From the ¶ Veterans Affairs Chicago Health Care
System-Lakeside Division and the Department of Pathology
and Feinberg Cardiovascular Research Institute, Northwestern University
Medical School, Chicago, Illinois 60611 and the § Institute
of Neuroscience and Department of Molecular Pharmacology and Biological
Chemistry, Northwestern University Medical School,
Chicago, Illinois 60611
Received for publication, September 5, 2000, and in revised form, October 3, 2000
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ABSTRACT |
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Three families of phospholipase C
(PI-PLC Many hormones, neurotransmitters, and growth factors elicit
intracellular responses by activating a family of inositol
phospholipid-specific phospholipase C
(PLC)1 isozymes (1, 2). There
are three established families of PLC termed The mode of regulation differs considerably for members of the
different isoform families. The Activation of G protein-coupled receptors modulates various aspects of
cellular growth and proliferation, processes that are primarily
controlled by small Ras-related G proteins and their downstream
effector, the mitogen-activated protein (MAP) kinases (8, 9). There
appear to be multiple mechanisms involving heterotrimeric G Cloning of hPLC Creation of the Phosphodiesterase-deficient Mutant hPLC Northern Blot Analysis--
Human multiple-tissue Northern blot
I and blot IV membranes (CLONTECH) containing
approximately 2 mg of poly(A)+ RNA/lane were probed with
full-length cDNA of hPLC Transient Transfection of TSA201 Cells--
The hPLC Immunoblotting--
Samples were subjected to 6% or 15%
SDS-polyacrylamide gel electrophoresis and were electrophoretically
transferred to nitrocellulose as described previously (15). hPLC PLC Activity in TSA201 Cells--
TSA201 cells were transfected
with either pcDNA3 vector (control), hPLC Cell Free Assays--
TSA201 cells were transfected with either
vector (control) or hPLC MAP Kinase Assay--
TSA201 cells were co-transfected (12 mg
DNA/100 mm plate) with vector, hPLC Ras Pull-down Assays--
TSA201 cells were transiently
co-transfected as previously mentioned with either control plasmid
(pcDNA3), hPLC Isolation of the cDNA Encoding Human PLC
Structural analysis reveals that PLC Expression of PLC
PLC The Heterotrimeric G Protein G
PLC PLC
To determine whether PLC
The role of the Ras-associating or RA domains is presently
unknown. Proteins such as RalGDS that contain both Ras-GEF and RA
domains are assumed to serve as links between different Ras family
members. Although RA domains are known to bind to the effector loop of
activated members of the Ras superfamily, the RA domain seems to not be
necessary for regulation of GDP/GTP exchange by some proteins. For
example, activated Ras stimulates RGL (for RalGDS-like), which
exchanges GDP for GTP on Ral in vivo. In vitro, however, the RA domain of RGL is not necessary for GDP/GTP exchange on
Ral (34). It is presumed that the role of the RA domain in RGL
activation of Ral is to mediate redistribution of RGL to the membranes
where Ral is located. To differentiate the role of the RasGEF domain
from that of the RA domain of PLC
It is difficult to predict the function of the RA domains in PLC PLC
This work demonstrates that PLC,
, and
) are known to catalyze the hydrolysis
of polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate
(PIP2) to generate the second messengers inositol
1,4,5 trisphosphate and diacylglycerol, leading to a cascade of
intracellular responses that result in cell growth, cell
differentiation, and gene expression. Here we describe the founding
member of a novel, structurally distinct fourth family of PI-PLC.
PLC
not only contains conserved catalytic (X and Y) and regulatory
domains (C2) common to other eukaryotic PLCs, but also contains two
Ras-associating (RA) domains and a Ras guanine nucleotide exchange
factor (RasGEF) motif. PLC
hydrolyzes PIP2, and this
activity is stimulated selectively by a constitutively active form of
the heterotrimeric G protein G
12. PLC
and a mutant (H1144L) incapable of hydrolyzing phosphoinositides promote formation of GTP-Ras. Thus PLC
is a RasGEF. PLC
, the mutant H1144L, and the
isolated GEF domain activate the mitogen-activated protein kinase
pathway in a manner dependent on Ras but independent of PIP2 hydrolysis. Our findings demonstrate that PLC
is a
novel bifunctional enzyme that is regulated by the heterotrimeric G protein G
12 and activates the small G protein
Ras/mitogen-activated protein kinase signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(~150 kDa),
(~145 kDa), and
(~ 85 kDa) (3). All three families of the
PI-PLC family are able to recognize phosphatidylinositol (PI),
phosphatidylinositol 4-phosphate, and phosphatidylinositol
4,5-bisphosphate (PIP2) and to carry out the
Ca2+-dependent hydrolysis of these inositol
phospholipids. It is presumed that the primary substrate for hydrolysis
is PIP2, which yields the second messengers inositol
1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).
IP3 releases intracellular Ca2+ from the
endoplasmic reticulum via interaction with a specific receptor located
on the surface of the endoplasmic reticulum. DAG, as well as increased
intracellular Ca2+, activate protein kinase C leading to a
cascade of intracellular events including regulation of cellular
growth, smooth muscle contraction, and cardiac hypertrophy (2).
isoforms are regulated by large
heterotrimeric G proteins. After activation by agonists such as
epinephrine,
1 adrenergic receptors are able to couple to G
subunits from the Gq/G11 family and
stimulate hydrolysis of phosphatidyl inositol lipids via PLC
isoforms. For some
isoforms, Gq alone is sufficient for
activation, whereas for others the coordinated action of both
Gq and
is necessary. The
isoforms of PLC contain
SH2 and SH3 domains; hence, they are activated by both receptor and
nonreceptor tyrosine kinases. Until recently, the mode of regulation of
the
class was unknown. Work from our laboratory and that of others
has determined that this class is regulated in vitro by
lipid ligands and ionized free calcium (regulation by calcium via
C2 domain is described by Lomasney et
al.)2 (4-7).
and
subunits by which G protein-coupled receptors regulate small
monomeric G protein function (10). G
subunits can induce
phosphorylation of the Shc adapter protein leading to association with
the Grb2 docking protein and eventual stimulation of Ras guanine
nucleotide exchange activity (11). G
i and
G
o subunits can lead to activation of MAP kinase via a
protein kinase C-dependent pathway (12). Recently, a novel
molecule, p115 RhoGEF, has been identified that serves as a direct link
between the heterotrimeric G
subunit G
13 and the
small G protein Rho (13, 14). G
13 stimulates the
nucleotide exchange activity of p115 RhoGEF for Rho. p115 RhoGEF also
serves as a GTPase-activating protein for G
13 and
G
12. This is the first example of a protein that is able
to directly link large and small G protein pathways. In this report we
identify a novel fourth class of PI-PLC that we designate PLC
and
demonstrate that PLC
interacts with large and small G proteins,
although it is very different from p115 RhoGEF.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
cDNA--
A computer search of the human
GenBankTM expressed sequence tag (EST) data base was
conducted using three relatively short amino acid sequences from the
conserved X and Y domains of the mammalian PLCs. An EST clone,
zb59f12.s1, showed a high degree of homology and contained a putative
open reading frame. Screening of a human placental cDNA library
with the EST cDNA probe yielded a larger (3.0 kilobases) but still
incomplete cDNA fragment. The full-length PLC
cDNA was
generated by 5' rapid amplification of cDNA ends utilizing the
Marathon cDNA amplification kit (CLONTECH). The cDNA was reverse transcribed from poly(A)+ mRNA
obtained from human heart (CLONTECH), using the
cDNA synthesis primer provided with the kit. The PCR
amplification was carried out using adaptor primer 1 (5'-CCATCCTAATACGACTCACTATAGGGC-3') and a gene specific reverse primer,
5'-TCCACCGTCTGCCACCAAACAACTCCACA-3'. The first round PCR was performed
according to the recommendations of the manufacturer of 94 °C for 1 min, five cycles at 94 °C for 5 s and 72 °C for 4 min,
another five cycles at 94 °C for 5 s and 70 °C for 4 min,
followed by 25 cycles at 94 °C for 5 s and 68 °C for 4 min.
The second round of PCR was performed under the same conditions
using a 1:200 dilution of the first round PCR amplification mixture as
the template, adaptor primer 2 (5'-ACTCACTATAGGGCTCGAGCGGC-3') and a
nested gene-specific primer
(5'-AGCAGCGGGCAGAGAGGTGTGTGTCC-3'). Southern blot
analysis using an end-labeled internal oligonucleotide (5'-TGTCAACAGCATCTTTCAGGTCATCC-3') 5' from the PCR primers confirmed that the PCR product contained the expected sequence. The PCR product
was subcloned into the PCR2.1 TA cloning vector (InVitrogen) according
to the recommendations of the manufacturer. Positive clones were
identified by hybridizing filter lifts with the same labeled
oligonucleotide that was described for the Southern blots. Clones
containing the expected size DNA fragment were then sequenced along
both strands using the Taq dye terminator method at the University of Georgia Molecular Genetics Facility, Department of
Genetics, using Applied Biosystems 373 and 377 automatic sequencers. The entire hPLC
cDNA was obtained from heart cDNA by PCR
using the following primers: 5'-ATGGTTTCAGAAGGAAGTGCAGCAGGAA-3'
(sense) and 5'- TCACTGTCGGTAATCCATTGTGTCACTGG-3' (antisense).
The PLC
cDNA was subcloned in-frame into pBluescript KS+.
The sequence of the inserted DNA was determined as previously mentioned.
H1144L--
The phosphodiesterase-deficient mutant of hPLC
, PLC
H1144L, was generated by changing the histidine residue at position 1144 to leucine using the QuickChange Kit (Stratagene). Briefly, the
fragment of PLC
containing the mutation was amplified with Pfu Turbo Polymerase (Stratagene) using sense
(5'-GCTTGACGGCGCCTCCGG-3') and antisense
(5'-CTACCCTACGGGTAGTAAATAGAACCTGTATGCGACTGTTGGTTC-3') primers
containing the mutation changing the histidine (CAT) to a leucine
(CTT). The PCR was performed according to the recommendations of the
manufacturer of 95 °C for 30 s, 12 cycles at 95 °C for 30 s, 1 min at 55 °C and 4 min at 68 °C. A second fragment
overlapping the first was amplified using the sense primer,
5'-GATGGGATGCCCATCATTTATCTTGGACATACGCTGACAACCAAG-3' and an
antisense primer (5'-AACGGGGAGGGGGCACGG-3') corresponding to
pcDNA3 vector, 3' from the multiple cloning site of the vector. The
PCR was also performed according to the recommendations of the
manufacturer of 95 °C for 30 s, 12 cycles at 95 °C for
30 s, 1 min at 55 °C, and 6 min at 68 °C. The overlapping
products were then used to amplify the entire PLC
cDNA from
nucleotide1278-6507 using the sense primer 5'-GCTTGACGGCGCCTCCGG-3'
and the antisense primer corresponding to the vector just 3' of the
multiple cloning site. The PCR conditions were 95 °C for 30 s,
12 cycles at 95 °C for 30 s, 55 °C for 1 min, and 10 min at
68 °C. After ligation and plasmid preparation, the mutant insert was
digested with SacII and XbaI. The mutated
fragment was gel purified using the QIAquick gel extraction kit
(Qiagen) and ligated into pcDNA3 in place of the wild type
fragment. The sequence was then verified for the presence of the H1144L
mutation and the absence of any additional mutations.
or
-actin according to the
manufacturer's instructions. Briefly, 30 ng of hPLC
or
-actin
were random-primed with [
-32P]dCTP (<6000 Ci/mmol;
PerkinElmer Life Sciences) using the Prime-It II random primer labeling
kit (Stratagene). Unincorporated radionucleotide was removed from the
probe by using Bio-Spin 30 chromatography columns (Bio-Rad). The
radiolabeled probes were then denatured by boiling for 5 min and placed
on ice. Membranes were prehybridized in 15 ml of Express hyb solution
(CLONTECH) with continuous shaking at 68 °C for
1 h. The prehybridization buffer was replaced with 10 ml of fresh
ExpressHyb solution containing the radiolabeled probe. After a 1-h
incubation at 68 °C with continuous shaking, the blots were quickly
rinsed several times with 2× SSC, 0.05% SDS at room temperature and
then washed twice for 10 min at room temperature in the same buffer.
This was followed by two 20-min washes with fresh 0.1× SSC, 0.1% SDS
at 50 °C and continuous shaking. The blots were then wrapped in
plastic wrap and exposed to HyperFilm MP x-ray film (Amersham Pharmacia
Biotech) at
80 °C with two intensifying screens for 4-24 h before
developing the film. Blots exposed to multiple probes were stripped of
the first probe as suggested by the manufacturer's protocols.
cDNA
was subcloned in-frame into the unique HindIII and
NotI sites of the mammalian expression vector pcDNA3
(InVitrogen) downstream of the cytomegalovirus promotor. A Kozak
consensus sequence (TAAT) and a Myc tag
(5'-ATGGAGCAGAAGCTGATCAGCGAGGAGGACCTG-3') were incorporated in frame
before the start codon. Plasmids were purified for transfection using
Qiagen kits. TSA201 cells were cultured in complete Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) containing
glutamine, high glucose, 10% fetal bovine serum (Life Technologies,
Inc.), and 50 mg/ml gentamicin (Life Technologies, Inc.) at 37 °C in
a humidified 5% CO2 incubator. The cells were transfected
with Myc-hPLC
or pcDNA3 empty vector using LipofectAMINE (Life
Technologies, Inc.) according to manufacturer's instructions with
slight modifications. Cells were harvested 48 h after transfection.
was
detected by Western blot analysis using a primary anti-Myc tag antibody
(InVitrogen). G
* subunits were detected using specific anti-G
antibodies. Alkaline phosphatase or horseradish peroxidase-conjugated
secondary antibody (IgG) was used for detection.
, hPLC
H1144L,
G
s*, G
i*, G
12*,
G
13* cDNA, or Ras (2 mg/35-mm plate) alone or in
combination using LipofectAMINE (Life Technologies, Inc). To maintain
uniform amount of transfected DNA, empty vector was added to the
transfection mixture when necessary. The cells were labeled with
[3H]myo-inositol (PerkinElmer Life Sciences)
for 24 h and harvested 48 h after transfection. The amount of
inositol phosphates (inositol 1-phosphate, inositol
1,4-bisphosphate, and IP3) was determined using anion
exchange chromatography (16). The percentage of PI hydrolyzed is
expressed as the total inositol phosphates formed relative to the
amount of [3H]myo-inositol incorporated into
the phospholipid pool.
cDNA (12 mg DNA/100-mm plate). Cells
were harvested 48 h after transfection and homogenized in ice-cold
lysis buffer (50 mM Tris, pH 7.5, 30 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, 1 mM aprotinin, and 1 mM pepstatin). Cytosolic and particulate fractions were
isolated as described previously (15). Cytosolic and membrane fractions
were then assayed for PLC activity using a vesicle assay previously
described with slight modification (17). Substrate was provided as
mixed phospholipid vesicles containing phosphotidylinositol-4,5-bis phosphate, PIP2 (28 mM), and
phosphatidylethanolamine (280 mM) in a ratio of 1:10 with
25,000- 35,000 cpm of [3H]PIP2/assay. PLC
assays were performed at 37 °C for varying time periods in a mixture
(70 ml) containing 50 mM HEPES, pH 7.3, 3 mM
EGTA, 0.2 mM EDTA, 1 mM MgCl2, 20 mM NaCl, 30 mM KCl, 4 mM dithiothreitol, 0.1 mg/ml acetylated bovine albumin, 1.6 mM
sodium deoxycholate, and 1 mM CaCl2 The
reactions were started by addition of the transfected cell membranes
and terminated by adding 350 µl of chloroform/methanol/concentrated
HCl (500:500:3, v/v/v). The samples were vortexed, and 100 µl of 1 M HCl containing 5 mM EGTA was added. Phases
were separated by centrifugation and assayed for radioactivity by
liquid scintillation counting.
, hPLC
H1144L, or RasGRF2 in
combination with HA-tagged MAP kinase (2 mg). 24 h after
transfection the cells were serum starved over night. Cells were lysed
48 h after transfection as described previously with slight
modifications (18). Briefly, cells were washed twice with ice-cold
phosphate-buffered saline (Life Technologies, Inc.) and lysed by
addition of one volume 10× MAP kinase lysis buffer (5.5% Triton
X-100, 0.2 mM phenylmethanesulphonyl fluoride, 0.7 mg/ml
pepstatin A, 10 mg/ml leupeptin, 2 mg/ml aprotinin, 20 mM
sodium orthovanadate, and 20 mM sodium pyrophosphate) to 10 volumes phosphate-buffered saline. The cells were incubated for 10 min
at 4 °C and centrifuged at 4 °C for 15 min at 10,000 × g. Activated MAP kinase was immunoprecipitated by incubating the cells for 2 h with anti-HA antibodies (Upstate
Biotechnology Inc., 0.4 mg/ml). At the end of the incubation period
precleared protein A beads (50% slurry) were added, and the samples
were rotated 2 h at 4 °C. The ability of MAP kinase to
phosphorylate myelin basic protein was measured following the
manufacturer's protocol (Upstate Biotechnology Inc.). Protein samples
were separated on 6% (PLC
and H1144L) and 15% SDS-polyacrylamide
gel and subsequently transferred to nitrocellulose at room temperature
for 1 or 2 h at 5 or 15 V, respectively, using a semidry transfer
cell (Bio-Rad). The HA monoclonal antibody (clone 12CA5; Roche
Molecular Biochemicals) was used to detect HA-MAP kinase, anti-FLAG M2
antibody (Sigma) was used to detect FLAG-tagged RasGRF2, and anti-Myc
antibody (InVitrogen) was used to detect hPLC
and the H1144L mutant.
The horseradish peroxidase-coupled goat anti-mouse antibody (1:2,000; Sigma) was used as the secondary antibody. The blots were developed by
ECL (Amersham Pharmacia Biotech).
, hPLC
H1144L, or RasGRF2 and Ha-Ras. The cells
were serum-starved for 24 h. 48 h after transfection the
cells from a 10-cm dish were lysed and scraped in 1 ml of RIPA buffer
containing 50 mM Tris, pH 8.0, 150 mM NaCl,
0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 0.1 mM aprotinin, 1 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged
at 14,000 rpm for 8 min at 4 °C to remove nuclei. The desired amount
of bacterial lysate containing the expressed GST-Ras-binding domain
(GST-RBD) of Raf1 (prepared as described by de Rooij and Bos (19)) was
thawed on ice and incubated with glutathione-agarose beads at 4 °C
for 1 h. The beads were isolated by centrifugation and washed
three times with RIPA buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1%
SDS, 0.1 mM aprotinin, 1 mM leupeptin, 1 mM pepstatin, and 1 mM phenylmethylsulfonyl
fluoride at 4 °C. Cell lysates were added to GST-RBD precoupled to
glutathione-agarose beads and incubated at 4 °C for 1 h. Beads
were collected by centrifugation, washed three times with RIPA buffer,
and resuspended in SDS sample buffer. GTP bound Ras was identified by
precipitation with GST-Raf RBD followed by immunoblotting as described
previously by de Rooij and Bos (19). The protein samples were separated
on 6% (PLC
and H1144L) and 15% SDS-polyacrylamide gel and
subsequently transferred to nitrocellulose at room temperature for
1 h at 5 V using a semidry transfer cell (Bio-Rad). The monoclonal
antibody Y13-259 (1:500, Transduction Labs) was used to detect Ras and
anti-Myc tag antibody was used to detect hPLC
and the H1144L mutant.
The horseradish peroxidase-coupled goat anti-mouse antibody (1:2,000,
Sigma) was used as the secondary antibody. The blot was developed by
ECL (Amersham Pharmacia Biotech).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, Determination of
Expression in Human Tissues, and Identification of Structural
Features--
X and Y domains are regions of ~170 and ~260 amino
acids, respectively, which share 60% to 40% amino acid identity among
all PLC isoforms. The X and Y domains are necessary for
phosphodiesterase activity of PLC. We selected three relatively short
amino acid sequences from the conserved X and Y domains to use in a
BLAST search. The Basic Local Alignment Search Tool (BLAST) is an
extremely powerful tool that allows one to simultaneously search
multiple nucleotide sequence data bases. Using the BLAST server at the National Center for Biotechnology Information, we were able to identify
an EST clone that partially encoded for a novel PLC isoform. Using the
EST cDNA as a probe, a human placental cDNA library was
screened yielding a larger but still incomplete cDNA. A full-length cDNA clone was ultimately generated with 5' rapid amplification of
cDNA ends PCR using human heart mRNA as template that we termed PLC
. The full-length cDNA (Fig.
1A) possesses an open
reading frame of 6.05 kilobases encoding a 1994-amino acid protein with a calculated molecular mass of 230,000, making this the largest PLC
isolated to date. The next largest would be a member of the
family
at ~1300 amino acids and ~150 kDa. Hybridization of human multiple
tissue Northern blots with PLC
cDNA revealed a ~7.5-kilobase message corresponding to PLC
expressed in a wide variety of tissues including brain, lung, kidney, testis, and colon with highest expression detected in the heart (Fig.
2). An additional transcript of larger
size (~9.5 kilobases) could be observed in most tissues, suggesting
the possibility of an alternatively spliced form of PLC
or
differential polyadenylation. Results from Southern blotting indicate
that isoforms likely do not exist (data not shown).
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Fig. 1.
Structure and comparison of
PLC . A, Full-length amino acid
sequence of human PLC
. Highlighted residues indicate the
identified protein domains. X and Y, X and Y
domains, catalytic core regions of PI-PLCs; PH, pleckstrin
homology domains; C2, calcium-regulated lipid binding
domain; RA, Ras-associating domain. B, schematic
comparison drawn to scale of the PLC family functional domains.
X and Y, X and Y domains, catalytic core regions;
PH, pleckstrin homology domains; C2,
calcium-regulated lipid binding domain; SH, Src homology
domain; RA, Ras-associating domain. C, evolutionary
comparison depicting the relationships among all known isoforms of
eukaryotic PLC and PLC
. Clustal X was used to generate
alignments of the primary structures which were then displayed with
TreeView. The scale bar is used to quantitate branch
lengths. A scale of "0.1" means 0.1 amino acid
substitutions/residue.
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Fig. 2.
Tissue distribution of
hPLC mRNA. Multiple human tissue
Northern blots were analyzed as described under "Experimental
Procedures." Each lane contains approximately 2 mg of
poly(A)+ RNA. Blots were hybridized with the full-length
cDNA or
-actin probe. The hybridized membrane was exposed to
x-ray film for 24 h. kb, kilobases.
contains the conserved
catalytic X and Y domains, thus identifying this isoform conclusively as a Pl-PLC (Fig. 1B). Like members of the three other PLC
families, PLC
also contains the regulatory C2 domain. Unlike other
eukaryotic PLCs, PLC
appears not to contain a pleckstrin homology
domain. A phylogenetic comparison of all known mammalian PLC isoforms (Fig. 1C) demonstrates that PLC
shares little homology
with other PLC families (
,
, and
) and therefore constitutes a
distinct family of PLCs. PLC
is most similar to PLC210, a largely
uncharacterized isoform from the nematode Caenorhabditis
elegans (20). PLC210 was first identified by open reading frame
prediction of genomic sequences generated from the C. elegans genome sequencing project. Although there are significant
similarities, human PLC
also differs considerably from PLC210.
PLC
is considerably larger than PLC210 (~200 amino acids) and
differs extensively in the primary structure of the C terminus and
portions of the N terminus. We predict that the C terminus of PLC
will mediate protein-protein interactions that are completely different
from those of PLC210. Interestingly, both PLC
and PLC210 share
structural domains that are not present in any other PLC, further
suggesting the existence of a novel PLC family. Both PLCs contain a Ras
binding motif denoted as the RA domain (21). Although the functional
role of RA domains is presently unknown, a recent report has
demonstrated that PLC210 binds to Ha-Ras via this domain, suggesting a
possible role of this isoform in Ras signaling (20). A number of other
RA domain-containing proteins such as RalGDS and RGL are also known to
bind to Ras (21). PLC
has two RA domains at the C terminus (amino
acids 1688-1792 and 1813-1916), suggesting that it might interact
with the effector region of Ras (Fig. 1B). PLC
also
contains domains in the N terminus, which suggests that it may interact
with small G proteins of the Ras superfamily. Very significant homology
(p < 0.00001) is found with aimless RasGEF from
Dictyostelium, CDC25 RasGEF from yeast (both Candida
albicans and Saccharomyces cerevisiae), human RasGEF
homolog Sos1, human RasGEF H-GRF55, and son-of-sevenless RasGEF from
mouse. The area of homology is encompassed by the RasGEF catalytic
domain signature
(G/A/P)CVP(F/Y)X4(L/I/M/F/Y)X(D/N)(L/I/V/M) PROSITE121 (22). No other eukaryotic PLC contains these Ras binding
motifs. This strongly suggests that PLC
activates Ras signal
transduction pathways.
in TSA201 Cells and Characterization of
Polyphosphoinositide Hydrolysis--
Western blot analysis of cell
lysates from TSA201 cells (a clone of human embryonic kidney 293 cells
stably expressing simian virus 40 large T antigen) revealed the
presence of a single protein of approximately 230 kDa from cells
transfected with PLC
cDNA inserted into the mammalian expression
vector pcDNA3 but not from control cells (Fig.
3A) (23). The observed
apparent molecular mass of 230 kDa is consistent with the
predicted molecular mass of PLC
. Extensive PLC
immunoreactivity
resides in the particulate fraction of transfected TSA201 cells (data
not shown) similar to eukaryotic PLC
isoforms but unlike PLC
and
PLC
isozymes that are primarily localized in cytosolic fractions
(24, 25).
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Fig. 3.
Expression of PLC in
TSA201 cells reveals a PI-PLC. A, Western blot analysis
reveals a band of approximately 230 kDa corresponding to Myc-tagged
PLC
(arrow) only in cells lysates (50 mg) obtained from
TSA201 cells transfected with PLC
, whereas none is found in lysates
(50 mg) obtained from TSA201 cells transfected with control (vector).
B, membranes (10 mg) isolated from TSA201 cells expressing
PLC
(filled circles) exhibit time dependent increases in
InsP formation when reconstituted with mixed phospholipid vesicles
containing [3H]PIP2 as described under
"Experimental Procedures." Data represent the averages ± S.E.
of duplicate samples. Similar results were obtained in three
independent experiments. Where error bars are not shown, the
range was less than the size of the point. MWt., molecular
mass.
has Pl-PLC activity, measured by its ability to hydrolyze
exogenous PIP2, a selective substrate of PI-PLCs. As can be seen in Fig. 3B, 10 µg of plasma membranes obtained from
PLC
transfected TSA201 cells had a 2-3-fold (61-114 pmol
PIP2 hydrolyzed) greater PLC activity over a 15-min
incubation period than membranes of cells transfected with control
vector (20-46 pmol of PIP2 hydrolyzed).
12 Selectively
Stimulates PLC
-mediated Hydrolysis of Polyphosphinositides--
As
depicted in the phylogenetic tree (Fig. 1C), the closest
mammalian homolog of PLC
is PLC
, an effector for heterotrimeric G
protein G
and
subunits. Currently, there are no identifiable motifs for proteins that bind to heterotrimeric G proteins. However, truncation of the terminal 112 amino acids of PLC
1
(Gln1030-Leu1142) has been shown to totally
abolish regulation of this isoform by G
q, suggesting
that the C terminus is necessary for G
subunit interaction (26).
Like PLC
, PLC
has a long C terminus, suggesting the possibility
for regulation by heterotrimeric G proteins. The ability of eight
different constitutively active (GTPase-deficient) mutants of G protein
subunits (G
*) to stimulate PLC
activity in TSA201 cells was
determined. In the absence of any extracellular activators,
co-transfection of G
12* with PLC
augmented PLC
activity nearly 3-fold greater than control (Fig.
4A). Cells transfected with
G
12* alone did not have increased PLC activity.
Co-transfection with G
13* also led to an increase in PLC
activity; however, G
13 alone increased endogenous PLC
activity, suggesting that the effects obtained with G
13*
co-transfected with PLC
were in fact equal in magnitude to the sum
of the individual effects of PLC
and G
13* alone. Because
co-expression of G
13* with PLC
tended to decrease PLC
expression somewhat, we cannot be sure that G
13* has no
effect. In many systems G
12 and G
13 are
interchangable, that is they both regulate the same effectors. A clear
exception has been described for p115RhoGEF. Although both
G
12 and G
13 can bind to this novel RhoGEF
(p115RhoGEF serves as a GAP for both G
subunits), only
G
13 can activate the GEF activity toward Rho (14).
G
s* and G
i* had no effect on PLC
activity. The ability of other G
subunits: G
i2*,
G
z*, and G
o* to regulate PLC
were also
determined; however, none of these G
subunits increased PLC activity
when co-transfected with PLC
(data not shown). G
q*
expressed in cells greatly increased basal PLC activity most likely by
activation of endogenous PLC
found in these cells. Co-transfection
of PLC
with G
q* did not increase PLC activity above
G
q* transfected cells, suggesting that this G
subunit does not activate PLC
. PLC
activity was obtained under conditions where similar amounts of PLC
and G
subunits were expressed (Fig. 4, B and C).
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Fig. 4.
Overexpression of GTPase-deficient
heterotrimeric G protein subunits stimulates
PLC
enzymatic activity. A, PI
hydrolysis (InsP formation) is stimulated by co-expression of
G
12 with PLC
in TSA201 cells. Each value represents
the mean ± S.E. of four experiments each carried out in
duplicate. B, protein (100 mg) obtained from cells
transfected with either control (vector), G
subunits, and PLC
alone or in combination with G
subunits were separated by
SDS-polyacrylamide gel electrophoresis on an 6% gel and transferred
unto nitrocellulose. Levels of expression of G
subunits were
analyzed by Western blotting using antisera directed against
G
s, G
i, G
12, and
G
13 as described under "Experimental Procedures."
This figure is a representative of four different experiments with
similar results. C, protein (100 mg) obtained from cells
were separated on a 6% gel and transferred unto nitrocellulose. Levels
of expression of PLC
were analyzed using anti-Myc antibody as
described under "Experimental Procedures." This figure is a
representative of four different experiments with similar
results.
is one of very few known effectors for G
12,
because the G
12/G
13 family of
heterotrimeric G proteins is currently poorly characterized (27).
G
12 and G
13 appear to play an important role in regulating cellular and cytoskeletal changes and may themselves be regulated by the receptors for thrombin and lysophosphatidic acid
and other mitogenic agonists (9). G
12 is in fact a
highly oncogenic G
subunit. A GTPase deficient mutant
G
12 Q229L fully transforms NIH 3T3 cells. The
transformed cells form foci, grow in semisolid medium, and form tumors
in nude mice (28). G
12 has also been found to regulate
extracellular signal-regulated kinase and c-Jun kinase pathways (29,
30). It appears that G
12 stimulates c-Jun via activation
of Ras; however, the mechanism by which G
12 activates
Ras is unknown (31). Perhaps PLC
can act as a direct link between
G
12 and Ras.
Activates Ras and a Downstream Serine/Threonine Kinase MAP
Kinase--
The presence of a RasGEF motif in the N terminus of PLC
suggests that PLC
can activate Ras by acting as an exchange factor by promoting the exchange of GTP for bound GDP. Ras mediates its effects on cellular growth and transformation mainly by activating a
cascade of serine/threonine kinases including Raf, MEK, and MAP kinase
(32). Because MAP kinase (ERK1/2) is a downstream effector of Ras
signaling, the ability of PLC
to activate Ras was determined by
measuring phosphorylation of MAP kinase. To determine whether PLC
could activate Ras and the MAP kinase pathway independent of PI
hydrolysis, the ability of an X domain phosphodiesterase deficient
mutant of PLC
was examined. X and Y domains are regions of ~170
and ~260 amino acids, respectively, which share 60% to 40% amino
acid identity among all eukaryotic PI-PLCs. The X and Y domains are
necessary for phosphodiesterase activity and make up the catalytic core
of the enzyme. Bacterial PLCs contain only the X domain. We have
previously demonstrated through extensive site-directed mutagenesis of
PLC
1 that the X domain is responsible for the catalytic hydrolysis
of polyphosphoinositides, whereas the Y domain is responsible for
substrate binding (33). Mutation of amino acid residues
Arg338, Glu341, and His356 in the X
domain of PLC
1 lead to cleavage defective enzymes (33). These
residues are absolutely conserved in all eukaryotic PLCs including
PLC
. A cleavage-defective PLC
was created by mutating the
conserved histidine at position 1144 to leucine. This residue is
analogous to the histidine at position 356 of PLC
1. PLC
H1144L
is expressed normally as determined by Western blot using anti-Myc
antibodies (Fig. 5B) but does
not hydrolyze substrate (data not shown). Thus, any effects on Ras
mediated by PLC
H1144L would be due to the Ras binding domains of
PLC
rather than due to indirect effects of PI hydrolysis and the
production of the second messengers DAG and IP3.
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Fig. 5.
PLC activates the
MAP kinase pathway. A, cell lysates (~3-5 mg of
total protein) were acquired from control, PLC
, PLC
H1144L, and
RasGRF2 transfected cells, and MAP kinase activity was assayed as
described under "Experimental Procedures." The data are the
means ± S.E. of two to six experiments using separate
transfections. B, Western blot analysis of cell lysates (100 mg), using HA antibody 12CA5 for MAP kinase, anti-Myc for PLC
, or
anti-FLAG antibody for RasGF2 as described under "Experimental
Procedures." Antibodies were used at a dilution of 1:1000. This
figure is a representative of three to five different experiments
repeated with similar results.
and PLC
H1144L could indeed activate the
MAP kinase pathway, TSA201 cells were co-transfected with either
pcDNA3 vector (control), PLC
, PLC
H1144L, or RasGRF2 (as a
control for activation of the Ras/MAP kinase pathway) and HA-tagged MAP
kinase. Immunoprecipitates obtained from cells expressing PLC
show a
3-fold (454 pmol/min/mg total protein) increase in phosphorylation of
MAP kinase relative to control immunoprecipitates (139 pmol/min/mg)
obtained from cells transfected with vector alone (Fig. 5A).
Expression of PLC
H1144L also stimulates phosphorylation of MAP
kinase, indicating that PI hydrolysis is not necessary for activation
of the Ras effector MAP kinase. In fact, PI hydrolysis seems to inhibit
the activation of MAP kinase, because H1144L-stimulated MAP kinase
approximately 2-fold greater than wild type PLC
(907 and 454 pmol/min/mg, respectively). The expression of wild type PLC
and
H1144L was nearly identical (31.9 versus 29.3 relative units, respectively) in TSA201 cells, as was the level of HA-MAP kinase. Therefore, levels of expression did not account for the differences in activity (Fig. 5B). The products of PI
hydrolysis IP3 and DAG could act as indirect negative
regulators of the RasGEF activity and thereby act as a negative
feedback loop. For example, stimulation of protein kinase C by DAG
might lead to phosphorylation of PLC
and inhibition of the RasGEF
activity. The known RasGEF, RasGRF2, gave the most robust stimulation
(1231 pmol/min/mg) yet was comparable with the stimulation by H1144L
and PLC
, demonstrating that PLC
is a fairly robust activator of
MAP kinase.
in stimulating MAP kinase, a
Myc-tagged construct PLC
-GEF was made of the N-terminal 600 amino
acids of PLC
. This construct contains the entire RasGEF domain but
lacks the X and Y phosphodiesterase domains and both of the C-terminal
RA domains. PLC
-GEF was able to stimulate MAP kinase to a level
comparable with the wild type holo enzyme (Fig. 6), suggesting that the RasGEF domain is
sufficient for activation of the Ras/MAP kinase pathway by PLC
and
that the C-terminal RA domains are not necessary. This result is
consistent with the previously described findings for RGL (34).
View larger version (33K):
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Fig. 6.
The GEF domain of PLC
is sufficient to activate MAP kinase. A, cell
lysates (~3-5 mg of total protein) were acquired from control,
PLC
, PLC
-GEF transfected cells, and MAP kinase activity assayed
as described under "Experimental Procedures." The data are the
means ± S.E. of three experiments using separate transfections.
B, Western blot analysis of cell lysates (100 mg), using HA
antibody 12CA5 for MAP kinase, and anti-Myc for PLC
and PLC
-GEF
as described under "Experimental Procedures." Antibodies were used
at a dilution of 1:1000. This figure is a representative of three
different experiments with similar results.
from these studies. They do not seem to be involved with membrane
association, because the PLC
-GEF construct is highly targeted to
membranes (data not shown). The domains might be closely associated
with the phosphodiesterase catalytic domains (X and Y) because when one
or more of the RA domains are truncated, the resulting enzyme has very
little phosphodiesterase activity, suggesting that these domains are
necessary for proper folding of the enzyme (data not shown). This is
similar to truncation of the
1 isoform of PLC, where deletion of
even a few C-terminal residues leads to inactivation of
phosphodiesterase activity.3
Because of the potential interaction of the RA domains with the X and Y
domains, the ability of activated Ras to regulate the phosphodiesterase
activity of PLC
was assessed by co-transfection. As can be seen from
Fig. 7A, v-Ras does not
stimulate but may inhibit PLC
in vivo. Transfection of
cells with PLC
alone increased PI hydrolysis 3.5-fold, whereas
co-transfection with PLC
and v-Ras lead to an increase of only
2-fold. Expression levels of v-Ras and PLC
did not vary
substantially among different conditions. Actually, PLC
expression
was slightly greater when co-transfected with v-Ras. Overall, these
results suggest a potential role for PLC
in regulating Ras
activation and thus activation of the MAP kinase pathway in a manner
dependent upon the RasGEF domain and independent of PI hydrolysis.
View larger version (23K):
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Fig. 7.
Activated Ras inhibits PLC
phosphodiesterase activity in vivo.
A, PI hydrolysis (InsP formation) is assayed in whole cells
after transfection in TSA201 cells. Each value represents the
means ± S.E. of two experiments each carried out in duplicate.
B, Western blot demonstrating expression of v-Ras and
PLC
. This figure is a representative of two different experiments
with similar results.
Activates Ras--
More direct evidence of the ability of
PLC
to activate Ras comes from experiments in which GTP-Ras is
trapped using the Ras effector Raf1. The minimal RBD of Raf1 (amino
acids 51-131) binds very tightly and specifically to the GTP-bound
form of Ras (Kd 20 nM), whereas the
affinity for RasGDP is 3 orders of magnitude lower (35). To determine
whether PLC
could indeed act as an exchange factor for Ras, TSA201
cells were transiently co-transfected with either pcDNA3 (control),
PLC
, PLC
H1144L, or RasGRF2 (as a GEF control for activation of
Ras) and Ha-Ras. The cells were harvested after 48 h and
RasGTP was identified by precipitation with GST-RBD and immunoblotting
using anti-Ras antibody. Fig. 8A demonstrates that GST-RBD
bound RasGTP was increased significantly in cells transfected with
PLC
, PLC
H1144L, and the known RasGEF, RasGRF2. The fold increase
in RasGTP for PLC
, H1144L, and RasGRF was 4.2-, 9.3-, and 4.1-fold,
respectively. Although levels of expression for PLC
and the
phosphodiesterase deficient mutant H1144L were very similar (Fig.
8B), H1144L was 2.2-fold more potent for activation of Ras.
As discussed in the previous section, H1144L was also more potent in
activating MAP kinase. The increase in H1144L activity suggests that
the phosphodiesterase activity may serve as a negative feedback
regulator of the RasGEF activity. Levels of expression for Ha-Ras were
similar for each of the experimental conditions (Fig. 8B).
There was no appreciable RasGTP detected in control cells. Basal levels
of RasGTP were reduced significantly by preincubation of the cells in
serum-free medium for 12 h. Taken together, these results provide
more direct evidence that PLC
indeed activates Ras by acting as a
RasGEF.
View larger version (38K):
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Fig. 8.
Effect of PLC and
H1144L on Ras activation. A, cells were transfected
with either vector (control), PLC
, PLC
H1144L, or RasGRF2 in
combination with Ha-Ras. Cell lysates were prepared, and the amount of
protein was determined. GTP-Ras was isolated by binding to GST-RBD and
visualized by Western blot Analysis as described under "Experimental
Procedures." Approximately equal amounts of protein were loaded per
lane. This figure is a representative of three to five different
experiments with similar results. B, Western blot analysis
of cells lysates (100 mg), using anti-Myc, anti-HA, anti-FLAG, or
anti-Ras antibodies. Antibodies were used in dilutions of 1:1000.
is a widely expressed unique PLC
enzyme that constitutes a new PLC family possessing bifunctional activity, both PLC and RasGEF activity. As such, PLC
may mediate the
effects of G protein-coupled receptors, especially those coupled with
G
12/G
13 through two divergent pathways
involving phosphatidylinositol hydrolysis as well as direct activation
of the Ras/MAP kinase pathway. Thus, this new member of the PLC family
may play a vital role in transducing signals from the plasma membrane
to the nucleus through multiple pathways to modulate cytoskeletal
changes, cell growth, and mitogenesis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Tatyana A. Voyno-Yasenetskaya for
kindly providing the constitutively active Gs*,
G
i*, G
12*, G
13*,
G
z*, G
o*, and G
16* and
David R. Manning for providing antibodies against G
s,
G
12, and G
13. We also thank Dr. Alan
Wolfman and Johannes L. Bos for kindly providing GST-Raf1 minimal
Ras-Binding Domain (GST-RBD), Mike Moran for providing RasGRF2, and
Mark Marshall for v-Ras G12V and Ha-Ras. We also thank John Campion for
technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by a Merit Review Grant from the Department of Veterans Affairs (to J. W. L.), by National Institute of Health Grants HL03836 (to I. L.) and HL55591 and HL03961 (to J. W. L.), by the Robert H. Lurie Comprehensive Cancer Center's American Cancer Society Institutional Research Grant IRG-93-037-06 (to I. L.), and by American Heart Association Grant-in-Aid 9951330Z (to I. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF170071.
To whom correspondence should be addressed: Dept. of Pathology
and Feinberg Cardiovascular Research Institute, Tarry Bldg. 12-703, Northwestern University Medical School, 303 E. Chicago Ave., Chicago,
IL 60611. Tel.: 312-503-0450; Fax: 312-503-0137; E-mail:
j-lomasney@northwestern.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M008119200
2 J. W. Lomasney and K. King, submitted for publication.
3 K. King and J. W. Lomasney, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PLC, phospholipase C; PI-PLC, PI-PLC; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; MAP, mitogen-activated protein; RasGEF, Ras guanine nucleotide exchange factor; RA, Ras-associating; RBD, Ras-binding domain; PI, phosphatidylinositol; EST, expressed sequence tag; PCR, polymerase chain reaction; HA, hemagglutinin; GST, glutathione S-transferase.
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