Rho GTPases stimulate
PLC
2 with a rank order of potency of Rac2
Rac1 > Cdc42Hs and that, among the PLC
isoforms tested,
PLC
2 is most sensitive to stimulation by activated
Rac2.

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Fig. 4.
Stimulation of
PLC 1,
PLC 2, and
PLC 3 by Rac2. Aliquots of
soluble fractions of Sf9 cells expressing PLC 1
(0.3 µg of protein/sample), PLC 2 (0.5 µg of
protein/sample), or PLC 3 (4.0 µg of protein/sample)
were incubated at increasing concentrations of Rac2 extracted from
membranes of baculovirus-infected insect cells with buffer containing
sodium cholate. Phospholipase C activity was measured with phospholipid
vesicles containing PI-4,5-P2. The incubation was performed
for 10 min at 150 nM free Ca2+ either in the
presence of 100 µM GDP (empty circles) or in
the presence of 100 µM GTP S (filled
circles).
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Comparison of Full-length and C-terminally Deleted PLC
Isozymes--
A truncated PLC
isozyme related to
PLC
3 has previously been reported to be remarkably
sensitive to activation by 
dimers (36). Similarly, C-terminal
truncation of PLC
3 from human platelets by calpain has
been shown to result in a marked augmentation of 
stimulation
(37). PLC
1 has been shown to be cleaved by calpain between residues 880 and 881, generating two fragments of 100 and 45 kDa, respectively (38). The presence of ~45- to 50-kDa proteins in
the preparations of recombinant PLC
isozymes, which are likely to
represent C-terminal proteolytic fragments of the enzymes
(cf. Fig. 1), raised the possibility that the observed order
of PLC
stimulation by Rac2 was a consequence of C-terminal proteolysis of the PLC
isozymes. To challenge this hypothesis, we
examined and compared the ability of Rac2 to stimulate the activity of
C-terminal deletion mutants of PLC
1,
PLC
2, and PLC
3. A schematic
representation of the wild-type and mutant PLC
isozymes is shown in
Fig. 5A. As shown in Fig.
5B, all three mutants were expressed in baculovirus-infected
insect cells as soluble proteins and migrated on SDS-polyacrylamide
gels at the expected molecular weights. The deletion mutants displayed
Ca2+ sensitivities indistinguishable from those of their
wild-type counterparts (not shown). Reconstitution of the wild-type and mutant PLC
isoforms with GTP
S-activated Rac2 (500 nM)
or
1
1 (300 nM) showed that,
surprisingly, the deletion of the C-terminal regions of each PLC
isozyme did not change the efficiency of
1
1 to cause PLC
stimulation (Fig.
6). In agreement with earlier findings
(20), the removal of the C-terminal part of PLC
2
enhanced the extent of Rac2-mediated stimulation of the enzyme.
Importantly, however, no enhancement of Rac2-mediated stimulation was
observed for the C-terminally deleted variants of PLC
1
and PLC
3. Therefore, removal of the C-terminal regions
of PLC
isozymes does not change the rank order of specificity of
PLC
stimulation by both Rac2 and G protein 
subunits.

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Fig. 5.
Schematic representation of wild-type
phospholipases C-
(PLC ) and their C-terminally
deletion mutants (PLC ) and
expression of the deletion mutants in Sf9 cells.
A, the structural organization of the PLC isozymes, each
made up of a putative pleckstrin homology domain (PH), four
EF-hand motifs (4EF), the catalytic subdomains X and Y, and
a C2 domain (C2), is based on the crystal structures of
mammalian PLC 1 (57, 58). The antibody recognition site
(AB) and the position of the calpain cleavage site in
PLC 1 (arrow) are indicated. B,
aliquots of soluble fractions (40 µg of protein/sample) of Sf9
cells expressing PLC 1 (lane 1),
PLC 2 (lane 2), and PLC 3
(lane 3) were subjected to SDS-PAGE and immunoblotting using
specific antisera reactive against the respective PLC
isozymes.
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Fig. 6.
Stimulation of full-length
PLC enzymes and C-terminally deleted
PLC enzymes by G protein
 dimers and Rac2. Left
panels, soluble fractions of insect cells expressing
PLC 1 (1.3 µg of protein/sample),
PLC 1 (0.25 µg of protein/sample),
PLC 2 (0.5 µg of protein/sample),
PLC 2 (1.4 µg of protein/sample),
PLC 3 (1.5 µg of protein/sample), or
PLC 3 (1.0 µg of protein/sample) were incubated with
Rac2 (500 nM) extracted from membranes of
baculovirus-infected insect cells with buffer containing sodium
cholate. The incubations were performed for 8 min (upper left
panel), 10 min (middle left panel), and 30 min
(lower left panel) in the absence (empty bars) or
in the presence (hatched bars) of 100 µM
GTP S. Right panels, soluble fractions of insect cells
expressing PLC 1 (1.3 µg of protein/sample),
PLC 1 (0.25 µg of protein/sample),
PLC 2 (1.0 µg of protein/sample),
PLC 2 (1.0 µg of protein/sample),
PLC 3 (2.1 µg of protein/sample), or
PLC 3 (1.0 µg of protein/sample) were incubated
either in the absence (empty bars) or in the presence of 300 nM 1 1 (hatched
bars) for 6 min (upper right panel), 7 min
(middle right panel), or 4 min (lower right
panel). Phospholipase C activity was measured at 150 nm free
Ca2+ in the presence of phospholipid vesicles containing
PI-4,5-P2. The results are given as the percentage of the
basal activities, which were set to 100%. For any given pair of
wild-type and mutant PLC isozyme, the basal activities varied by
less than a factor of two (not shown).
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Simultaneous Stimulation of PLC
2 by Rac2 and 
Subunits--
The next experiments were designed to examine whether
the different specificities of PLC
isozyme stimulation by Rho
GTPases and 
dimers may reflect independent regulation of
PLC
2 by both stimulators. We have previously shown that
5
2-His is a more potent stimulator of
PLC
2
than is
1
2-His and
that the latter, but not the former 
dimer, activates
PLC
3 (33). A comparison of the effects of
5
2-His and
1
2-His on the activity of wild-type PLC
1, PLC
2, and PLC
3 is
shown in Fig. 7. Among the PLC
isoforms tested, PLC
3 was the isoform most sensitive to
stimulation by
1
2-His, followed by
PLC
2 and PLC
1, which was hardly activated by this 
preparation. In marked contrast,
5
2-His proved to be a potent and
efficacious activator of full-length PLC
2, but it did
not affect full-length PLC
1 or PLC
3.
Because activation of PLC
2 by
5
2 occurred at low concentrations
(EC50: ~10 nM) and reached saturation within
the range of 
dimer concentrations tested,
5
2-His was chosen as the 
dimer to
compare the effects of Rac2 and 
dimers on PLC
2
activity when added alone or in combination at maximally effective
concentrations (Fig. 8). Because stimulation of PLC
2 by 
dimers is sensitive to
high concentrations of detergent (32), detergent-free Rac2-LyGDI
heterodimers were used as a source of Rac2. In our hands, Rac2-LyGDI
heterodimers and monomeric Rac2 stimulated PLC
2 with
equal potency and efficacy in the presence of phospholipid vesicles
containing the phospholipase C substrate PI-4,5-P2 (31).
Fig. 8 shows that addition of GTP
S allowed activation of Rac2 from
the heterodimer (EC50: ~20 nM) leading to an
3-fold stimulation of PLC
2
and that stimulation of
the enzyme was observed both in the absence and in the presence of
5
2-His (EC50: ~20
nM). The extent of stimulation by GTP
S-activated Rac2-LyGDI was additive with that obtained by
5
2-His, suggesting independent
stimulation of PLC
2 by Rho GTPases and 
subunits. Interestingly, partial inhibition of
5
2-His-mediated PLC
2
stimulation by Rac2-LyGDI was measured in the presence of GDP. Because
there was no effect of Rac2-LyGDI on basal PLC
2
activity, this result may indicate that inactive Rac2-LyGDI may
interact with
5
2-His and/or
noncompetitively interfere with
5
2-His-mediated PLC
2 activation.

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|
Fig. 7.
Stimulation of PLC
isozymes by
5 2
and
1 2
dimers. Soluble proteins of insect cells expressing
PLC 1 (0.3 µg of protein/sample), PLC 2
(0.5 µg of protein/sample), or PLC 3 (4.0 µg of
protein/sample) were reconstituted with increasing amounts of purified
recombinant 1 2-His (filled
triangles) and 5 2-His (filled
squares). PLC activity was measured for 30 min at 150 nM free Ca2+ in the presence of phospholipid
vesicles containing PI-4,5-P2.
|
|

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|
Fig. 8.
Effect of G protein
5 2
dimers on stimulation of
PLC 2 by
Rac2-LyGDI. Purified PLC 2 (0.5 µg/sample) was
incubated in the absence (empty and filled
triangles) or presence (empty and filled
circles) of 30 nM 5 2-His
and increasing concentrations of purified Rac2-LyGDI with phospholipid
vesicles containing PI-4,5-P2. The incubation was performed
in the presence of 100 µM GDP (empty symbols)
or 100 µM GTP S (filled symbols).
|
|
Structural Requirements of PLC
Stimulation by Rho GTPases
versus 
Dimers--
The independent stimulation of
PLC
2 by 
subunits and Rho GTPases prompted us to
delineate the structural elements of PLC
2 required for
enzyme activation. The site of interaction of PLC
2 with

was localized by others to the region between the catalytic subdomain Y residues Glu574 and Lys583 (39,
40). However, 
dimers have also been reported to bind to the
isolated PH domains of PLC
1 and PLC
2
(41). To identify the sites on PLC
2 relevant for
activation by Rac2 and 
, we took advantage of the fact that both
stimulators elicited only a slight effect on PLC
1
,
but markedly activated PLC
2
. The effects of the two
stimulators were examined on the activity of PLC
1-PLC
2 chimera in which N-terminal
portions of PLC
2
had been replaced by the
corresponding regions of PLC
1
(Fig.
9). Two chimera, designated A and B,
carrying the putative PH domain and the PH domain together with the
four EF-hand motifs of PLC
1
, respectively, were
analyzed. A third chimera, comprising the PH domain, the EF-hand
motifs, the catalytic subdomain X of PLC
1
, and the
catalytic subdomain Y of PLC
2
, was catalytically
inactive and hence not used for further analysis. Although chimera A
was expressed in baculovirus-infected insect cells at levels
considerable lower than were PLC
2
and chimera B, the
two chimeras and PLC
2
were indistinguishable in terms
of the dependence of their catalytic activity on Ca2+ (not
shown). Fig. 10 shows that substitution
of the N-terminal portions of PLC
2
for the
corresponding regions of PLC
1
led to reduction of the
degree of
1
1-mediated stimulation.
Specifically, at the highest concentration of
1
1 tested (4 µM), the
degrees of stimulation were 129-, 88-, 66-, and 8-fold for
PLC
2
, chimera A, chimera B, and
PLC
1
(Fig. 10A). Because the effects of
1
1 did not reach saturation within the
range of concentrations tested, it is currently unclear whether the
reduced stimulation is due to a decrease in the potency or efficacy of
1
1. Very interestingly, substitution of
the putative PH domain of PLC
2
for its counterpart of
PLC
1
caused an almost complete (>95%) loss of
stimulation by GTP
S-activated Rac2 (Fig. 10B). In
additional experiments (not shown), we found that chimeras A and B were
also resistant to stimulation by GTP
S-activated Cdc42Hs. Taken
together, these results not only show that the structural requirements
of PLC
2 stimulation by 
dimers and by the Rho
GTPases Rac2 and Cdc42Hs are distinct but also suggest that the
putative PH domain of PLC
2 is critically involved in
mediating its activation by Rho GTPases.

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Fig. 9.
Linear representation of
PLC 2 ,
chimera A, chimera B, and
PLC 1 .
The amino acid sequences were aligned using the ClustalW program
contained in the PC/GENE software package (Intelligenetics, Mountain
View, CA). Chimeras A and B are composed of the first 142 and 307 residues, respectively, of PLC 1, followed by the 695 and
530 C-terminal residues, respectively, of PLC 2 .
AB, antibody recognition site.
|
|

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Fig. 10.
Stimulation of
PLC 2 ,
chimera A, chimera B, and
PLC 1
by  dimers and Rac2.
A, aliquots of soluble fractions of insect cells expressing
PLC 2 (filled circles), chimera A
(filled triangles), chimera B (filled squares),
or PLC 1 (filled diamonds) were incubated
at increasing concentrations of 1 1 with
phospholipid vesicles containing PI-4,5-P2. The incubation
was performed for 6 min at 150 nM free Ca2+. In
B, PLC activity was measured as in A, except that
the assay was performed at increasing concentrations of Rac2 in the
presence of either 100 µM GDP (open symbols)
or 100 µM GTP S (filled symbols). Prior to
this experiment, the amounts of the samples containing soluble PLC
isoforms had been adjusted to give similar basal activities in the
presence of 150 nM free Ca2+ and 2 mM sodium cholate (not shown) (PLC 2 , 2 µg of protein/sample; chimera A, 60 µg of protein/sample; chimera
B, 3 µg of protein/sample; PLC 1 , 0.3 µg of
protein/sample). There was no effect of
1 1 (1 µM) or Rac2 (1 µM) on phospholipase C activity of soluble fractions (60 µg of protein/sample) of non-infected insect cells or insect cells
infected with baculovirus encoding E. coli
-galactosidase.
|
|
 |
DISCUSSION |
Specificity of PLC
Stimulation by Rho GTPases--
We have
previously shown that the Rho GTPases Rac1 and Cdc42Hs, but not RhoA,
stimulate the activity of PLC
2 (21, 31). In this study,
we demonstrate that the PLC
isozymes PLC
1,
PLC
2, and PLC
3 are differentially
sensitive to stimulation by Rho GTPases and G protein 
dimers.
Activated Rac2 is shown to stimulate PLC
isozymes with the rank
order of potency and efficacy of PLC
2 > PLC
3
PLC
1. This rank order is clearly
different from the order observed for G protein 
dimers, which is
PLC
3 > PLC
2 > PLC
1 for
most 
dimers, e.g.
1
1 or
1
2-His (cf. Figs. 2 and 7 and
Refs. 9, 42, and 43) and PLC
2 > PLC
1
>>> PLC
3 for
5
2-His (cf. Fig. 7).
Furthermore, the results reported here show that both Rac1 and Rac2 are
more potent activators of PLC
2 than Cdc42Hs. All three
PLC
-stimulating Rho GTPases, Rac2, Rac1, and Cdc42Hs, preferentially
activate PLC
2 (PLC
2 > PLC
3
PLC
1) (not shown). RhoA did not
stimulate any of the three PLC
isozymes tested under the same
conditions (not shown). Notably, the specificity of PLC
activation
by Rho GTPases described here did not depend on the presence of
detergents, which could conceivably differentially affect the
activities of the PLC
isozymes, because purified detergent-free Rac2-LyGDI heterodimers (31) stimulated the PLC
isozymes studied here with the same rank order as did detergent-solubilized monomeric, C-terminally processed Rac2 (not shown). Interestingly, the rank order
of PLC
stimulation by Rho GTPases also differs from that reported
for activation of these enzymes by members of the
q subfamily of G protein
subunits: PLC
1
PLC
3 > PLC
2 (3, 4). Together with the
observation that the C-terminal region of PLC
isozymes is required
for the stimulation by
q subunits (43, 44), but not for
stimulation by Rho GTPases (20 and Fig. 6), this finding suggests that
the stimulation of PLC
by
subunits of heterotrimeric G proteins
differs mechanistically from stimulation of this enzyme by Rho GTPases.
Thus, PLC
isozymes can be activated, in the nanomolar concentration
range, by both subunits of G proteins and by Rho GTPases but with
distinct PLC
isozyme specificities.
The Role of the C-terminal Regions of PLC
s in Their Regulation
by Rho GTPases and 
--
Proteolytic cleavage of
PLC
3 by calpain at a site upstream of the C2 domain has
been suggested to enhance 
-mediated stimulation (36, 37). Our
data show, however, that truncation of recombinant PLC
1,
PLC
2, and PLC
3 at a site corresponding to
the calpain cleavage site in PLC
1 had no effect on their
sensitivity to 
stimulation. The finding that the deletion of the
C-terminal region of PLC
3 did not enhance 
stimulation suggests that the increased PLC
3 stimulation
following calpain cleavage reported by Banno and coworkers (37) may not
simply result from the removal of an inhibitory constraint built up by
the C-terminal amino acids of PLC
3. Instead, it is more
likely that the C-terminal region generated by treatment of
PLC
3 with calpain still interacts with the remaining
part of the enzyme, to inhibit its activity, whereas it is absent from
the deletion mutant studied here. Consistent with earlier results (20),
Rac2 stimulation of PLC
2 was enhanced in the absence of
the C-terminal region of the enzyme. However, because this effect was
not observed in the case of PLC
1 and PLC
3, the PLC
2 specificity described here
is clearly not a consequence of C-terminal proteolysis of the enzymes.
Simultaneous Stimulation of PLC
Isozymes by Rho GTPases and
G Proteins--
The fact that the stimulatory effects of
5
2 and GTP
S-activated Rac2 on
PLC
2 were strictly additive at saturating concentrations of the two activators suggests that there are separate sites on PLC
2 for the interaction with 
and Rac2. Similar
observations have been made previously for the activation of
PLC
2 and PLC
3 by
q and

(9, 45). In additional experiments, we have found that neither

-mediated activations nor
q-mediated activations of PLC
3 or PLC
1 were influenced by
GTP
S-activated Rac2. Collectively, these results suggest that PLC
isozymes can be isozyme-specifically activated by three different
stimulators, G protein
subunits, G protein 
dimers, and Rho
GTPases, via independent regulatory sites.
Structural Requirements of PLC
Stimulation by Rho GTPases
versus 
Dimers--
The generation of chimeric PLC
enzymes
made up of portions from an isoform stimulated only poorly by 
dimers and Rho GTPases, PLC
1
, together with the
remaining portions of PLC
2
, an isoform markedly
sensitive to both activators, allowed to delineate the structural
elements of PLC
2 required for the regulation by 
and Rac2. The fact that chimeras A and B were still markedly activated by 
dimers suggests that the catalytic subdomains of
PLC
2 are both necessary and sufficient for 
stimulation. This is consistent with a previous report showing that a
region within the catalytic Y domain of PLC
2 contains
the stimulatory 
interaction site (40). The lower extent of
stimulation of chimeras A and B by 
relative to
PLC
2
is in line with the suggestion that an
additional binding site for 
dimers may exist in the putative PH
domain of PLC
2 (41), which, albeit not absolutely
required for 
stimulation, may increase the affinity of the

-PLC
2 interaction. In addition, our results
suggest that the region corresponding to the putative PH domain of
PLC
1, an isozyme barely activated by 
, is capable
of substitute for the corresponding region of PLC
2. This
is consistent with the recent report describing interaction of an
isolated PH domain of PLC
1 with 
dimers (41).
Interestingly, construction of a chimera consisting of the putative PH
domain of PLC
2 and the catalytic subdomains X and Y of
PLC
, an enzyme that is not regulated by 
dimers, resulted in a

-regulated enzyme (46). This suggests that 
may interact
with multiple sites in phospholipase C isozymes and that these sites
can be provided even by isozymes that are poorly (e.g.
PLC
1) or not at all (e.g.
PLC
1) sensitive to 
stimulation. An important outcome of our experiments on chimeric
PLC
1-PLC
2 enzymes is the observation that
the substitution of the putative PH domain of PLC
2 by
the corresponding region of PLC
1 abolished Rac2-mediated stimulation of the enzyme. This result not only demonstrates different structural requirements of PLC
2 stimulation by 
and Rac2, but also shows, for the first time, that the putative PH
domain of PLC
2 is specifically and critically involved
in mediating the regulation of the activity of the PLC
isozyme. It
is currently unknown whether Rac2 directly binds to the PH domain of
PLC
2 or whether Rac2 induces a conformational change of
the enzyme involving the PH domain. Interestingly, the PH domain of
PLC
2 was found to be required for both membrane
targeting and catalytic activity of recombinant PLC
2 in
transfected COS-7 cells (44). The functional role of the region
corresponding to the putative PH domain in PLC
isozymes is poorly
understood. The isolated PH domain of PLC
1 has been
reported to bind inositol phospholipids (PI-3-P > PI-4,5-P2, PI-3,4,5-P3) (47). A cooperative
mechanism involving phosphatidylinositol 3-phosphate and 
subunits has been proposed to regulate plasma membrane localization and
activation of PLC
1 through the putative PH domain of
this enzyme. A similar scenario involving Rac2 and 
could be
depicted in the case of PLC
2. Thus, PLC
isozymes seem
to act as a point of convergence of transmembrane signaling:
PLC
1 integrating signals emanating from inositol
phospholipid 3-kinase and G protein
q subunits, PLC
3 those from G protein
q and 
subunits, and PLC
2 those from G protein 
subunits
and Rac/Cdc42Hs.
The finding that the activity of PLC
isozymes can be specifically
regulated by three different stimulators, G protein
subunits, G
protein 
dimers, and the Rho GTPases Rac and Cdc42Hs also enhances the cellular repertoire to coordinate, both spatially and
temporally, responses to extracellular signals acting through stimulation of PLC
isozymes and thereby to enhance the degree of
signal specificity. An intriguing possibility is that the Rho GTPases
Rac and Cdc42Hs act as organizers to mediate recruitment of
PLC
2 to allow activation by G protein-coupled receptors
only at specific sites within the cell and/or only within a specific time frame during or after receptor activation. The mechanisms involved
in the recruitment of PLC
isozymes to their phospholipid are still
not known. It seems clear, however, that the known activators,
q and 
, are not involved in this process (48, 49).
Interestingly, although PLC
3 is strongly activated by

dimers in cell-free systems, this isoform is, in marked contrast
to PLC
2, not stimulated by chemoattractant receptor
activation or exogenous 
dimers in transiently transfected COS-7
cells (17). These results support the notion that each PLC
isoform
may require distinct additional components either for recruitment or
stimulation of the catalytic activity. For example, activated members
of the
q subfamily of G protein
subunits have been
shown to permit PLC
stimulation by receptors acting through 
subunits of pertussis toxin-sensitive G proteins (50). The results
presented herein suggest that Rac1, Rac2, and Cdc42Hs may contribute to
the specificity and/or efficacy of PLC
signaling. Interestingly,
both PLC
2 and Rac2 are the major representatives of the
PLC
and Rac GTPase subfamilies, respectively, in myeloid cells (16,
51). Both proteins are activated in response to activation of
chemoattractant receptors in a pertussis-toxin sensitive manner (17,
52, 53, 54). The recent finding, that expression of a
dominant-interfering form of Cdc42Hs in myeloid-differentiated HL-60
cells drastically reduced the formyl peptide receptor mediation of
(a) formation of inositol 1,4,5-trisphosphate,
(b) increase in the concentration of intracellular
Ca2+, and (c) rapid activation of Rac2 (55),
supports our hypothesis that Rac2 and possibly Rac1 and Cdc42Hs are
critically involved in receptor-mediated regulation of
PLC
2 activity in intact cells. Although the mechanisms
by which chemoattractant receptors stimulate Rac/Cdc42 are not defined
and the functional relevance of phosphatidylinositol 3-kinase to this
process is a subject of debate, the recently identified 
- and
PI-3,4,5-P3-dependent Rac exchanger P-Rex 1 appears to fill the gap between chemoattractant receptors and Rac
GTPases in leukocytes (56).
In conclusion, our results demonstrate that the specificity of PLC
stimulation by the Rho GTPases Rac and Cdc42Hs differs from the
specificity observed for
q/11 subunits and 
dimers of heterotrimeric G proteins and that PLC
2 represents
the PLC
isozyme most sensitive to stimulation by the Rho GTPases Rac
and Cdc42 among the PLC
isozymes investigated in this study.
Moreover, there are separate sites on PLC
isozymes for the
regulation by Rho GTPases, G protein
subunits, and 
subunits
allowing independent activation by these stimulators.
 |
ACKNOWLEDGEMENTS |
We are grateful to Norbert Zanker, Renate
Straub, and Susanne Gierschik for excellent technical assistance. We
thank Drs. Geurts van Kessel and Martien van Asseldonk for the generous
gift of the cDNA-encoding PLC
3.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft (Grant SFB 497).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-731-5002-3874; Fax: 49-731-5002-3872; E-mail:
daria.illenberger@medizin.uni-ulm.de.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208282200
 |
ABBREVIATIONS |
The abbreviations used are:
PLC, phospholipase C;
LyGDI, a Rho guanine nucleotide dissociation inhibitor
originally identified in lymphocytes, but subsequently also found in
other cells;
GTP
S, guanosine
5'-O-(3-thiotriphosphate);
PH, pleckstrin homology;
PI-3-P, phosphatidylinositol 3-phosphate;
PI-4, 5-P2,
phosphatidylinositol 4,5-bisphosphate;
PI-3, 4,5-P3,
phosphatidylinositol 3,4,5-trisphosphate.
 |
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