A Novel Phospholipase C
4 (PLC
4) Splice Variant as a
Negative Regulator of PLC*
Kohji
Nagano
,
Kiyoko
Fukami
,
Tetsuya
Minagawa
,
Yutaka
Watanabe§,
Choichiro
Ozaki§, and
Tadaomi
Takenawa
¶
From the
Department of Biochemistry, Institute of
Medical Science, University of Tokyo, Tokyo 108-8639, and the
§ Faculty of Technology, Ehime University,
Matsuyama 790-8577, Japan
 |
ABSTRACT |
It has been reported that there are two
alternatively spliced variants of phospholipase C-
4 (PLC
4),
termed ALT I and II, that contain an additional 32 and 14 amino acids
in their respective sequences in the linker region between the
catalytic X and Y domains (Lee, S. B., and Rhee, S. G. (1996)
J. Biol. Chem. 271, 25-31). We report here the
isolation and characterization of a novel alternative splicing isoform
of PLC
4, termed ALT III, as a negative regulator of PLC. In ALT III,
alternative splicing occurred in the catalytic X domain,
i.e. 63 amino acids (residues 424-486) containing the C-terminal of the X domain and linker region were substituted for 32 amino acids corresponding to the insert sequence of ALT I. Although the
expression level of ALT III was found to be much lower in most tissues
and cells compared with that of PLC
4, it was significantly higher in
some neural cells, such as NIE-115 cells and p19 cells differentiated
to neural cells by retinoic acid. Interestingly, recombinant ALT III
protein did not retain enzymatic activity, and the activity of PLC
4
overexpressed in COS7 cells was markedly decreased by the co-expression
of ALT III but not by ALT I or II. Moreover, N-terminal pleckstrin
homology domain (PH domain) of ALT III alone could inhibit the increase of inositol-1,4,5-trisphosphate levels in PLC
4-overexpressing NIH3T3
cells, whereas a PH domain deletion mutant could not, indicating that
the PH domain is necessary and sufficient for its inhibitory effect.
The ALT III PH domain specifically bound to phosphatidylinositol (PtdIns)-4,5-P2 and PtdIns-3,4,5-P3 but not
PtdIns, PtdIns-4-P, or inositol phosphates, and the mutant R36G, which
retained only weak affinity for PtdIns-4,5-P2, could not
inhibit the activity of PLC
4. These results indicate that
PtdIns-4,5-P2 binding to PH domain is essential for the
inhibitory effect of ALT III. ALT III also inhibited PLC
1 activity
and partially suppressed PLC
1 activity, but not PLC
1 in
vitro; it did inhibit all types of isozymes tested in
vivo. Taken together, our results indicate that ALT III is a
negative regulator of PLC that is most effective against the PLC
-type isozymes, and its PH domain is essential for its function.
 |
INTRODUCTION |
Phospholipase C (PLC)1
plays a crucial role in the inositol phospholipid signaling by
hydrolyzing phosphatidylinositol (PtdIns)-4,5-bisphosphate. This
reaction produces two intracellular second messengers, inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and diacylglycerol,
which cause the increase of intracellular calcium concentration and the
activation of protein kinase C (PKC), respectively (1). These cascades are thought to be terminated by the dephosphorylation of
Ins-1,4,5-P3 by inositol phosphate phosphatases (2) and the
phosphorylation of diacylglycerol by diacylglycerol kinases (3).
However, since two signals are generated simultaneously by one enzyme
(an enzyme of PLC), there might be also a mechanism that turns off
these cascades simultaneously, i.e. a negative regulation of PLC.
The PLC family is comprised of 10 subtypes found in mammalian species,
and on the basis of their structure, they have been divided into three
classes,
(
1-4),
(
1 and 2), and
(
1-4) types (4).
Positive regulation mechanisms of PLC by association with membrane
receptors are well characterized in
- and
-type isozymes.
-Type isozymes are activated by the G
or G
subunit released
from heterotrimeric G

proteins after ligand stimulation (4-7).
-Type isozymes are activated by the phosphorylation of specific tyrosine residues through the activation of receptor or
nonreceptor tyrosine kinases (4, 8, 9). The mechanism by which
-type
isozymes are coupled to membrane receptors remains unclear.
Several recent studies indicate that the pleckstrin homology (PH)
domains of PLC isozymes are also important for the activity of the
isozymes. First, PLC
1 was shown to be activated by the binding of
PtdIns-4,5-P2 to its PH domain and inhibited by the binding
of Ins-1,4,5-P3 to the same domain (10-12). On the basis of the information obtained from the three-dimensional structure of
PLC
1, a catalytic mechanism comprising two steps, tether and fix,
was proposed (13); the PH domain tethers the enzyme to the membrane by
specific binding to PtdIns-4,5-P2, and the C2 domain fixes
the catalytic domain in a productive orientation on the membrane.
Therefore, the PH domain plays a important role for the first contact
to the plasma membrane. Second, more recent studies indicate that the
PtdIns-3,4,5-P3 binding to the PH domain is involved in the
protein tyrosine kinase-independent activation of PLC
1. It has been
reported that the overexpression of PtdIns-3 kinase, which produces
PtdIns-3,4,5-P3, results in the activation of PLC
1 by
the binding of PtdIns-3,4,5-P3 to the N-terminal PH domain
(14). Furthermore, SH2-containing inositol-5'-phosphatase, which
removes 5'-phosphates from phosphoinositides, was shown to eliminate
the PtdIns-3,4,5-P3-dependent activation of
PLC
1 in B cells, resulting in a decrease in Ins-1,4,5-P3
formation and decreased calcium mobilization (15, 16). Therefore,
SH2-containing inositol-5'-phosphatase functions as a negative
regulator of PLC
1 by inhibiting
PtdIns-3,4,5-P3-dependent activation, at least in B cells.
Negative regulations of PLC by protein kinase C (PKC) and
cAMP-dependent protein kinase are also known (4, 17-21).
The proposed targets for phosphorylation by these kinases include
cell-surface receptors, G proteins, and PLC itself.
cAMP-dependent protein kinase directly phosphorylates the
serine residues of PLC
2 and -
3 and inhibits the stimulation of
their activity by G
or G
q, respectively (17, 18).
PLC
1 is phosphorylated by PKC
, resulting in the inhibition of its
activation by G
/
(21). It was also reported that gelsolin and
profilin, which bind to PtdIns-4,5-P2, compete with PLC for
PtdIns-4,5-P2, resulting in the inhibition of the activity
of PLC
and -
, respectively (22, 23). However, the negative
regulation mechanism of PLC
-types remains unknown.
Here, we report that a novel spliced variant of PLC
4, termed ALT
III, functions as a negative regulator of PLC through its PH domain.
ALT III inhibited the activity of PLC
most effectively among the PLC
isozymes in vivo and in vitro. This is the first demonstration of a negative regulator of PLC
-type isozymes.
 |
EXPERIMENTAL PROCEDURES |
Materials
[3H]PtdIns-4,5-P2,
[3H]Ins-1,4,5-P3, and
[3H]Ins-1,3,4,5-P4 were obtained from NEN
Life Science Products. Phosphatidylethanolamine, phosphatidylserine,
phosphatidylcholine, and phosphatidylinositol (PtdIns) were from Doosan
Sedary Research Laboratories, Ins-1,4,5-P3, Ins-1,3,4,5-P4, and bradykinin were from Sigma, and
platelet-derived growth factor (PDGF) was from Boehringer Mannheim
(Mannheim, Germany). PtdIns-4,5-P2 and PtdIns-4-P were
purified from bovine spinal cords. PtdIns-3,4,5-P3 (both
acyl groups are palmitoyl) was chemically synthesized. The transfected
cell selection kit, the MACSelect4 and the MACS high-gradient magnetic
separation columns were purchased from Miltenyl Biotec
(Bergisch-Gladbach, Germany). The Ins-1,4,5-P3 assay kit
was obtained from Amersham Pharmacia Biotech. The rabbit polyclonal
anti-PLC
4 antibody was raised against the C-terminal 157 amino
acid-tagged 6xHis using the vector pQE-31. PLC
1, -
1, and -
1
were purified from bovine brain by using a three-step column
chromatography technique (24).
Methods
Isolation of a Novel Spliced Variant of PLC
4, Termed ALT
III--
Total RNA was isolated from rat tissues by the guanidinium
isothiocyanate method and from culture cells by the Nonidet P-40 method. A reverse transcription-polymerase chain reaction (RT-PCR) was
carried out using rat testis total RNA as described previously (25). In
brief, the entire coding region of PLC
4 cDNA was amplified with
two sets of primers, both of which corresponded to the 5'- or
3'-untranslated regions of the cDNA by two consecutive rounds of
PCR. The second-round PCR products were subcloned into the vector
pBluescript (KS
). The nucleotide sequences were determined by the
autocycle sequencing method using a DSQ-1000 DNA sequencer (Shimadzu,
Kyoto, Japan). Among these clones, two clones coding a novel splice
variant of PLC
4, termed ALT III, were identified.
Measurement of mRNA Level by RT-PCR--
To determine the
tissue distribution of PLC
4 isoforms, first strand cDNA was
amplified with PLC
4-specific primers (nucleotides 1187-1206 and
1658-1677) to produce 491- (PLC
4), 587- (ALT I), 533- (ALT II), and
398-base pair (ALT III) fragments. PCR was performed for 24 cycles of
15 s at 94 °C, 15 s at 60 °C, and 30 s at 72 °C
with 10 µCi of [
-32P]dCTP, and the products were
detected by autoradiography. We confirmed that these products were
linearly amplified during this cycle.
Immunoblotting--
Lysates from various tissues and cells were
subjected to 7.5% SDS-polyacrylamide gel electrophoresis, and proteins
were transferred to polyvinylidene difluoride membranes. The membranes
were incubated with anti-PLC
4 antibodies and stained with alkaline
phosphatase-conjugated goat anti-rabbit IgG antibody. Alternatively,
primary incubation using anti-PLC
4 antibody was followed by the
incubation in horseradish peroxidase-conjugated goat anti-rabbit IgG.
Membranes were developed using an enhanced chemiluminescence system
(ECL, Amersham Pharmacia Biotech) according to the method recommended
by the manufacturer.
Cell Culture, Transfection, and Selection of Transfected Cells
Using the MACSelect 4 Kit--
COS7, N1E-115, and Bosc23 cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal bovine serum (FBS). NIH3T3 cells were maintained in
DMEM with 10% calf serum. PC12 cells were maintained in DMEM with 10%
horse serum and 5% FBS. C6 cells were maintained in Ham's F-10
nutrient medium (Life Technologies, Inc.) with 10% horse serum and
2.5% FBS. NG108-15 cells were maintained in DMEM supplemented with
10% FBS, 0.1 mM hypoxanthine, 1 µM
aminopterin, and 16 µM thymidine. p19 cells were
maintained in
-minimal essential medium with 10% FBS. The induction
of the differentiation of p19 cells with retinoic acid was performed as
described previously (26). NG108-15 and p19 cells were kindly provided
by Dr. T. Michikawa (University of Tokyo). PLC
4, ALT I, II, and III
were subcloned into the expression vector pcDNA3 (kindly provided
from Dr. F. Shibasaki (Tokyo Metropolitan Institute of Medical
Science)). The pMACS4 vector carrying cDNA of CD4 was
co-transfected with pcDNA3 carrying cDNAs encoding PLC
4, ALT
I, II, and III in COS7 cells. Transfections were performed by
electroporation (Bio-Rad). The cells were incubated at 37 °C for 2 days and harvested. The cells were then applied on the MACS high
gradient magnetic separation columns to collect the cells expressing
CD4 protein, and after 3 washes, the cells bound to the column were
eluted according to the manufacturer's protocol. Lysates from the
harvested cells were then used for the PLC assay.
Retrovirus Expression--
The retrovirus vector, pMx, was
kindly provided by Dr. T. Kitamura (University of Tokyo). cDNAs of
PLC
4, ALT III, and ALT III PH (nucleotides 1-376),
PH domain
(nucleotides 202-3'-end), and PLC
1 were subcloned into this vector.
Recombinant retroviruses were generated and infected to NIH3T3 cells as
described (27). In brief, cDNAs of interest were transfected in
Bosc23 cells, in which the recombinant retrovirus is produced, and the
cells were incubated for 2 days. The supernatant of the cells that
contained the recombinant virus was then collected, and exponentially
growing NIH3T3 cells were infected in the virus solutions for 2 days. After that, the cells were harvested. Alternatively, cells were further
incubated in starvation medium (DMEM supplemented with insulin/transferrin/sodium selenite supplement (ITS) and bovine albumin
serum (Boehringer Mannheim)) for 24 h, stimulated by 1 µM bradykinin or 50 ng/ml PDGF for the indicated time
(see figure legends) and harvested. Cells infected with the recombinant
viruses containing cDNA of green fluorescent protein were used as
the control.
Purification of PLC
4 and ALT III Protein Generated by an
Sf9/Baculovirus Expression System--
Recombinant PLC
4 and
ALT III proteins were prepared using a baculovirus expression system.
cDNA of PLC
4 and ALT III was subcloned into the vector pFastbac
(Life Technologies, Inc.). Recombinant, clonal baculoviruses were
generated according to the protocol described by Life Technologies,
Inc. Sf9 cells were grown at 28 °C in Sf-900 II SFM (Life
Technologies, Inc.) containing 10% FBS. Baculoviruses were used to
infect at a density of 1 × 106 cells/ml. The
proteins were purified according a method described previously
(25).
Measurement of PLC Activity--
PLC activity was assayed with
[3H]PtdIns-4,5-P2 as the substrate. The
PtdIns-4,5-P2-hydrolyzing activity was measured with mixed
phospholipid micelles containing 40 µM
phosphatidylethanolamine, 5 µM PtdIns-4,5-P2,
and 1 µCi/ml [3H]PtdIns-4,5-P2. The lipids
in chloroform were dried under a steam of nitrogen gas, suspended in
assay buffer (20 mM HEPES, pH 7.0, 120 mM NaCl,
2 mM MgCl2, 40 or 100 µM
CaCl2, and 1 mg/ml bovine albumin serum (Bayer, Leverkusen,
Germany)), and subjected to sonication. All proteins added to the
reaction mixture were dialyzed overnight against the assay buffer.
Incubation was performed for 10 min at 37 °C in a 50-µl reaction
mixture containing lipid micelles (5 µM
[3H]PtdIns-4,5-P2, 20,000 dpm). The reaction
was stopped by adding 2 ml of chloroform:methanol (2:1, v/v). Inositol
trisphosphates were extracted with 0.5 ml of 1 N HCl, and
the radioactivities in the upper aqueous phase were measured.
Assay of [3H]PtdIns-4,5-P2 Binding to
the ALT III PH Domain--
Recombinant glutathione
S-transferase (GST) fusion proteins containing the ALT III
PH domain (nucleotides 1-860) and R36G ALT III PH were constructed.
Site-directed mutagenesis by a two-stage PCR method was performed as
described (28). These proteins were expressed in Escherichia
coli, and the lysates were mixed with glutathione-Sepharose beads.
After three washes, proteins were eluted and dialyzed overnight against
binding buffer (20 mM HEPES, 120 mM NaCl, 2 mM MgCl2, pH 7.4). Purified fusion proteins
were mixed with micelles containing 2 µM
[3H]PtdIns-4,5-P2, 20,000 dpm, 6 µM phosphatidylserine, and 18 µM phosphatidylcholine in 50 µl of the binding buffer for 1 h at room temperature. Then 50 µl of glutathione-Sepharose beads were added, and the tubes were incubated for 15 min at room temperature with
occasionally mixing. The tubes were centrifuged at 2,000 rpm for 2 min,
and the radioactivity bound to beads was then measured.
Assay of Inositol Phosphate Binding--
Recombinant GST fusion
proteins containing the ALT III PH domain or PLC
1 PH domain
(nucleotides 1-880) were expressed in E. coli and purified.
Ins-1,4,5-P3 and Ins-1,3,4,5-P4 binding assays
using [3H]Ins-1,4,5-P3 and
[3H]Ins-1,3,4,5-P4 were performed as
described elsewhere (29).
Measurement of Ins-1,4,5-P3
Level--
Ins-1,4,5-P3 levels were assayed using a
commercial Ins1,4,5-P3 assay kit (Amersham Pharmacia
Biotech) according to the manufacturer's protocol.
 |
RESULTS |
Isolation of ALT III, a Novel Alternatively Spliced Variant of
PLC
4--
PLC
4 is an enzyme that is inducible in response to
mitogenic stimuli, and its expression is restricted in several tissues and cells (30). In addition to such characters, two alternatively spliced variants of PLC
4, termed ALT I and ALT II, are also known to
exist (25). To understand the specific function of PLC
4 and the
mechanisms of its regulation, we tried to obtain another isoform of
PLC
4. The entire coding region of PLC
4 cDNA was amplified by
RT-PCR as described by Lee and Rhee (25), subcloned into the vector
pBluescript KS(
), and sequenced. The complete sequence of these
products revealed that a novel splice variant termed ALT III could be
obtained in addition to the reported isoforms, including PLC
4, ALT
I, and ALT II. In ALT III, 63 amino acids containing the C-terminal of
the catalytic X domain and linker region between the catalytic X and Y
domains were substituted for 32 amino acids corresponding to the insert
sequence of ALT I (Fig. 1A).
ALT III protein was highly expressed in some neural cells (Fig.
2C), indicating that it exists
in living cells and is not an artifact obtained by PCR. An analysis of
the genomic DNA sequence of PLC
4 revealed that PLC
4, ALT I, II,
and III were generated from a single gene by alternative
splicings.2 ALT I and II
mRNA are comprised of exons IX, X, XI and new exons X' or X"
between exons X and XI of the gene, respectively. A splicing donor site
for the exon X was not recognized in ALT III, and ALT III is comprised
of exons IX, XI, and a new exon, X'. Thus, ALT III contains amino acids
VMKCPMSCLLICVHVLAQAPNSIPESILLPKR instead of the region from 424 to 486 (Fig. 1B).

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Fig. 1.
Splicing isoforms of phospholipase
C 4. A, schematic
representation of PLC 4 splicing isoforms. A PLC 4 gene
produces four isoforms, PLC 4, ALT I, II, and III. ALT I and II
contains 32 and 14 additional amino acids in their respective sequences
in the linker region between the catalytic X and Y domains, whereas in
the case of ALT III, 63 amino acids containing the C-terminal of the X
domain and linker region (424-486, the numbers
denote the positions of the peptide sequence) were substituted for 32 amino acids corresponding to the insert sequence of ALT I. B, schematic representation of PLC 4 genomic structure in
the region at which splice differences occurred. Exons of PLC 4 are
indicated as IX, X, X', X", and XI. ALT I and II contain an additional
new exon, X' or X", respectively. ALT III also contains exon X' but
does not possess exon X.
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Fig. 2.
Distribution of PLC 4
isoforms. A, immunoblotting analysis of lysates from
rat tissues with anti-PLC 4 antibodies. Lane 1, brain;
lane 2, testis; lane 3, thymus; lane
4, heart; lane 5, lung; lane 6, spleen;
lane 7, liver; lane 8, kidney; lane 9, intestine. B, RT-PCR analysis of PLC 4 total RNA from rat
tissues with PLC 4-specific primers. Lane 1, brain;
lane 2, thymus; lane 3, heart; lane 4, lung; lane 5, spleen; lane 6, liver; lane
7, kidney; lane 8, intestine; lane 9, testis; lane 10, negative control ( template).
C, immunoblotting analysis of lysates from various neural
culture cells with anti-PLC 4 antibodies. Lane 1, brain;
lane 2, glia; lane 3, C6, rat glioblastoma cells;
lane 4, N1E-115, mouse neuroblastoma cells; lane
5, PC12, rat pheochromocytoma cells; lane 6, NG108-15,
mouse neuroblastoma cells; lane 7, p19, mouse embryonal
carcinoma cells; lane 8, p19 differentiated to neural cells
by retinoic acid (5 × 10 7 M).
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Distribution of PLC
4 Isoforms in Tissues and Cells--
We next
examined the distribution of PLC
4 isoforms in rat tissues and
culture cells. Lysates from rat tissues and culture cells were
subjected to an immunoblot analysis with affinity purified polyclonal
antibodies to PLC
4 (Fig. 2, A and C). The
expressions of PLC
4 isoforms mRNA were also examined by RT-PCR
with a pair of PLC
4-specific primers (Fig. 2B). Both
analyses indicated that PLC
4 was expressed in the brain and testis,
and weak signals were observed in the heart and lung. The expressions
of ALT I and II were abundant in the testis. A strong signal for ALT
III was detected by immunoblotting in the heart. Some differences between the results of the RT-PCR and those of the immunoblotting were
observed. For instance, although the expression of PLC
4 mRNA was
detected by RT-PCR in the spleen and kidney, the proteins were not
detected. Also, ALT II and III proteins were detected by immunoblotting
in the liver, and PLC
4 protein was detected in the thymus, but the
respective mRNAs were not detected. Although the ALT III expression
was at quite a lower level compared with that of PLC
4 in most
tissues and cells, it was abundant in NIE-115 cells (a mouse
neuroblastoma cell line) and p19 cells differentiated to neural cells
by 5 × 10
7 M retinoic acid (Fig.
2C). In C6 cells, a rat glioblastoma cell line, PLC
4, and
ALT III were quite rich. We also detected PLC
4 and ALT III mRNA
in these cell lines by RT-PCR (data not shown). It was noteworthy that
the expression level of ALT III was significantly higher in
differentiated neural p19 cells compared with that in undifferentiated
embryonic p19 cells.
Inhibition of PLC
4 Activity by ALT III Caused by the PH
Domain--
Since ALT III lacks a part of the X domain and almost the
entire linker region, we expected that ALT III would not retain enzymatic activity. To investigate this, we subcloned the full-length of all PLC
4 isoforms into the vector pcDNA3, and the encoded genes were co-expressed with the vector carrying CD4 gene in
COS7 cells. Transfected cells were separated by magnetic beads to
collect the cells expressing CD4 protein. At least 30% of the cells
expressing CD4 protein were co-transfected with PLC
4 (data not
shown). Therefore, PLC
4-overexpressing cells were concentrated
enough to detect the activity of PLC
4 in their lysates. Fig.
3A shows that PLC
4 and ALT
II retained their activity up to 2.7- and 2.8-fold, respectively, compared with the control, whereas the activity of ALT I only reached
approximately 1.5-fold. ALT III does not have PLC activity at all. We
also confirmed that recombinant ALT III protein generated by the
Sf9/baculovirus system did not retain PLC activity (data not
shown). Moreover, the activity of PLC
4 was markedly decreased by the
co-expression of ALT III, to less than 40%, but not by ALT I (Fig.
3B). On the other hand, ALT II is catalytically as active as
PLC
4, but PLC
4 activity was rather suppressed by the co-expression of ALT II, slightly (Fig. 3B). We do not know
why it happened. However, we confirmed that the activity of recombinant PLC
4 protein was not inhibited by the addition of the recombinant ALT II protein generated by Sf9/baculovirus system (data not
shown). Therefore, this partial suppression by ALT II may be caused by some factors that affect the function of ALT II in vivo.

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Fig. 3.
Enzymatic activity of
PLC 4 was inhibited by ALT III, and the PH
domain was necessary and sufficient for its inhibitory effect.
A, activity of PLC 4 isoforms. PLC 4 isoforms were
co-expressed with a vector carrying the CD4 gene in COS7
cells. Transfected cells were separated by magnetic beads that could
harvest the cells expressing CD4 protein. cDNA of CD4 alone was
also overexpressed, and transfected cells were separated and used for
the control. PLC assays were performed using their lysates, and the PLC
activity is presented as the fold increase against control lysates.
B, enzymatic activity of PLC 4 was inhibited by the
co-expression of ALT III but not by ALT I or II. PLC 4 splicing
isoforms were co-expressed with PLC 4 and CD4, and lysates from
transfected cells harvested by magnetic beads were used for the PLC
assay. Data represent inhibition (%) against PLC 4-overexpressing
cell lysates. C, the PH domain alone could inhibit the
activity of PLC 4, whereas the PH domain deletion mutant could not.
Various constructs (indicated in the figure) were overexpressed by a
retrovirus expression system in NIH3T3 cells, and
Ins-1,4,5-P3 levels were measured with an assay kit. Data
represent inhibition (%) against Ins-1,4,5-P3 levels in
PLC 4-overexpressing cells. All data are means ± S.E.(n = 4).
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In order to clarify how PLC
4 is inhibited by ALT III in
vivo, we next investigated whether the PH domain alone or the PH domain deletion mutant of ALT III could inhibit the activity of PLC
4. PLC
4, ALT III, the PH domain coding region, and PH domain deletion mutant cDNAs were expressed by a retrovirus system in NIH3T3 cells. The cells expressing various constructs were then harvested, and the Ins-1,4,5-P3 level was measured. As a
result, ALT III did not retain enzymatic activity and inhibited the
activity of PLC
4 in vivo (Fig. 3C). Moreover,
the ALT III PH domain alone could completely block the activity of
PLC
4, whereas the PH domain deletion mutant could not (Fig.
3C). These results indicate that the ALT III PH domain is
necessary and sufficient for its inhibitory effect.
The ALT III PH domain specifically binds to
PtdIns-4,5-P2 and PtdIns-3,4,5-P3 but not
PtdIns, PtdIns-4-P, or inositol phosphates--
We then analyzed the
binding of inositol phospholipid to the ALT III PH domain. A
recombinant GST fusion protein containing the ALT III PH domain and the
mutant R36G were expressed in E. coli and purified, and an
inositol phospholipid binding assay was performed. Since Arg-36 of ALT
III corresponds to Arg-40 of PLC
1, which is essential for the
Ins-1,4,5-P3 binding and catalytic activity of PLC
1
(31), we constructed the R36G mutant as a mutant that could not bind to
PtdIns-4,5-P2. The ALT III PH domain bound to
PtdIns-4,5-P2 in phospholipid vesicles in a
dose-dependent manner (Kd = 5.8 µM) (Fig. 4A and
data not shown). Since the PLC
1 PH domain bound to
PtdIns-4,5-P2 (Kd = 3.4 µM) in our system (data not shown), the affinity of the
ALT III PH domain to PtdIns-4,5-P2 was comparable to that
of the PLC
1 PH domain. Unexpectedly, the R36G mutant could also bind
to PtdIns-4,5-P2 with quite low affinity. Next, to
determine the binding specificity of the ALT III PH domain to inositol
phospholipid, we examined the competitive effect of various unlabeled
inositol phospholipids on the binding of
[3H]PtdIns-4,5-P2 to the PH domain. We found
that the binding of [3H]PtdIns-4,5-P2 was
inhibited to 60% by the addition of a 10-fold excess amount of
unlabeled PtdIns-4,5-P2 and to 50% by
PtdIns-3,4,5-P3, but not PtdIns or PtdIns-4-P, in a
dose-dependent manner (Fig. 4B), showing that
the ALT III PH domain has strong binding affinity for
PtdIns-4,5-P2 and PtdIns-3,4,5-P3. We also
examined the binding of the ALT III PH domain to inositol phosphates.
Interestingly, the ALT III PH domain did not bind to
Ins-1,4,5-P3 or Ins-1,3,4,5-P4 at all (Fig. 4,
C and D).

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Fig. 4.
Binding of the ALT III PH domain to inositol
phospholipids and inositol phosphates. A, binding of
PtdIns-4,5-P2 to the ALT III PH domain and the mutant R36G. Recombinant GST fusion
proteins were expressed in E. coli. Eluted proteins were
mixed with various amounts of
[3H]PtdIns-4,5-P2, and then
glutathione-Sepharose beads were added to the reaction mixture. After
centrifugation, radioactivities bound to beads were measured.
B, binding specificity of the ALT III PH domain to inositol
phospholipids. Binding assays of the ALT III PH domain were performed
with 100 pmol of [3H]PtdIns-4,5-P2 in the
presence of various amounts of competitive inhibitors. The
concentrations of competitive unlabeled lipids are indicated as the
relative molar ratio of [3H]PtdIns-4,5-P2.
C and D, binding of the ALT III PH domain to
Ins-1,4,5-P3 and Ins-1,3,4,5-P4. Recombinant
GST fusion proteins containing the PLC 1 PH domain or ALT III PH
domain were mixed and incubated on ice in the presence of various
amounts of [3H]Ins-1,4,5-P3 and
[3H]Ins-1,3,4,5-P4. Ins-1,4,5-P3
and Ins-1,3,4,5-P4 binding were determined by the method of
polyethylene glycol precipitation (33). Data represent duplicate
determinations in one of two experiments; error bars give
the range of duplicates.
|
|
Inhibitory Effect of ALT III on PLC Activity in Vitro--
To
investigate the importance of the PH domain for the inhibitory effect
of ALT III on PLC activity in vitro, the activities of
recombinant PLC
4 protein generated by the Sf9/baculovirus system were measured in the presence of recombinant ALT III protein generated by the Sf9/baculovirus system, and the PH domain, X-Y domain, and R36G mutant proteins generated by the bacteria system. As
in the in vivo experiments (Fig. 3C), the
activity of recombinant PLC
4 protein was inhibited to 70% by the
addition of equal amounts of ALT III and PH domain protein and to 40 and 55% by 2-fold excess amounts of ALT III and PH domain proteins,
respectively (Fig. 5A). The
activity of PLC
4 was suppressed by a 2-fold excess amount of X-Y
domain protein. The mutant R36G, of which the potency for PtdIns-4,5-P2 binding is markedly reduced, did not inhibit
the activity at all, indicating that PtdIns-4,5-P2 binding
to PH domain is essential for the inhibitory effect of ALT III. Next,
we investigated the effect of ALT III protein on the other PLC
activities, such as PLC
1, -
1, and -
1 purified from bovine
brain. As shown in Fig. 5B, ALT III inhibited the activity
of PLC
1 most effectively. The activity of PLC
1 was partially
suppressed, and PLC
1 was not affected at all. It is difficult to
compare the sensitivity of PLC
1 and that of PLC
4, because the
sources of the proteins are different, i.e. PLC
1 is
purified from bovine brain whereas PLC
4 is a recombinant protein.
However, at least these data show that ALT III is more effective
against PLC
-type isozymes than PLC
1 and -
1 in
vitro.

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|
Fig. 5.
Inhibitory effect of ALT III in
vitro. A, effect of various constructs of
ALT III on the activity of PLC 4. PLC assays were performed as
described under "Experimental Procedures" in the presence of
various amounts of ALT III generated by the Sf9/baculovirus
system and GST fusion proteins containing the ALT III PH domain, ALT
III X-Y domain (nucleotides 202-1844), and R36G mutant generated by
bacteria system. The amounts of proteins are indicated as the molar
ratio against PLC 4 protein. B, effect of ALT III on the
activity of PLC 1, - 1, and - 1 in vitro. The activity
of purified PLC 1, - 1, and - 1 was assayed in the presence of
various amounts of recombinant ALT III protein in vitro. The
amounts of ALT III protein are indicated as the molar ratio against
each PLC isozymes. Data represent duplicate determinations in one of
two experiments; error bars give the range of
duplicates.
|
|
The Suppression of Ins-1,4,5-P3 Production by ALT III
in Vivo--
To determine which PLC isozymes were inhibited by ALT III
in vivo, we next investigated whether the overexpression of
ALT III in NIH3T3 cells affects the Ins-1,4,5-P3 production
generated by the stimulation of bradykinin, PDGF, or the overexpression of PLC
1. As shown in Fig. 6,
A and B, in contrast to the results of the
present in vitro experiments, the PLC activities stimulated by bradykinin and PDGF were inhibited by ALT III, although it was
demonstrated that bradykinin and PDGF activate mainly PLC
1 and
PLC
1, respectively. Moreover, the increase in the production of
Ins-1,4,5-P3 by the overexpression of PLC
1 in NIH3T3
cells reverted to the control level by the co-expression with ALT III (Fig. 6C), indicating that the activity of PLC
1 is
inhibited by ALT III in vitro and in vivo.

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|
Fig. 6.
ALT III effectively inhibited the activity of
PLC 1 and suppressed the activity of
PLC and - in
vivo. A-C, effects of ALT III on the
activities of PLC , - , and - 1 in vivo. ALT III was
overexpressed using a retrovirus expression system in NIH3T3 cells, and
then the cells were serum-starved in starvation medium for 24 h,
stimulated by bradykinin (1 µM) (A) or PDGF
(50 ng/ml) (B) for the indicated times, and harvested. The
Ins-1,4,5-P3 level was measured with an
Ins-1,4,5-P3 assay kit. C, PLC 1 was
co-expressed with ALT III. Data represent duplicate determinations in
one of two experiments; error bars give the range of
duplicates.
|
|
 |
DISCUSSION |
We demonstrated that a novel alternatively spliced variant of
PLC
4 acts as a negative regulator of PLC isozymes. Although some of
the genes that encode PLC isozymes are also known to produce splice
variants, such as norpA (32) and plc21 (33), both
of which encode phospholipase C in Drosophila,
PLC
1 (34) and -
4 (35), all of their
splicing differences occur outside of the catalytic X and Y domains.
Therefore, ALT III is the first example of a splice variant in which
the difference occurs inside the catalytic domain. Although the
expression level of ALT III was quite lower than that of PLC
4 in
most of the tissues and cells examined, it was significantly higher in
N1E-115 cells, a rat neuroblastoma cell line (Fig. 2). Remarkably, p19
cells, a rat embryonal carcinoma cell line, normally expressed ALT III
at a low level, but the expression was higher compared with that of PLC
4 when p19 cells were differentiated to neural cells by retinoic acid (Fig. 2C). These results suggest that ALT III is
expressed and functions specifically in some neural cells.
Since ALT III lacks the C-terminal of the X domain and linker region,
we first studied whether this molecule had PLC activity; we found that
it does not (Fig. 3A). We next examined the biological functions of this molecule, since it is presumed that ALT III inhibits
PLC isozymes by competition for PtdIns-4,5-P2. As shown in
this report, ALT III inhibited the activity of PLC
4 and -
1 in vitro and in vivo and PLC
and -
in
vivo (Figs. 3, 5, and 6). Unexpectedly, the overexpression of ALT
III did not result in the inhibition of endogenous PLC activity in
unstimulated control cells (Fig. 3, A and C).
Since the PLC activity in the control cells was not so high without
agonist stimulation, it may be not sensitive to ALT III. Besides, the
ALT III PH domain alone inhibited the PLC activity, whereas the PH
domain deletion mutant did not (Figs. 3C and 5A).
These results indicate that ALT III is a negative regulator of PLC
-type isozymes and that the PH domain is necessary and sufficient
for the inhibitory effect. Furthermore, since the mutant R36G, the
binding potency to PtdIns-4,5-P2 of which is very low, did
not inhibit the activity of PLC
4 (Fig. 5A),
PtdIns-4,5-P2 binding to the ALT III PH domain, resulting
in the competition for PtdIns-4,5-P2 with the other PLCs,
is essential for the inhibition by ALT III.
Different PH domains have different binding specificities for various
inositol phospholipids and are divided into four groups based on their
selectivities (36, 37). The first group is the PH domains that bind to
PtdIns-3,4,5-P3 with high affinity rather than to
PtdIns-4,5-P2, such as the PH domains of Bruton's tyrosine
kinase-1, general receptor for phosphoinositides-1, N-terminal T
lymphoma invasion and metastasis protein-1, and son of sevenless-1. The
second group is the PH domains that bind to PtdIns-4,5-P2 with high affinity and to PtdIns-3,4,5-P3 with relatively
weak or the same affinity, such as those of PLC
1,
-adrenergic
receptor kinase, spectrin, and oxysterol-binding protein-1. The third
group of PH domains bind specifically to PtdIns-3,4-P2,
including that of Akt/protein kinase B. The fourth group of PH domains
bind with low affinity and no selectivity to inositol phospholipids. In regard to the PLC PH domain, PLC
1 belongs to the second group with
high affinity for PtdIns-4,5-P2 (11, 38-40), whereas the PLC
1 PH domain would be in the first group with high affinity for
PtdIns-3,4,5-P3 (14, 15). Since the PLC
1 PH domain is known to bind lipids nonspecifically (39), it might belong to the
fourth group.
Our present results showed that the ALT III PH domain can specifically
bind to PtdIns-4,5-P2 and PtdIns-3,4,5-P3 but
not PtdIns or PtdIns-4-P (Fig. 4B), suggesting that the ALT
III PH domain belongs to the second group, to which the PLC
1 PH
domain belongs. Interestingly, despite the sequence similarity of ALT
III PH domain with the PLC
1 PH domain which binds to
PtdIns-4,5-P2 and Ins-1,4,5-P3 with high
affinity, the ALT III PH domain could not bind to
Ins-1,4,5-P3 or Ins-1,3,4,5-P4 (Fig. 4,
C and D). One possible reason is that the binding
of the ALT III PH domain to PtdIns-4,5-P2 and
PtdIns-3,4,5-P3 is required for additional contact with a
hydrophobic region such as the acyl chain of lipids. This explanation
is not inconsistent with our finding that the R36G mutant still bound
to PtdIns-4,5-P2 with low affinity. In fact, some of the
proteins, such as Bruton's tyrosine kinase, bind to inositol
phospholipid with higher affinity than to inositol phosphate, and it is
considered that the natural ligand of such protein is the lipid rather
than the inositol phosphate (37).
Our results showed that ALT III inhibited the activity of PLC
-types
the most efficiently and were less effective against PLC
1 and -
1
(Fig. 5B) in vitro. The different effects on PLC isozymes imply that the inhibition is not caused by the simple competition for PtdIns-4,5-P2 as a substrate with the PLC.
It is known that the activity of PLC
1 is activated by the binding of
PtdIns-4,5-P2 to its PH domain (10), but PLC
1 and -
1
are not known to be activated by PtdIns-4,5-P2 (39, 41).
Therefore, PLC
1 needs more PtdIns-4,5-P2 than the other
isozymes for binding to the PH domain and for a substrate, whereas
PLC
1 and -
1 need PtdIns-4,5-P2 only as a substrate.
The specificities of the inhibitory effect on PLC
1 are reflected by
the specificities of the binding affinity of its PH domain for
PtdIns-4,5-P2. Therefore, the competitive binding of ALT
III to PtdIns-4,5-P2 may cause the inhibition of PLC
1
activation which is induced through the binding of
PtdIns-4,5-P2 to its PH domain, i.e. the feature
of PLC
1 that is activated through the binding of
PtdIns-4,5-P2 to its PH domain may cause the sensitivity to
ALT III. In contrast, the low sensitivity of PLC
1 and -
1 to ALT
III are reflected by their activation mechanism which is independent of
the additional binding of PtdIns-4,5-P2 and may be caused
by no specificity of their PH domain binding to
PtdIns-4,5-P2. Alternatively, PLC
1 and -
1 may
hydrolyze PtdIns-4,5-P2 bound to ALT III, whereas PLC
1
and PLC
4 may not do so in vitro.
In contrast to the results of the present in vitro
experiments, the overexpression of ALT III suppressed the activity of
PLC
and -
in vivo (Fig. 6, A and
B), and the increase in the intracellular calcium
concentration stimulated by bradykinin was also suppressed (data not
shown). Unfortunately, since the level of the transient calcium
increase stimulated by PDGF was not enough high to analyze the
inhibitory effect of ALT III, we do not know whether ALT III could
suppress it or not. However, the Ins-1,4,5-P3 production stimulated by PDGF was suppressed more efficiently than was that stimulated by bradykinin (Fig. 6, A and B),
indicating that ALT III is more effective against PLC
than PLC
in vivo. Since the activities of PLC
1 and -
1 were not
suppressed by ALT III in vitro, we do not think that ALT III
inhibits their activity directly. One possible explanation for the
suppression in vivo is that PLC
and -
are suppressed
by ALT III indirectly, i.e. competition for
PtdIns-4,5-P2 with the other membrane targeting molecules that affect the PLC
and -
activity causes the suppression of them
by ALT III in vivo. However, in the case of PLC
, there
still remains a possibility that competitive binding of ALT III to
PtdIns-3,4,5-P3 causes the direct inhibition of PLC
activation which is induced through binding of
PtdIns-3,4,5-P3 to its PH domain. Thus, the suppressions of
PLC
and -
by ALT III in vivo are meaningful events in
the physiological condition and may be suppressed not directly but
indirectly, and specific targets for the direct inhibition by ALT III
are thought to be PLC
-type isozymes.
 |
ACKNOWLEDGEMENTS |
We thank Dr. T. Kitamura for providing the
retrovirus vector; Dr. F. Shibasaki for pcDNA3 vector; Dr. T. Michikawa for NG108-15 and p19 cells; Maiko Fukuoka for important
contributions to isolating mRNA and proteins from rat tissues;
Kyoko Takahashi for purifying PLC
1, -
1, and -
1.
 |
FOOTNOTES |
*
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: Dept. of
Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5510; Fax: 81-3-5449-5417; E-mail: takenawa{at}ims.u-tokyo.ac.jp.
The abbreviations used are:
PLC, phospholipase
C; PtdIns, phosphatidylinositol; Ins, inositol; PH, pleckstrin
homology; PDGF, platelet-derived growth factor; RT-PCR, reverse
transcription-polymerase chain reaction; GST, glutathione
S-transferase; FBS, fetal bovine serum; DMEM, Dulbecco's
modified Eagle's medium.
2
K. Fukami, K. Takenaka, A. Ito, K. Nagano, and
T. Takenawa, submitted for publication.
 |
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