 |
INTRODUCTION |
Phospholipase C
isoforms (
1,
2,
3 and
4) at the
plasma membrane are regulated by G protein-coupled seven-transmembrane receptors which activate heterotrimeric G

protein complexes upon ligand stimulation (1-3). The hydrolysis of phosphatidylinositol 4,5-bisphosphate
(PIP2)1 by PLC
s generates two well recognized second messengers, inositol 1,4,5-trisphosphate and DG. Inositol 1,4,5-trisphosphate evokes release of Ca2+ from intracellular stores, while DG alone,
or in concert with Ca2+, activates some isoforms of PKC
(4). Mounting evidence suggests that an analogous phosphoinositide
(PI) signaling, which is distinct from classic PI signaling at plasma
membrane, exists in the nucleus (5-7). The presence of such a nuclear
PI cycle has been demonstrated in several different cell lines (8-11,
12), and has been shown to be important for both cell proliferation
(13) and differentiation (14).
The key enzyme for the initiation of nuclear PI signaling is
phospholipase C (PLC)
1, which is the major isoform present in the
nucleus of 3T3 cells (15) as well as other cell lines (16-19). It
exists as two alternatively spliced subtypes, PLC
1a (150 kDa) and
PLC
1b (140 kDa) which differ only in a short region of their
carboxyl termini (20). Recent studies suggest that the
1b form
predominantly localizes in the nucleus while the
1a form distributes
equally between the nucleus and plasma membrane (21). A nuclear
localization motif has been mapped to a cluster of lysine residues
(between 1055 and 1072) which is common to both subtypes (22).
It has recently been demonstrated that ablation of PLC
1 with
antisense abolished the mitogenic response of Swiss 3T3 cells to IGF-I,
indicating the pivotal role of this enzyme in cell proliferation (13).
Overexpression of both subtypes of PLC
1 alone, even in the absence
of growth factors, is sufficient to elevate expression of cyclin D3 and
cdk4 (23). This in turn leads to hyperphosphorylation of retinoblastoma
protein (pRb) and activation of E2F-1 transcription factor, thus
enhancing cell cycle G1/S progression. The importance of
this enzyme in the cell cycle is further strengthened by a recent
finding that in Saccharomyces cerevisivae nuclear PLC1 (homologous in function to the mammalian PLC
1), and two inositol polyphosphate kinases constitute a nuclear signaling cascade that is
directly involved in RNA transport and transcriptional regulation (24,
25).
One of the critical downstream targets for nuclear PLC
1 is protein
kinase C, which is involved in progression through the G1/S
and G2/S checkpoints of the cell cycle (26, 27). Nuclear DG
has been shown to be critical for the activation of nuclear PKC
II
during G2/M phase transition (28). It also acts as a chemoattractant to induce nuclear translocation of PKC
by a mechanism which is not clearly understood (9, 29). In Swiss 3T3 cells,
the IGF-I evoked production of nuclear DG, nuclear translocation of PKC
and DNA synthesis were completely blocked by the selective
phospholipase C inhibitor Et-18-0-CH3, but not by a PLD
inhibitor (30).
Nuclear PLC
1 is under separate control from that of PLC
s at
plasma membrane. Bombesin, a G-protein coupled-receptor agonist, only
activates PLC
isoforms at the plasma membrane and has no effect on
nuclear PLC activity (9). Conversely, stimulation of cells with IGF-I,
which acts through a tyrosine kinase receptor, caused a rapid,
2~3-fold increase in enzyme activity of nuclear PLC
1, while the
PLC activity at plasma membrane was not affected (15). The
IGF-I-stimulated activation of nuclear PLC
1 is transient and the
enzyme activity returns to basal level within 30 min (15, 31). However,
the molecular mechanisms for the termination of this activation signal
are currently unclear. Here, we demonstrated that a negative feedback
regulation of nuclear PLC
1 by PKC
accounts for the termination
of IGF-I induced activation of this enzyme. PKC
was shown to
interact with PLC
1 in the nucleus, and evoke phosphorylation at
serine 887, a putative PKC phosphorylation site which is located within
the long, characteristic carboxyl tail of PLC
1. Overexpression of
either a PLC
1 variant (S887A) or dominant-negative PKC
caused a sustained elevation of nuclear PLC activity following IGF-I stimulation.
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EXPERIMENTAL PROCEDURES |
Materials--
Polyclonal antibodies against PKC
or
, a monoclonal antibody which recognizes PKC
, PKC
1, and PKC
2, PKC inhibitor calphostin C, Cy3-conjugated goat anti-rabbit IgG,
aprotinin, and leupeptin were obtained from Sigma. Isotopes
([
-32P]ATP, [32P]orthophosphate, and
[3H]PIP2) were purchased from ICN. Protein
kinase C substrate peptide QKRPSQRSKYL was obtained from Upstate
Biotechnology Inc. LipofectAMINE Plus, G418, Dulbecco's modified
Eagle's medium and phosphate-free Dulbecco's modified Eagle's medium
were the product of Life Technologies Inc. MEK inhibitor PD98059 was
purchased from New England Biolabs, Inc. Protein kinase C
(human,
recombinant, Spodoptera frugiperda), PKC
inhibitor
Go6976, and PKC
inhibitor rottlerin were from Calbiochem. The
parental mammalian expression vector pCIN4 which encodes a neomycin
resistance gene, and pCIN4 DN PKC
, which expresses
dominant-negative PKC
(DN PKC
), are the generous gifts from Dr
Albert Descoteaux (Université du Québec, Canada) (32). pCIN
DN PKC
contains a dominant-negative version of the gene in which
the conservative lysine residue (Lys338) in the ATP-binding
domain was replaced by aspartic acid.
Site-directed Mutagenesis and Construction of Expression
Vector--
The cDNA corresponding to wild-type PLC
1 (33) was
amplified by PCR with forward and reverse primers containing
BamHI and EcoRI restriction sites, respectively.
Following digestion with the restriction enzymes, the DNA fragment was
ligated into the pcDNA3.1 eukaryotic expression vector, which
contains a cytomegalovirus promoter. Construction of the vector
expressing histidine-tagged PLC
1 (referred to as PLC
1
(His)6) is similar to the above except that the upstream
primer contains a DNA fragment encoding six histidine residues.
Mutation of the putative PKC phosphorylation site S887A was performed
by sequential PCR mutagenesis as follows. A PCR reaction was performed
using primer I (5'-CAGCATATGAGGAAGGAGGCAAATTTATTG-3') which
spans a unique NdeI site at nucleotides 2236-2252) and
primer II (5'-TGCCTTCACAGCCCCTGGAGCAGG-3') which includes the S887A
mutation in non-coding strand) as a forward and a reverse primer,
respectively. Another PCR reaction utilized primer III
(5'-CAGGGGCTGTGAAGGCACCCGCCA-3'), which partially overlaps with primer
II and also contains the Ser887 (TCT) to alanine (GCT)
mutation in coding strand and primer IV (5'-CATCTGCAGCTTGGGCTTCTCATCCAGGAT-3'), which spans a unique PstI (in bold) site at nucleotides 3424-3430) as a forward
and reverse primer. The resultant product of these two PCR reaction was
purified, annealed, and used as template for a second round of PCR with
primer I and primer IV as a forward primer and reverse primer. The
1191-base pair PCR product was then digested with NdeI and
PstI and inserted into the corresponding sites in the wild-type PLC
1 expression vector. The resultant clone was confirmed by DNA sequencing and is referred as pcDNA.PLC
1 S887A.
Cell Culture, Transfection, and in Vivo 32P
Labeling--
Swiss 3T3 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. Cells were
transfected with the above expression vectors using LipofectAMINE
according to the vendor's instruction. Stable transfectants were
generated by selection with 0.6 mg/ml G418. For in vivo
32P labeling, the cells were starved in phosphate-free
Dulbecco's modified Eagle's medium for 1 h to deplete ATP
metabolic pool, and subsequently incubated with 0.2 mCi/ml
[32P]orthophosphate for 4 h. Cells were then
subjected to different treatments as indicated.
Isolation of Nuclei and Analysis of PLC Activity--
Nuclei
were purified as previously described (15). Briefly, 5 × 106 cells were lysed in 400 µl of nuclear isolation
buffer (10 mM Tris-HCl, pH 7.8, 1% Nonidet P-40, 10 mM mercaptoethanol, 0.5 mM phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin and leupeptin, 10 µg/ml soybean trypsin
inhibitor, 15 µg/ml calpain inhibitor-1 and 2 (Roche Molecular
Biochemicals), 2.0 mM Na3VO4, and 5 mM NaF) for 3 min on ice. 400 µl of MilliQ water were
then added to swell cells for 3 min. The cells were sheared by 8 passages through a 23-gauge hypodermic needle. Nuclei were recovered by centrifugation at 400 × g and 4 °C for 6 min, and
washed once in 400 µl of washing buffer (10 mM Tris-HCl,
pH 7.4, 2 mM MgCl2, plus protease and
phosphatase inhibitors as above). This method has been shown to yield
nuclei which lack both inner and outer nuclear membranes and are
essentially free of cytoplasmic contamination (13, 15).
The activity of nuclear PLC was analyzed as outlined previously (15).
10 µg of nuclear proteins were incubated with 100 mM MES
buffer, pH 6.7, plus 150 mM NaCl, 0.06% sodium
deoxycholate, 3 nmol of [3H]PIP2 (specific
activity 30,000 dpm/nmol) for 30 min at 37 °C. Hydrolysis was
stopped by adding chloroform/methanol/HCl, and the amount of inositol
1,4,5-trisphosphate in the upper phase was quantified by liquid
scintillation counting.
In Vitro Nuclear PKC Activity Assay--
The PKC activity in
isolated nuclei was measured as previously outlined by Neri et
al. (30). Briefly, 10 µg of nuclear proteins were incubated at
30 °C for 10 min with 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 µM ATP, 5 µCi of
[32P]ATP, 1.2 mM CaCl2, 40 µg/ml phosphatidylserine, 3.3 mM dioleoylglycerol, plus
10 µg of PKC substrate peptide. The reaction was stopped by adding 15 µl of acetic acid and spotted onto Whatman p81 paper. The filter
paper was then washed with 0.75 mM
H3PO4 and counted for radioactivity assay.
Immunoprecipitation and Purification of PLC
1
(His)6 from Nuclei--
Purified nuclei were
solubilized in lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA and EGTA, 50 mM NaCl, 50 mM
NaF, 30 mM sodium pyrophosphate, 10% glycerol, 1% Triton
X-100, plus protease inhibitor mixture as above) for 20 min at 4 °C
with shaking. Cell debris was removed by centrifugation at 12,000 × g and 4 °C for 5 min. The supernatants were incubated
with 50 µl of a 50% slurry of protein A/G-agarose beads for 1 h. The cleared lysates were then incubated with 5 µg of mouse
anti-PLC
1 antibody (34) for 16 h. The immunocomplexes were
recovered by adding 50 µl of protein A/G-agarose beads for another
hour, and released by boiling in 50 µl of 1 × SDS buffer for 5 min. Samples were then separated by 8% SDS-PAGE, and the protein
phosphorylation was analyzed by autoradiography and quantified by
ImageTM software (Amersham Pharmacia Biotech).
To purify nuclear PLC
1 (His)6 or PLC
1
(His)6 mutant S887A, nuclei were purified from cells
transfected with the corresponding expression vectors constructed as
above, and solubilized in lysis buffer (50 mM Tris-HCl, pH
8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1%
Triton X-100, plus protease and phosphatase inhibitor mixture as
above). The lysates were centrifuged at 10,000 × g and
4 °C for 10 min. The supernatant was applied to a pre-equilibrated column packed with Ni-NTA resin. After washing the column, PLC
1
(His)6 was eluted using lysis buffer plus 100 mM imidazole and 10% glycerol. The elutes were pooled,
concentrated, and analyzed by SDS-PAGE and autoradiography.
Phosphorylation of Nuclear PLC
1 by Recombinant PKC
--
Equal amounts of PLC
1 (His)6 purified as
above were incubated with 50 ng of recombinant PKC
or PKC
in
the presence of 2.5 µCi of [
-32P]ATP in a total
volume of 30 µl of reaction mixture (20 mM HEPES, pH 7.4, 100 mM CaCl2, 10 mM
MgCl2, 50 µg/ml phosphatidylserine, 40 µg/ml
diacylglycerol, 0.03% Triton X-100) at 30 °C for the times
specified. The reactions were terminated by adding 6 × Laemmli sample buffer and boiling for 5 min. Following separation by 10% SDS-PAGE, the phosphoprotein was visualized and analyzed by phosphorimaging.
In-gel Trypsin Digestion and Two-dimensional Phosphopeptide
Mapping--
In vivo 32P-labeled PLC
1
(His)6 was purified from nuclei as above, separated by
SDS-PAGE. The bands corresponding to this protein were excised from the
gels, minced, and in-gel digested as previously described (35). The
tryptic mixtures were lyophilized and solubilized in 10 µl of
electrophoresis buffer (1% pyridine, 10% acetic acid, pH 3.5) and
applied to the middle of a thin layer chromatography (TLC) plate. The
samples were then subjected to first dimensional electrophoresis,
followed by second dimensional chromatography as previously detailed
(36). The tryptic phosphopeptides were visualized by autoradiography.
Immunoblotting, Immunostaining, and Confocal Imaging
Microscopy--
Nuclear proteins were separated by SDS-PAGE,
transferred to nitrocellulose membranes, blocked with 10% fat-free
milk, and incubated with the various primary and secondary antibodies
as described in the text. The immunoreactive proteins were detected using ECL reagents according to the manufacturer's instructions.
For immunostaining, The cells grown on coverslips were starved for
24 h in serum-free medium, stimulated without or with 40 ng/ml
IGF-I, or IGF-I plus 50 µM calphostin C as above. Cells were then stained for PKC
using rabbit anti-PKC
(1:1000)
antibody, followed by goat anti-rabbit antibody conjugated with Cy3
(1:250). The specimens were then examined using a Leica TCS 4D confocal laser scanning microscopy (Lasertechnik, Heidelberg, Germany) fitted
with a mercury vapor lamp and a mixed gas krypton-argon laser.
 |
RESULTS |
The Effect of Protein Kinase C Inhibitors on
IGF-I-dependent Activation of Nuclear PLC--
Several
recent studies have found that PKC can regulate PLC activity in
vitro or at the plasma membrane (37-40). Thus, we investigated the effect of PKC on the activity of nuclear PLC using several selective PKC inhibitors. As shown in Fig.
1, the nuclear PLC activity in Swiss 3T3
cells was significantly increased following IGF-I treatment, in
agreement with a previous report (31). After 5 min of stimulation, the
enzyme activity increased 3.1-fold over the basal level. This level of
activity persisted for up to 15 min, and then began to decrease,
reaching basal level after 30 min. By contrast, treatment of cells with
Go6976, a specific inhibitor for
isoform of PKC (41), caused a
sustained activation of nuclear PLC activity. Under this treatment the
enzyme still retained maximal activity at 30 min after IGF-I
stimulation. It remained 1.9-fold higher after 60 min and only returned
to basal level after 2 h (data not shown). The IGF-I
dependent activation of nuclear PLC was also sustained by treatment of
the cells with 40 µM calphostin C (a specific inhibitor
of PKC (42, 43)), but not by 15 µM rottlerin (a specific
inhibitor for
isoform of PKC (44)) (data not shown). This result
indicates that PKC
is probably involved in the attenuation of the
IGF-I-evoked activation of nuclear PLC.

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Fig. 1.
Treatment of cells with Go6976 causes
sustained elevation of nuclear PLC following IGF-I stimulation.
Quiescent Swiss 3T3 cells were pretreated without or with 0.5 µM Go6976 for 30 min, and then incubated with 40 ng/ml
IGF-I for the indicated time periods. The purified nuclei were assayed
for PLC activity as described under "Experimental Procedures." The
results are from three independent experiments and expressed as
mean ± S.D.
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A previous study showed that among the several PKC isoforms detected in
Swiss 3T3 cells only PKC
is present in the nucleus (29). In
response to IGF-I stimulation, PKC
was selectively translocated
into the nucleus from the cytosol (30). This nuclear translocation
process occurs within the same time frame for the activation of nuclear
PI cycle by IGF-I. Western blot analysis of the nuclear proteins from
Swiss 3T3 cells revealed that the concentration of PKC
in the
nucleus starts to increase after 15 min of IGF-I stimulation, and
reaches a maximal level after 30 min (Fig.
2A). In agreement with this
result, stimulation of cells with IGF-I also caused a progressive
increase in nuclear PKC activity which reached a maximum at 30 min
(Fig. 2B). Significantly, the activity of nuclear PKC
inversely correlates with the activity of nuclear PLC, further
suggesting the involvement of PKC
in the termination of nuclear PI
cycle signaling.

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Fig. 2.
Time course analysis for nuclear
translocation of PKC and nuclear PKC activity
in IGF-I-treated cells. A, cells were treated without
or with 40 ng/ml IGF-I. Nuclear proteins were separated by 8% SDS-PAGE
and probed with an anti-PKC polyclonal antibody. B,
aliquots of the same nuclear samples were analyzed for PKC
activity as described under "Experimental Procedures." The results
are from three independent experiments and expressed as mean ± S.D.
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We next investigated the effect of Go6976 on nuclear translocation of
PKC
and nuclear PKC activity in cells treated with IGF-I.
Immunofluorescent analysis revealed that pretreatment of cells with
Go6967 does not affect the IGF-I induced nuclear translocation of PKC
(Fig. 3). However, the IGF-I-induced
increase in nuclear PKC activity was completely inhibited under these
conditions (data not shown).

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Fig. 3.
The effect of Go6976 on IGF-I-induced nuclear
translocation of PKC . Quiescent cells
grown on coverslips were pretreated without or with 0.5 µM Go6976 for 30 min, and then incubated with 40 ng/ml
IGF-I for another 30 min. Cells were then fixed, stained for PKC ,
and analyzed using confocal laser scanning microscopy.
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Time-dependent Association of PLC
1 and PKC
within the Nucleus following IGF-I Stimulation--
It has previously
been shown that PLC
1, a major isoform of PLC family residing in the
nucleus, was responsible for the initiation of nuclear PI cycle (13).
To investigate the direct involvement of PKC
in the regulation of
nuclear PLC
1 activity, nuclear proteins from cells overexpressing
PLC
1 were subjected to immunoprecipitation using an anti-PLC
1
antibody. The precipitated immunocomplex was separated by SDS-PAGE and
probed with either anti-PLC
1 or anti-PKC
. A direct interaction
between PLC
1 and PKC
within the nucleus was detected at 15 min
after stimulation with IGF-I (Fig. 4).
This association reached a maximum after 30 min, and persisted for up
to 1 h. Again, the time course of interaction between these two
enzymes strongly correlates with the decrease in nuclear PLC activity
(Fig. 1). A similar analysis failed to detect association between PLC
1 and several other isoforms of PKC (
1,
2,
1, and
2) (data not
shown), suggesting that the interaction between PLC
1 and PKC is
specific for the
isoform.

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Fig. 4.
Time course of interaction between PKC
and PLC 1 in the nucleus
following IGF-I stimulation. Nuclear proteins from cells
overexpressing PLC 1 were immunoprecipitated with an anti-PLC 1
monoclonal antibody. The immunocomplexes were separated by 8% SDS-PAGE
and then probed with either anti-PLC 1 or anti-PKC .
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PKC
Evokes Phosphorylation of PLC
1 at Serine 881 within the
Nucleus--
Previous in vitro studies have shown that PKC
can phosphorylate PLC
1 at serine 887 (45). However, the
physiological relevance of this phosphorylation is uncertain. Here, we
tested whether or not the PKC-mediated PLC
1 phosphorylation on
serine 887 is the direct consequence of IGF-I stimulation. To this end,
Swiss 3T3 cells were transfected with a plamid which express either PLC
1 (His)6, or PLC
1 (His)6 mutant S887A.
These proteins were then radiolabeled with 32P in
vivo and purified from nuclei as described under "Experimental Procedures." Two-dimensional phosphopeptide mapping analysis of 32P-labeled nuclear PLC
1 (His)6 revealed a
single, prominent tryptic phosphopeptide for PLC
1 overexpressed in
quiescent 3T3 cells, indicating that a constitutive phosphorylation of
PLC
1 occurs within this peptide (Fig.
5A). Stimulation of cells with
IGF-I for 30 min caused the production of two extra tryptic
phosphopeptides, referred to as phosphopeptides 2 and 3, respectively
(Fig. 5B). The IGF-I-dependent production of
phosphopeptide 2 was inhibited by MEK inhibitor PD98059 (Fig.
4D), indicating that MEK/MAP kinase is involved in
phosphorylation of this peptide. The significance of this observation
is currently under separate investigation.

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Fig. 5.
PKC-mediated phosphorylation of PLC
1 at serine 887 occurs in the nucleus following
IGF-I stimulation. Cells were transiently transfected with a
plasmid expressing His6-tagged PLC 1 (A-D) or
PLC 1 mutant S887A (E and F) and rendered
quiescent by starvation in serum-free medium after 24 h of
transfection. Cells were then radiolabeled with 32P for
4 h, and then treated without (A and E), or
with 40 ng/ml IGF-I (B-D and F) for another 30 min. In C and D, cells were pretreated with 0.5 µM Go6976 or 50 µM PD98059 for 30 min,
respectively, before adding IGF-I. His6-tagged PLC 1 or
PLC 1 mutant S887A protein were then purified from nuclei as
described under "Experimental Procedures." Proteins were separated
by 8% SDS-PAGE and visualized by autoradiography. The bands
corresponding to 32P-PLC 1 (His)6 or
32P-PLC 1 (His)6 mutant S887A were excised
from the gel, in-gel digested with trypsin, and analyzed by
two-dimensional phosphopeptide mapping as described under
"Experimental Procedures." Note that IGF-I-dependent
phosphopeptide 3 was absent in samples treated with Go6976 or
overexpressing PLC 1 (His)6 mutant S887A.
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Phosphopeptide 3 could not be detected in cells treated with PKC
inhibitor Go6976 (Fig. 5C), or cells overexpressing PLC
1 (His)6 mutant S887A in which the phosphorylation site
serine 887 was replaced by alanine (Fig. 5, E and
F). The production of phosphopeptide 3 was also inhibited by
calphostin C, but not rottlerin (data not shown). This result
demonstrates that PKC-mediated phosphorylation at serine 887 occurs
in vivo within the nucleus in response to IGF-I.
To further establish that nuclear PLC
1 is the direct target of PKC
, nuclear PLC
1 or PLC
1 mutant S887A purified as above was
subjected to in vitro phosphorylation by recombinant PKC
. In the presence of PKC
, phosphorylation of nuclear PLC
1
was observed and increased with time of incubation (Fig.
6). Maximal phosphorylation was achieved
within 30 min. Phosphorylation was not detected in the absence of
recombinant PKC
, demonstrating that PLC
1 purified from nuclei
was free of contamination of other potential kinases. Recombinant PKC
did not cause phosphorylation of the mutated PLC
1 S887A within
the time frame observed, further confirming S887 to be the
phosphorylation site of PKC
.

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Fig. 6.
Recombinant PKC phosphorylate nuclear PLC 1 at serine
887 in vitro. PLC 1 (His)6 or PLC
1 (His)6 mutant S887A was purified from nuclei of
transiently transfected cells as described in the legend to Fig. 5. An
equal amount of these proteins was incubated without or with 50 ng of
recombinant PKC and 2.5 µCi of [ -32P]ATP for
different periods as described under "Experimental Procedures." The
reaction was terminated by adding 6 × Laemmli loading buffer.
Equal aliquots of samples were separated by 8% SDS-PAGE and analyzed
by phosphorimaging or probed with anti-PLC 1 antibody.
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The Sustained Responsiveness of PLC
1 Mutant S887A to IGF-I
Stimulation--
To evaluate the effect of PKC-mediated PLC
1
phosphorylation on enzyme activity, stable transfectants expressing
wild type PLC
1 or PLC
1 mutant S887A were generated as described
under "Experimental Procedures." The clones in which the level of
immunoreactive PLC
1 was significantly higher than that of
endogenous PLC
1 were selected by immunoblotting, and used for
further experiments. As shown in Fig.
7A, a similar level of
immunoreactive PLC
1 was detected in the nuclei of cells
overexpressing wild type PLC
1 and cells overexpressing PLC
1
mutant S887A, indicating that the nuclear localization of PLC
1
mutant S887A was not affected. Quantitative analysis showed that the
level of immunoreactive PLC
1 in both of these cell lines is about
3-fold above that of control cells.

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Fig. 7.
Mutation of S887A abolishes PKC-mediated
inhibition of nuclear PLC 1 in IGF-I-treated
cells. Stable transfectants which overexpress wild type PLC 1
or PLC 1 mutant S887A were selected as described in the text. 40 µg of nuclear proteins from control cells or cells overexpressing
wild-type PLC 1 or PLC 1 mutant S887A were separated by 8%
SDS-PAGE and probed with anti-PLC 1 polyclonal antibody.
B, quiescent cells overexpressing PLC 1 or PLC 1
mutant S887A were treated without or with 40 ng/ml IGF-I for the
indicated period. The nuclei from these cells were analyzed for PLC
activity (n = 4, expressed as mean ± S.D.). The
figure shows the result of a typical experiment, and similar results
were obtained from another two separate experiments using independent
stable transfectant which express wild type PLC 1 or PLC 1 mutant
S887A.
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Consistent with the above result, analysis of nuclear PLC activity
revealed a similar basal level of enzyme activity between cells
overexpressing wild type PLC
1 and cells overexpressing S887A, both
of which were about 3-fold higher than that of control cells. In both
of these cell lines the nuclear enzyme activity increased by about
2.7-fold after 5 min of IGF-I stimulation (Fig. 7B). In
cells overexpressing wild-type PLC
1, the level of enzyme activity
decreased by 37.4% after 20 min and returned to basal level after 30 min. In contrast, the elevated activity of nuclear PLC from cells
overexpressing S887A was sustained for a much longer period following
IGF-I stimulation. It was still maximal after 30 min (Fig.
7B), only decreasing about 43% after 60 min, and returning
to basal level by 150 min (data not shown). This result suggests that
PKC-mediated PLC
1 phosphorylation at serine 887 is critical for the
attenuation of nuclear PLC activity induced by IGF-I.
The Effect of DN PKC
on IGF-I-dependent Activation
of Nuclear PLC--
To confirm further that IGF-I-induced activation
of nuclear PLC
1 is terminated by PKC
, a kinase-deficient mutant
of this isoenzyme was introduced into Swiss 3T3 cells by transfecting with pCIN DN PKC
. Such a catalytically inactive mutant has been shown to act as a dominant-negative molecule by competing with the
corresponding endogenous isoenzyme (32, 46). Following selection with
500 ng/ml G418, Western blot analysis was performed for clones selected
from each independent population of stable transfectants to evaluate
the expression level of PKC
. Two clones (termed DN PKC
A and DN
PKC
B), in which the immunoreactive PKC
is significantly higher
than endogenous expression level, were chosen for further experiment
(Fig. 8A). The expression of DN PKC
in these two clones was also verified by taking advantage of
the fact that PKC degradation and down-regulation in response to
tumor-promoting phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) is dependent on
an active kinase (47). Following TPA treatment for 24 h, PKC
in control cells decreased to an undectable level. In contrast, the
level of PKC
in the two clones expressing DN PKC
was reduced
only slightly, consistent with the expression of the kinase-deficient
DN PKC
(Fig. 8A).

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Fig. 8.
Expression of DN PKC inhibits PKC-mediated nuclear PLC 1
phosphorylation at serine 887 and causes sustained activation of
nuclear PLC in IGF-I-treated cells. A, Swiss 3T3 cells
were transfected with pCIN DN PKC or empty vector pCIN4. Following
selection with 500 ng/ml G418, 80 µg of cell lysates from two clones
(DN PKC A and DN PKC B) expressing the dominant-negative PKC
mutant and two control clones (pCIN4 A and pCIN4 B) were analyzed
for the level of immunoreactive PKC before and after treatment with
TPA (400 nM, 24 h). TPA treatment causes degradation
of the endogenous wild-type PKC , but not the kinase-deficient
mutant (47). B, the clones selected in A were
transiently transfected with a plasmid expressing
(His)6-tagged PLC 1. Following in vivo
32P labeling and IGF-I treatment (40 ng/ml, 30 min),
32P-PLC 1 (His)6 was purified from nuclei
and the tryptic peptide mixtures was analyzed by two-dimensional
phosphopeptide mapping as described in the legend to Fig. 5. Note that
phosphopeptide 3 was not detected in two clones overexpressing DN PKC
. C, the clones selected above were treated with IGF-I
and the nuclei from there cells were subjected to analysis for PLC
activity as described in the legend to Fig. 1. The results are from
three independent experiments and expressed as mean ± S.D.
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In both clones expressing DN PKC
, the PKC-mediated phosphorylation
of PLC
1 at serine 887 was not detected in IGF-I-treated cells,
suggesting PKC
is the major isoform responsible for this phosphorylation (Fig. 8B). A time course analysis for
nuclear PLC activity revealed a sustained activation of nuclear PLC in both cell lines that express DN PKC
(Fig. 8C). At 30 min
after IGF-I stimulation, the activity of nuclear PLC is still
maintained at the maximally activated level. It decreased only by 43%
after 60 min and returned to basal level after 150 min (data not
shown). We thus conclude that
isoform of PKC is the major isoenzyme responsible for the termination of IGF-I dependent activation of
nuclear PLC.
 |
DISCUSSION |
Agonist-induced desensitization is an important regulatory process
in the phosphoinositide signaling pathway (48). Compelling evidence
suggests that PKC activation attenuates agonist-stimulated PLC
activity, thus providing a negative feedback regulatory mechanism to
control the magnitude and duration of the signal transmitted (37, 40,
49-51). Treatment of a variety of cells with the PKC activator phorbol
12-myristate 13-acetate inhibits both G
q- and G
i-coupled receptor stimulated PtdIns 4,5-P2
hydrolysis (49, 51, 52). This phorbol 12-myristate
13-acetate-dependent inhibitory action was prevented by
prior incubation of cells with PKC inhibitors. PKC has also been shown
to decrease the catalytic activity of PLC
isoforms in several
in vitro reconstitution assays (38, 53).
The targets of PKC involved in the desensitization of
agonist-stimulated PIP2 hydrolysis include
G-protein-coupled receptors, G proteins functionally coupled with PLC,
or PLC itself (for review, see Ref. 54). Direct phosphorylation of
several subtypes of PLC
isoforms by PKC has been reported, although
the functional consequences of these observations remains to be
established (37, 38, 53, 55). A recent study by Filtz and co-workers
(38) revealed that PLC
t, a turkey PLC
isoform which shares
highest homology with mammalian PLC
2, is phosphorylated by
conventional PKCs (37, 53, 55). This PKC-mediated phosphorylation
appears to decrease both basal activity of PLC
t and the activity
stimulated by GTP
S. A close correlation between PLC
3
phosphorylation and PKC-mediated inhibition of PIP2
hydrolysis has been observed (37). Yue et al. (56) have
recently demonstrated that conventional PKCs induce phosphorylation of
PLC
3 at serine 1105 both in vivo and in
vitro. Mutation of serine 1105 to alanine completely abolishes the
PKC-mediated inhibition of Gq-stimulated PLC
3 activity.
Despite the existence of distinct stimulatory controls for PI turnover
at the plasma membrane and in the nucleus (16), PKC appears to exert
similar inhibitory effects on PIP2 hydrolysis at the two
sites. In the case of the nucleus, it is responsible for the
termination of IGF-I dependent nuclear signaling. This conclusion is
supported by the following two findings. First, inhibition of PKC
activity by either calphostin C or Go6976 causes sustained activation
of nuclear PLC activity induced by IGF-I (Fig. 1). Second, within the
same time frame of IGF-I stimulation, the nuclear PKC activity
inversely correlates with the activity of nuclear PLC (Fig.
2B).
It was previously demonstrated that the PKC activator TPA induces
phosphorylation of PLC
1 in vivo, and phosphorylation of bovine brain PLC
1 by PKC in vitro resulted in a
stoichiometric incorporation of phosphate at serine 887 (45). However,
the physiological relevance of these findings has not been reported. Our two-dimensional tryptic peptide mapping analysis for
32P-labeled nuclear PLC
1 revealed that stimulation of
Swiss 3T3 cells with IGF-I for 30 min induced production of two
"extra" phosphopeptides (Fig. 5). One of these tryptic
phosphopeptides was not observed in either cells treated with PKC
inhibitor Go6976, or the cells overexpressing a PLC
1 variant in
which serine 887 was replaced by alanine. We thus conclude that
PKC-mediated phosphorylation of PLC
1 at serine 887 occurs within
the nucleus following IGF-I stimulation. The pivotal role of this
phosphorylation in PKC-mediated attenuation of PIP2
hydrolysis was confirmed by the observation that mutation of serine 887 to alanine causes sustained elevation of nuclear PLC activity in
IGF-I-treated cells (Fig. 7).
An explanation of how phosphorylation of PLC
1 at serine 887 modulates the enzyme activity in the nucleus remains to be defined. Phosphorylation of PLC
1 by PKC in vitro has no direct
effect on the enzyme activity (45). A recent report demonstrates that PKC-mediated phosphorylation inhibits the stimulation of PLC
1 activity by G
subunits, but does not affect G
-stimulated
enzyme activity (53). Whether or not the G
subunit plays a role
in IGF-I stimulated PLC activity in the nucleus is currently unclear. Nevertheless, evidence does exist for growth factor-induced
nuclear translocation of Gi (57, 58), which could in turn
lead to nuclear localization of 
subunit and subsequent
activation of putative PLC
1 in the nucleus.
PKC-promoted phosphorylation of PLC
s appears to be isoenzyme
specific (56). Our results strongly suggest that the
isoform of PKC
is responsible for phosphorylation of nuclear PLC
1 in Swiss 3T3
cells. This notion is supported by the following observations. (i) PKC
interacts specifically with PLC
1 in the nucleus of IGF-I-treated 3T3 cells (Fig. 4). (ii) Nuclear accumulation of PKC
closely correlates with the attenuation of nuclear PLC activity (Fig.
2). (iii) IGF-I dependent activation of nuclear PLC
1 is sustained
by treatment with the selective PKC
inhibitor Go6976 (Fig. 1) and
by introduction of DN PKC
(Fig. 8C). (iv) Recombinant PKC
can
directly phosphorylate PLC
1 purified from nuclei (Fig. 6). On the
other hand, expression of DN PKC
blocked phosphorylation of nuclear
PLC
1 at serine 887 (Fig. 8B). In agreement with this conclusion, a recent study has found that, among several PKC isoforms tested, PKC
is most effective in promoting phosphorylation of PLC
in vitro (53). Furthermore, Neri and co-workers (29) have demonstrated that of four PKC isoforms (
,
I,
, and
) detected in Swiss 3T3 cells, only PKC
is present in
the nucleus. Therefore, PKC activity in the nucleus of Swiss 3T3 cells
appear to be due solely to this isozyme.
In summary, the present study demonstrates for the first time the
existence of a negative feedback control mechanism to desensitize IGF-I
receptor-evoked activation of nuclear PI cycle, and that PKC
-promoted phosphorylation of nuclear PLC
1 at serine 887 plays a
key role in this regulation process. However, our study could not
exclude the possibility that PKC
may also exert its inhibitory
action on PLC
1 by phosphorylating other nuclear components involved
in the regulation of its activity. The potential downstream nuclear
targets of PKC
are currently under investigation in our laboratory.