In human erythroleukemia (K562) cells, the highly
related protein kinase C (PKC)
and PKC
II
isozymes serve distinct functions in cellular differentiation and
proliferation, respectively. Previous studies using two domain switch
PKC chimera revealed that the catalytic domains of PKC
and
II contain molecular determinants important for
isozyme-specific function (Walker, S. D., Murray, N. R.,
Burns, D. J., and Fields, A. P. (1995) Proc. Natl.
Acad. Sci. U.S.A. 92, 9156-9160). We have now analyzed a panel
of PKC chimeras to determine the specific region within the catalytic domain important for PKC
II function. A cellular assay
for PKC
II function was devised based on the finding
that PKC
II selectively translocates to the nucleus and
phosphorylates nuclear lamin B in response to the PKC activator
bryostatin. This response is strictly dependent upon expression of PKC
II or a PKC chimera that functions like PKC
II. We demonstrate that a PKC
/
II
chimera containing only the carboxyl-terminal 13 amino acids from PKC
II (
II V5) is capable of nuclear
translocation and lamin B phosphorylation. These results are consistent
with our recent observation that the PKC
II V5 region
binds to phosphatidylglycerol (PG), a potent and selective PKC
II activator present in the nuclear membrane (Murray,
N. R., and Fields, A. P. (1998) J. Biol.
Chem. 273, 11514-11520). Soluble
II V5 peptide
selectively inhibits PG-stimulated PKC
II activity in a
dose-dependent fashion, indicating that PG-mediated activation of PKC
II involves interactions with the
II V5 region of the enzyme. We conclude that
II V5 is a major determinant for PKC
II
nuclear function and suggest a model in which binding of PG to the
II V5 region stimulates nuclear PKC
II
activity during G2 phase of the cell cycle.
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INTRODUCTION |
Protein kinase C (PKC)1
is a family of serine/threonine kinases that play crucial roles in
various signaling processes, including cellular proliferation and
differentiation (1-4). Molecular cloning studies have shown that the
PKC family has at least 12 distinct members classified into three
groups according to their structure, calcium requirement, and cofactor
dependence (5, 6). The fact that PKC isotypes exhibit different
patterns of tissue expression, subcellular localization, and
activator/substrate specificity implies that there is functional
diversity among isotypes (6-10). Previous studies demonstrated that
PKC
and
II are involved in the differentiation and
proliferation of a variety of cell types (3, 10). In human
erythroleukemia (K562) cells, we determined that PKC
is involved in
PMA-induced cytostasis and megakaryocytic differentiation (10). PKC
II, on the other hand, is required for K562 cell
proliferation (10). PKC
II selectively translocates to
the nucleus during the G2/M phase transition of cell cycle
and leads to direct phosphorylation of the nuclear envelope polypeptide
lamin B at mitosis-specific sites involved in mitotic nuclear lamina
disassembly (9, 11-15).
In order to study the molecular basis of PKC
and
II
isozyme function, we expressed two domain switch chimeras between the regulatory and catalytic domains of PKC
and
II in
K562 cells (16). These chimeras demonstrated that the catalytic domains of PKC
and
II contain determinants that are critical
for isozyme-specific function (16). Specifically, a PKC
II/
chimera, consisting of the regulatory domain of
PKC
II and the catalytic domain of PKC
, exhibited a
phenotype resembling wild type PKC
(16). Conversely, a PKC
/
II chimera, composed of the regulatory domain of PKC
and the catalytic domain of PKC
II, behaved like
wild type PKC
II (16).
Based upon these findings, we wished to define the isozyme-specific
determinants within the catalytic domain of PKC
II. For this purpose, we constructed and expressed a series of
/
II PKC chimeras in which the variable and constant
regions within the catalytic domain of PKC
II were
replaced by the corresponding PKC
regions. Our results demonstrate
that the V5 region of PKC
II (
II V5),
consisting of the carboxyl-terminal 13 amino acids of PKC
II, contains the molecular determinant necessary for
nuclear translocation and activation of the enzyme. These results are interesting, in light of our recent studies demonstrating that the V5
region of PKC
II binds phosphatidylglycerol (PG), a
nuclear activator of the enzyme (17). A soluble peptide corresponding to
II V5 inhibits PG-stimulated PKC
II
activity, indicating that interactions between PG and the
II V5 region are important for PG-mediated activation of
PKC
II. Based on these observations, we propose a
model for the cell cycle-regulated activation of nuclear PKC
II.
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EXPERIMENTAL PROCEDURES |
Construction of PKC Chimeras--
In previous work, we
demonstrated that a chimera consisting of the regulatory domain of PKC
(
V1-V3) and the catalytic domain of PKC
II
(
II C3-V5) behaved like PKC
II (16).
Therefore, we constructed PKC
/
II chimeras in which
each of the constant and variable regions within the catalytic domain
of this chimera was replaced with the corresponding PKC
sequence
(Fig. 1). PKC chimeras were constructed by two-step polymerase chain
reaction (PCR) as described previously (16). The first step in the
chimera construction was to amplify the desired PKC
and PKC
portions of the chimera using the appropriate 5' and 3' end primers,
internal chimeric primers, and either PKC
or PKC
II
cDNA as a template. The internal primers were constructed so that
they would anneal in the second PCR step. Products from the first PCR
step were gel-purified and combined, along with 5' and 3' end primers,
and the complete chimera produced by extension and amplification. The
primers used for construction of the chimeras are presented in Table
I. The end primers contained restriction
sites (5' KpnI and 3' NheI) to facilitate
subsequent cloning. Completed chimeras were gel-purified and cloned
into the TA PCR cloning vector pCR 2.1 (Invitrogen). The chimeras were
restricted from pCR 2.1 using KpnI and NheI and
ligated into the KpnI and NheI sites within the
multiple cloning site of the episomal expression vector pREP4 (Invitrogen).
Transfection and Expression of PKC Chimeras in K562
Cells--
Human erythroleukemia K562 cells (ATCC) were maintained in
suspension culture as described previously (10). Cells were transfected with the pREP4 plasmids containing the PKC chimera constructs using
DOTAP lipofection reagent (Boehringer Mannheim) following the
manufacturer's protocol. 24 h after transfection, fresh medium containing 250 units/ml hygromycin B (Calbiochem) was added to the
cultures and transfectants selected for 3-4 weeks. K562 cell transfectants were maintained continuously in growth medium
supplemented with 250 units/ml hygromycin B. Expression of PKC chimeras
was determined by immunoblot analysis using previously characterized isotype-specific antibodies directed against the V5 regions of PKC
and
II (14). For immunoblotting, K562 cell transfectants were washed with cold phosphate-buffered saline, sonicated for 30 s in SDS sample buffer (18), and boiled for 5 min. Total cell extracts
from 1 × 105 cells were resolved by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose
(Schleicher & Schuell), and subjected to immunoblot analysis as
described previously (10). The chimeric nature of the chimeras was
confirmed by immunoblot analysis using isozyme-specific antibodies
against the V3 region of PKC
and
II as described
previously (16).
Drug Treatment, Isolation of Nuclear Envelopes, and Lamin B
Phosphorylation--
K562 cell transfectants carrying either control
vector (pREP4) or PKC chimera-containing pREP4 vectors were treated
with 30 nM PMA (LC Laboratories) or diluent (0.1%
Me2SO). Previous studies demonstrated that this PMA
treatment leads to loss of immunologically detectable PKC
II and loss of bryostatin-mediated lamin B
phosphorylation (10, 16). Following the 48-h PMA treatment, total cell
lysates were prepared from an aliquot of cells and subjected to
immunoblot analysis using isotype-specific PKC
V5 and
II V5 antibodies as described above. The remainder of
the cells were incubated in the presence and absence of bryostatin 1 (100 nM, LC Laboratories) for 30 min at 37 °C, followed
by nuclear envelope isolation and determination of lamin B
phosphorylation as described previously (16).
Assay of Protein Kinase C
II Activity in
Vitro--
In vitro protein kinase C assays were carried out using
purified baculovirus-expressed human PKC
II as described
previously (15). Briefly, reactions were performed in assay buffer
containing 50 mM Tris-HCl, pH 7.5, 10 mM
MgSO4, 1 mM dithiothreitol, 100 µM CaCl2, 100 µM ATP, 2 µCi
of [
-32P]ATP (Amersham Pharmacia Biotech), 40 µg/ml
(50 µM) phosphatidylserine (PS, Avanti Polar Lipids), 20 µM dioctanoylglycerol (DAG, Avanti Polar Lipids), and 10 µg of histone H1 for 15 min at 25 °C. In most reactions, 250 µg/ml (315 µM) dioleoyl PG (Avanti Polar Lipids) was
added to maximally stimulate PKC
II activity (17).
Synthetic peptides corresponding to
II V5
(CFVNSEFLKPEVKS) or
V5 (CQFVHPILQSSV) were added at the
concentrations indicated in the figure legends. Histone H1
phosphorylation was quantitated using phosphorimaging analysis as
described previously (12).
 |
RESULTS |
Construction and Expression of PKC Chimeras in K562
Cells--
Based on our previous finding that the catalytic domain of
PKC
II is crucial for PKC
II function
(16), we generated a series of PKC chimeras in which the variable and
constant regions within the catalytic domain of PKC
II
were exchanged with the corresponding sequences from PKC
by
two-step PCR as depicted in Fig. 1. To
investigate the biochemical properties of these chimeras in intact K562
cells, chimeric PKC constructs were subcloned into the pREP4 episomal
expression vector and transfected into K562 cells. Expression of the
PKC chimeras was confirmed by immunoblot analysis with antibodies
against the carboxyl-terminal V5 regions of PKC
and
II (Fig. 2). Expression of
each of the chimeras containing the
II V5 region
(
II V4,
II C4,
II V5,
II 1504, and
II 1851) was confirmed using
the PKC
II V5 antibody (Fig. 2A). Expression
of the
II
chimera, which contains the
carboxyl-terminal V5 region from PKC
, was confirmed using the PKC
V5 antibody (Fig. 2B). In each case, a band with
apparent molecular mass of 85 kDa was detected, corresponding in size
to intact PKC
and
II protein. The level of
expression for all chimeras was determined by densitometric analysis to
be between 2- and 3-fold that of the endogenous levels of PKC
or
PKC
II. The chimeric nature of each of the chimeras was
confirmed by immunoblot analysis using the appropriate PKC
and
II-specific V3 antibodies as described previously (Ref.
16; data not shown).

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Fig. 1.
Schematic representation of the constructed
PKC / II chimeras. PKC chimeras were constructed
between PKC and II by a two-step PCR method as
described under "Experimental Procedures." The schematic indicates
the regulatory and catalytic domains of PKC. The conserved (C1-C4) and
variable regions (V1-V5) regions of PKC are also indicated. Open
boxes represent PKC sequences, and hatched boxes
correspond to PKC II sequences.
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Fig. 2.
Expression of PKC chimeras in K562
cells. Total cell lysates from 1 × 105 K562
cells, transfected with vector containing the indicated PKC chimera,
were subjected to immunoblot analysis as described under
"Experimental Procedures." Panel A, immunoblot detection
using isotype-specific antibody against the V5 region of PKC
II. Cell transfectants expressing the following
expression vector constructs were subjected to immunoblot analysis:
pREP4 control vector ( Control), wild type PKC
II (+ Control), II V4,
II C4, II V5, II ,
II 1504, and II 1851 PKC chimeras.
Panel B, immunoblot detection using isotype-specific
antibody against the V5 region PKC . Lane Control,
pREP4 vector; lane +Control, wild type
PKC ;  II , PKC chimera.
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PKC Chimera Expression Persists after PMA Treatment--
Our
previous studies demonstrated that treatment of K562 cells with PMA for
48 h leads to an increase in PKC
expression and a loss of PKC
II expression (10). We therefore wished to assess the
fate of PKC chimeras expressed in K562 cells following treatment with
PMA. For this purpose, K562 cell transfectants were treated with PMA
(30 nM) for 48 h. Transfectants were harvested and
immunoblotted as described under "Experimental Procedures" (Fig.
3). As expected, endogenous PKC
II expression was dramatically reduced as a consequence
of PMA treatment (Fig. 3A, compare lanes 1 and
2) (10, 16). In contrast, the levels of PKC
were
increased as previously reported (10, 16) (Fig. 3B, compare
lanes 1 and 2). Interestingly, in contrast to
endogenous PKC
II, the expression of each of the
transfected PKC chimeras (
II V4,
II C4,
II 1504,
II 1851,
II V5,
and 
II
) persists after PMA treatment (Fig. 3,
A and B).

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Fig. 3.
Expression of PKC chimeras persists after
PMA-treatment in K562 cells. Panel A, K562 cells
transfected with either control vector or PKC chimera vectors were
treated with 30 nM PMA for 48 h. Following 48 h
treatment, total cell lysates from 1 × 105 cells were
immunoblotted with antibody against the V5 region of PKC
II as described under "Experimental Procedures."
Lane Control ( PMA), pREP4
vector in the absence of PMA; lane Control (+ PMA), pREP4 in the presence of PMA; lanes
IIV4, IIC4,
 II ; II1504,
II1851, and IIV5, PKC
chimeras after PMA treatment. Panel B, immunoblot analysis
as described under "Experimental Procedures" with
antibody against the V5 region of PKC . Lanes: controls
are the same as in A;  II , PKC chimera after
PMA treatment.
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Ability of PKC Chimeras to Reconstitute PKC
II
Function--
The observation that expression of PKC chimera persists
after chronic PMA treatment while endogenous PKC
II
levels are dramatically reduced suggested a strategy to specifically
determine whether the transfected PKC chimeras could function like PKC
II in intact K562 cells without the contribution of
endogenous PKC
II. This strategy is predicated upon
several key observations. First, PKC
II is selectively
translocated and activated at the cell nucleus in response to
bryostatin but not PMA (9, 11, 14). Second, bryostatin-mediated
translocation of PKC
II to the nucleus leads to direct
phosphorylation of the nuclear envelope polypeptide lamin B (11-15).
Third, the ability of bryostatin to stimulate nuclear lamin B
phosphorylation is strictly dependent upon the expression of PKC
II (16). Thus, when K562 cells are treated with PMA,
both PKC
II expression and bryostatin-mediated lamin B
phosphorylation is lost (16). Therefore, we assessed the ability of the
transfected PKC chimeras to reconstitute bryostatin-mediated lamin B
phosphorylation in PMA-treated K562 cell transfectants as described
under "Experimental Procedures" (Fig.
4). Treatment of K562 cells containing an
empty control vector with PMA leads to the expected loss in
bryostatin-mediated lamin B phosphorylation, whereas cells not treated
with PMA exhibited robust lamin B phosphorylation in response to
bryostatin (Fig. 4, compare panels labeled Control (
PMA) and Control (+PMA). K562 cells expressing
the
II V4,
II C4,
II 1504,
II 1851, and
II V5 chimeras were all
capable of reconstituting bryostatin-mediated lamin B phosphorylation
after treatment with PMA. In contrast, cells expressing the

II
chimera were incapable of reconstituting the
bryostatin-mediated response. From these results, we conclude that
chimeras containing the functional determinant important for PKC
II function are capable of reconstituting PKC
II-dependent lamin B phosphorylation.
Furthermore, our analysis localizes this functional determinant to the
extreme carboxyl-terminal 13 amino acids of PKC
II
(
II V5), since a chimera containing only these 13 amino
acid residues from PKC
II (PKC
II V5) is capable of functioning in this PKC
II-selective
pathway.

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Fig. 4.
PKC chimeras can reconstitute lamin B
phosphorylation. K562 cells transfected with either control vector
or the indicated PKC chimeras were harvested following 48 h PMA
treatment. After washing to remove the PMA, cells were incubated
without or with bryostatin 1 (bryo, 100 nM) for
30 min and assayed for lamin B phosphorylation as described previously
(16). The labels are the same as described in Fig. 3. Results are
representative of three independent experiments.
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PG-stimulated PKC
II Activity Involves Interactions
with the V5 Region of PKC
II--
The results from the
PKC chimera studies demonstrate that the carboxyl-terminal V5 region of
PKC
II represents a molecular determinant for PKC
II-selective function in intact K562 cells. In recent
studies, we identified a component of the nuclear membrane, termed
nuclear membrane activation factor (NMAF), which selectively activates
PKC
II (19). More recently, we identified NMAF as PG
(17). PG was found to be a potent and selective activator of PKC
II, stimulating the enzyme 3-5-fold above the level of activity seen in the presence of optimal concentrations of PS, DAG, and
calcium (19). We also demonstrated that PG binds selectively and
saturably to the carboxyl-terminal region of PKC
II
(17), suggesting that PG stimulates PKC
II activity
through interactions involving
II V5. To specifically
test this hypothesis, we assessed the effect of soluble
II V5 peptide on PG-stimulated PKC
II activity in vitro (Fig. 5). As
reported previously (17) addition of 315 µM PG stimulates
PKC
II more than 4-fold over the level in the absence of
PG. This stimulation is specific for PG since it is not observed in the
presence of 370 µM PS (data not shown). Inclusion of
II V5 peptide in the kinase assay leads to inhibition of
PG-stimulated PKC
II histone kinase activity (Fig.
5A, lane 5). Inhibition is specific for the
II V5 peptide, since no inhibition is observed using the
corresponding V5 peptide from PKC
(Fig. 5A, lane
4). Inhibition by
II V5 peptide is
dose-dependent with an apparent IC50 of ~100
µM (Fig. 5B), whereas significant inhibition was not observed with the corresponding
V5 peptide. Interestingly, the
II V5 peptide inhibits only the PG-stimulated
component of PKC
II activity and not calcium-, DAG-, and
PS-stimulated activity. These results are consistent with the
conclusion that PG mediates activation through the carboxyl-terminal V5
region of PKC
II, whereas calcium, DAG, and PS mediate
activation by binding to the C1 and C2 regions within the regulatory
domain of the enzyme.

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Fig. 5.
PG-stimulated PKC II activity
is inhibited by the II V5 peptide in a
dose-dependent manner. Recombinant human PKC
II was incubated in kinase buffer in the absence or
presence of the indicated concentration of either II V5
or V5 peptide for 15 min at 25 °C as described under
"Experimental Procedures." Activity was monitored by incorporation
of 32P into purified histone H1 (10 µg) as described
previously (12). Panel A, autoradiograph of PKC
II-mediated histone H1 phosphorylation by PKC
II. Reactions were as follows. Lane 1, no PKC
II added; lane 2, PKC II in
reaction buffer without PG; lane 3, PKC II in
reaction buffer with PG; lane 4, PKC II in
reaction buffer containing PG and 1 mM V5 peptide;
lane 5, PKC II in reaction buffer containing
PG and 1 mM II V5 peptide. Data are from a
representative experiment. Panel B,
dose-dependent inhibition of PG-stimulated PKC
II activity with either II V5 (open
circles) or V5 (closed circles) peptide. Results are
plotted as the percentage of the PG-stimulated PKC II
activity versus peptide concentration (µM).
Data represent the mean of five determinations ± S.D. Some
error bars are masked by the data point symbols.
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DISCUSSION |
The PKC family of serine/threonine kinases serves critical roles
in cellular function. Recent studies have begun to elucidate specific
roles for individual PKC isozymes (1, 3, 5). We have identified roles
for the three major PKC isozymes in human K562 leukemia cells. K562
cells express PKC
,
II, and
, and each of these
isozymes serve distinct functions in these cells. PKC
is important
in cellular differentiation (10), PKC
II is required for
cellular proliferation (10) and PKC
plays a critical role in
leukemia cell survival and resistance to apoptosis (20). However,
despite our knowledge of the importance of these isozymes in cellular
physiology, less is known about the specific pathways in which these
isozymes function, the relevant cellular targets of their action, and
the mechanisms by which they maintain isozyme identity within the
intact cell. We have addressed these questions in the case of PKC
II. PKC
II is required for cell cycle
progression through the G2/M phase transition (12, 13). PKC
II is activated at the nucleus during late
G2 phase just prior to mitosis in synchronized cells (12,
13). At the nucleus, PKC
II mediates direct
phosphorylation of the nuclear envelope polypeptide lamin B on sites
involved in mitotic nuclear lamina disassembly (12, 13, 15). Inhibition
of PKC
II activation leads to cell cycle arrest in
G2 phase, demonstrating the importance of nuclear PKC
II activation and lamin B phosphorylation in entry into
mitosis (13).
Nuclear PKC
II activation is regulated by several
factors. First, a nuclear phosphoinositide-specific phospholipase C
activity generates a peak of diacylglycerol at the nuclear membrane
during G2 phase that serves to activate nuclear PKC
II (21). Second, the nuclear membrane contains a potent
activator of PKC
II that stimulates its activity above
the level observed in the presence of optimal calcium, DAG, and PS (17,
19). This activator, originally termed NMAF, was recently identified as
the phospholipid PG (17). The selectivity of PG for PKC
II activation suggested that PG might function through
interactions within the carboxyl-terminal V5 region of PKC
II. Indeed, we have demonstrated that the
carboxyl-terminal region of PKC
II binds selectively and
saturably to PG-containing vesicles, but not to vesicles containing
other phospholipids (17).
In the present study, we wished to determine the molecular determinants
on PKC
II that allow it to translocate to the nucleus and phosphorylate lamin B in intact cells, a process that we have demonstrated requires PKC
II expression (16). We
previously demonstrated that a chimera between PKC
and PKC
II containing the catalytic domain of PKC
II was capable of translocating to the nucleus and
phosphorylating lamin B (16). In contrast, a chimera containing the
catalytic domain of PKC
was not capable of mediating this response
(16). From these data, we concluded that regions within the catalytic
domain of PKC
II serve as a molecular determinant of PKC
II function (16). We have now used further PKC
/
II chimeras to map this molecular determinant to the
extreme carboxyl-terminal 13 amino acids of PKC
II. The results of this study are interesting in light of our recent finding that the V5 region of PKC
II mediates binding to PG
(17). In fact, we now demonstrate that a
II V5 peptide
selectively inhibits PG-stimulated PKC
II activity in a
dose-dependent fashion. These results indicate that
interactions between PG and
II V5 are important for PG-
stimulated PKC
II activity.
The identification of the carboxyl-terminal region of PKC
II as an important region regulating isozyme specific
function is consistent with recent studies suggesting the importance of this region to PKC function. The V5 region of PKC
has recently been
shown to interact with PICK 1, a PKC-binding protein, through PDZ
domain-like interactions (22). PICK1 has been suggested to play a role
in targeting PKC
to appropriate intracellular sites where it can
transduce isozyme-specific signals (22). Likewise, we have
demonstrated that interaction of the V5 region of PKC
II, in this case with the phospholipid PG, is important for the nuclear translocation and activation of the enzyme at the
nuclear membrane, where it is required for nuclear lamina disassembly
and entry into mitosis (17). Finally, studies investigating the
differential regulation of PKC
I and
II
activity by calcium have led to the suggestion that the V5 region of
PKC
II interacts with the C2 region of the enzyme to
influence calcium- and PS-mediated enzyme activation (23).
Given our recent data on nuclear activation of PKC
II,
the proposed interdomain interactions involving the C2 and V5 regions of PKC
II, and related studies on the mechanism of PKC
membrane translocation and activation (24), we propose a working model for the cell cycle-regulated, nuclear activation of PKC
II as illustrated in Fig.
6. Soluble PKC
II is
stably phosphorylated on at least three known sites (24, 25). First,
PKC
II is phosphorylated by a putative protein kinase C
kinase at threonine 500 in the activation loop of the enzyme (24, 25).
Phosphorylation at threonine 500 makes PKC
II
catalytically competent and triggers subsequent autophosphorylation of
threonine 641 and serine 660 in the carboxyl-terminal region of the
enzyme. Phosphorylation of these sites stabilizes the catalytically
competent conformation of the enzyme and allows its release into the
cytosol (24, 25). In our model, a nuclear PI-PLC isozyme(s), whose
activity is linked to cell cycle by an unknown mechanism, is activated
during the G2 phase of cell cycle (21). Activation of
nuclear PI-PLC leads to generation of inositol trisphosphate
(IP3) and DAG. We propose that IP3 binds to
nuclear IP3 receptors to mobilize intracellular calcium
stores, leading to a rise in intracellular calcium concentrations. It
has been demonstrated that nuclei contain functional IP3
receptors (26) and that intracellular calcium concentrations rise in
synchronized cells just prior to mitosis (27-29). Furthermore,
inhibition of this rise in intracellular calcium, using calcium
chelators such as BAPTA, leads to cell cycle arrest in G2
phase, demonstrating the importance of calcium in entry into mitosis
(29). We propose that one function of elevated calcium levels is to
translocate PKC
II to the inner nuclear membrane. We
have not directly determined whether this step involves translocation
of PKC
II from cytosolic pools, or alternatively
involves translocation of inactive PKC
II from a
nucleoplasmic pool. However, based on our fractionation studies (9,
11-15) and the immunofluorescence studies of others in cultured
myocytes (30), we favor the hypothesis that this step represents
translocation of cytosolic PKC
II to the nucleus. As has
been previously demonstrated, binding of calcium to PKC increases its
membrane affinity leading to membrane translocation and an increase in
affinity of the enzyme for PS (31-33). Membrane bound PKC coordinately
binds multiple PS molecules, possibly within the C2 region of the
enzyme (34, 35). It has been demonstrated that PKC induces
calcium-dependent clustering of acidic phospholipids into
microdomains that may facilitate cooperative binding of phospholipid to
the C2 domain (36). We propose that nuclear PG coclusters with PS
during this phase of nuclear membrane binding. In the presence of DAG,
PKC activation occurs, through displacement of the pseudosubstrate
domain from the active site of the enzyme (37). It has recently been
proposed that the V5 region of PKC
II interacts with the
C2 region of the enzyme when it is in its active conformation (23). We
further propose that these V5-C2 interdomain interactions are mediated,
at least in part, by binding of the V5 region to PG that is clustered
in the same nuclear membrane microdomains containing the PS involved in
membrane/C2 domain interactions. Once optimally activated at the inner
nuclear membrane, PKC
II directly phosphorylates lamin B
at Ser-405 within its carboxyl-terminal globular domain (12, 15). Lamin
B is an ideal substrate for nuclear membrane-bound PKC
II since it is intimately associated with the inner
nuclear membrane surface by virtue of its carboxyl-terminal isoprenyl
groups (38). Phosphorylation of lamin B by PKC, and p34 cdc2/cyclin B
kinase, leads to nuclear lamina disassembly, a process required for
entry into mitosis (38). Current studies are focused on determining the
identity and mechanism of the cell cycle regulation of nuclear PI-PLC
and on characterizing the mechanisms by which PG and the V5 region of
PKC
II participate, along with the C1 and C2 domains, in
regulating nuclear PKC
II activity.

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Fig. 6.
A proposed model for the nuclear activation
of PKC II. Prior to activation, PKC
II is phosphorylated at multiple sites within the
catalytic domain (indicated by P) giving rise to a
catalytically competent enzyme that resides in the cytosol (Refs. 24
and 25; I). During G2 phase of cell cycle, a
nuclear PI-PLC is activated, leading to the generation of nuclear DAG
(21) and IP3. IP3 activates nuclear
IP3 receptors (26), leading to elevation of intranuclear
calcium concentrations just prior to mitosis (27-29). In the presence
of elevated calcium and acidic phospholipids, PKC interacts with the
nuclear membrane (II). Membrane binding leads to clustering
of acidic phospholipids including PS and PG to microdomains within the
membrane (III). The binding of DAG to the C1 region promotes
cooperative binding of PS to the C2 domain of PKC II,
thereby inducing a conformational change that releases the
pseudosubstrate domain of PKC II from the active site.
The carboxyl-terminal region of the enzyme is brought into
juxtaposition with the C2 domain via specific binding of
II V5 to clustered PG, leading to full activation of the
enzyme. Activated PKC II directly phosphorylates its
nuclear substrate lamin B, leading to mitotic nuclear lamina
disassembly and entry into mitosis (38).
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We thank B. Sun and Y. Wang for technical
assistance in the construction of PKC chimeras, N. R. Murray for
many helpful suggestions, and D. Schattenberg for preparation of the
PKC model (Fig. 6).