1 MRC Centre for Immune Regulation, Birmingham University, Birmingham B15 2TT,
UK
2 CRC Institute for Cancer Studies, Birmingham University, Birmingham B15 2TT,
UK
* Author for correspondence (e-mail: j.m.lord{at}bham.ac.uk )
Accepted 14 November 2001
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Summary |
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Key words: Cell cycle, Protein kinase C, Nucleus, Diacylglycerol, G2/M
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Introduction |
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The inositol 1,4,5 trisphosphate (Ins(1,4,5)P3)
signalling system has been shown to be increased during G2/M in regenerating
liver (Marino et al., 1992)
and local calcium transients have been shown to arise in the perinuclear
region prior to mitosis in sea urchin eggs
(Wilding et al., 1996
). Such a
localised increase in calcium flux prior to mitosis will provide the calcium
required for activation of the classical PKCs, but with no selectivity of
PKC-ß over
, or for nuclear localisation of the activated PKC.
Murray and Fields have more recently shown that phosphatidylglycerol (PG) is a
potent and selective activator of PKC-ßII, interacting with a region
within the C-terminal catalytic domain required for activation of this kinase
at the nucleus (Murray and Fields,
1998
). Moreover, PG increases PKC-ßII enzyme activity above
that seen with phosphatidylserine and DAG alone. However, nuclear PG levels do
not change through the cell cycle (Murray
and Fields, 1998
), leading the authors to suggest that PG is
involved in enhancing the selective association of PKC-ßII with the
nuclear membrane and sustained activation of the enzyme, while initial
activation is achieved by another lipid species, possibly nuclear DAG
(Sun et al., 1997
).
Diacylglycerols are potent activators of PKC that are generated as second
messenger molecules from the hydrolysis of membrane phospholipids, primarily
phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2)
(Noh et al., 1995
) and
phosphatidylcholine (Exton,
1996
). DAG serves as a hydrophobic anchor, localising PKC to the
membrane, as well as being involved in enzyme activation (Newton, 1995).
Several groups have shown that PtdIns(4,5)P2 hydrolysis
occurs within the nuclear matrix (Cocco et
al., 1987
; Irvine and Divecha,
1992
) and that the nuclear phosphoinositide cycle is distinct, and
regulated separately, from the classic plasma membrane cycle
(Cocco et al., 1989
). Moreover,
DAG production from phosphatidylcholine in the nuclear envelope was
demonstrated in IIC9 fibroblasts following
-thrombin stimulation
(Jarpe et al., 1994
). Nuclear
translocation of PKC isoenzymes, or the activation of pre-existing nuclear
PKCs, may therefore be mediated by the generation of DAG at the nucleus at a
specific stage in the cell cycle. The few studies that have attempted to
correlate DAG mass with PKC translocation, have not been informative. However,
analysis of individual DAG species has revealed that the component molecular
species are numerous and the generation of polyunsaturated species does
correlate with PKC activation (Pettitt and
Wakelam, 1999
).
Here we show that the activation of PKC-ßII through the cell cycle correlates with the generation of polyunsaturated DAG species at the nucleus, primarily 1-stearoyl, 2-arachidonyl glycerol (SAG). Moreover, SAG showed some selectivity for the activation of classical PKC isoenzymes over novel PKCs in an in vitro kinase assay and this diacylglycerol may represent a significant physiological activator of PKC-ßII at G2/M. We propose that the molecular composition of DAG generated by lipid hydrolysis will contribute to differential signalling through the PKC pathway in a variety of cell processes, including the cell cycle.
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Materials and Methods |
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Isolation of nuclei
Nuclei were isolated from U937 cells using a rapid method designed to
retain the double nuclear membrane and minimise loss of nuclear proteins
(Bunce et al., 1988). Briefly,
cells were suspended at 107/ml in buffer A (50 mM Tris-HCl pH 7.4,
250 mM sucrose, 50 mM KCl, 2 mM Mg2SO4, 1 mM
dithiothreitol) containing 2% Tween-40 and immediately snap frozen in liquid
nitrogen. Cells were then thawed slowly and homogenised with 25 strokes in a
Dounce homogeniser. The homogenate was layered onto a cushion of 30% (w/v)
sucrose in buffer A in a microfuge tube and spun at 11,000 g
for 1 minute (MSE microcentaur). The supernatant was discarded and the nuclear
pellet washed three times in buffer A. Purity of nuclei was checked using
marker enzymes as described previously
(Bunce et al., 1988
) and the
only contaminating element was endoplasmic reticulum, which was routinely less
than 6%.
Measurement of DAG mass and analysis of DAG molecular species
Lipids from whole U937 cells and from isolated U937 nuclei were extracted
using the method of Bligh and Dyer and total DAG was then measured using a DAG
kinase mass conversion assay (Priess et al., 1987) using DAG kinase purchased
from Boehringer-Mannheim. Changes in DAG saturation were measured using a
silver nitrate TLC methodology. Lipid extracts were
[32P]-phosphorylated as for the DAG kinase assay (Priess et al.,
1987) in a total volume of 90 µl. The reaction was stopped by addition of
705 µl chloroform/methanol/0.5 M HCl (150:300:20 by volume), left to
extract for 10 minutes then mixed with a further 225 µl chloroform followed
by 225 µl 0.1 M HCl to split the phases. The upper phase was discarded and
the lower organic phase (containing the [32P]phosphatidate now in
free acid form) washed with 600 µl chloroform/methanol/water (3:48:47 by
volume). After drying, the samples were methylated with a saturated solution
of diazomethane in diethyl ether (overnight at room temperature), dried again,
resuspended in 20 µl chloroform/methanol (2:1 v/v) and separated by silica
TLC developed three times with chloroform/methanol (98:2 v/v). The dimethyl
[32P]-phosphatidate band was eluted with chloroform/methanol (2:1
v/v). Finally separation based on double bond number was achieved using TLC
plates impregnated with 5% AgNO3, developed firstly to about half
way with chloroform/methanol (93:7 v/v), air dried and then developed to the
top with chloroform/methanol (97:3 v/v). Detection and quantification was
carried out by phosphorimaging using a Molecular Dynamics PhosporImager. For
analysis of DAG molecular species, lipids from a minimum of
5x106 cells and 1x107 nuclei were
derivatized with 3,5-dinitrobenzoyl chloride and separated by hplc as
described previously (Pettitt and Wakelam,
1993).
Assessment of PKC isoenzyme translocation during cell cycle
Cells in G1, S or G2/M, isolated by centrifugal
elutriation, were assessed for PKC isoenzyme activation/translocation by
measuring association of PKC with the particulate (membrane) fraction
(Pongracz et al., 1999).
Briefly cells were resuspended at 107 cells/ml in hypotonic lysis
buffer (20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM dithiothreitol, 5
mM EGTA, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 20 µg/ml leupeptin,
10 µg/ml aprotinin, 10 µg/ml pepstatin) and swollen on ice for 10
minutes. Cells were then homogenized with 25 strokes in a tight fitting Dounce
homogeniser. Nuclei were isolated at 1000 g for 10 minutes and
cytosol and particulate fractions prepared at 100,000 g for 45
minutes. Nuclear, cytosolic and particulate fractions were assessed for PKC
isoenzyme content by western blotting using isoenzyme-specific antibodies
(Santa Cruz) and HRP-conjugated secondary antibodies (Santa Cruz). Equal
loading of nuclear extracts was confirmed by reprobing blots with an
anti-lamin B antibody (N. Chaudhary, RPI, Boulder Co., USA). Blots were
developed using enhanced chemiluminescence (ECL, Amersham International).
In vitro PKC enzymatic assay
Activation of recombinant PKC isoenzymes was measured using an in vitro
micellar assay, essentially as described previously
(Lord and Ashcroft, 1984).
Brifely, 200 ng of recombinant human PKC-
, ßII,
or
(CN Biosciences, Nottingham, UK) was combined with lipid micelles containing
16 ug/ml phosphatidylserine (PS; Sigma) and 1.6 µg/ml sn1,2 stearoyl,
arachidonyl glycerol (Sigma) or PS alone, in PKC assay buffer (20 mM Tris-HCl
pH 7.4, 5 mM magnesium acetate, 0.2 mg/ml histone H1, 50 µM
CaCl2). The reaction was started by the addition of 20 µM ATP
and 1 µCi per assay tube of [
-32P]ATP (Amersham
International). The reaction was carried at 30°C and terminated after 10
minutes by the addition of ice-cold trichloroacetic acid. Incorporation of
radioactivity into histone was determined by scintillation counting and enzyme
activity was expressed as pmol of 32P incorporated per minute per
mg protein kinase C.
Indirect immunostaining for PKC-ßII
For analysis of PKC isoenzyme subcellular localisation, cells were
indirectly immunostained as cytospins, after air-drying for 2 hours and
fixation in ice-cold acetone, as previously described
(Pongracz et al., 1999).
Primary antibody was affinity purified and raised in rabbits against peptides
in PKC-ßII (Santa Cruz). Anti-rabbit IgG-FITC conjugated antibody (Dako,
UK) was used as the secondary antibody and fluorescence was visualised using a
confocal microscope (MRC 500, BioRad).
LC-MS analysis of phosphatidic acid (PA)
Lipids were extracted from cell or nuclei pellets by vigorous mixing with
methanol (1 ml containing 2 nmol 12:0/12:0-phosphatidic acid (PA) internal
standard) followed by chloroform (2 ml). After standing for 10 minutes, phases
were split by addition of 0.88% KCl in 0.1 M HCl (1 ml) and the upper aqueous
phase discarded. The lower organic phase was washed with 1 ml methanol/0.88%
KCl in 0.1 M HCl (1:1 v/v) then dried under a stream of nitrogen before
resuspending in a small volume of chloroform/methanol (2:1 v/v). The total
lipid extract was separated and characterized by LC-MS (QP8000alpha, Shimadzu)
using a Luna silica column (3µ, 2.0x150mm; Phenomenex, UK) with a
solvent gradient of chloroform/methanol/water/ammonia (90:9.5:0.5:0.32 by
volume) changing to chloroform/methanol/water/ammonia (50:48:2:0:32 by volume)
over 40 minutes at 0.35 ml/minute. PA was detected in negative electrospray
ionisation (ESI) mode (nitrogen flow; 4 l/minute, CDL; 300°C, probe high
voltage; -5 kV) with a retention time of approximately 30 minutes.
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Results |
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PKC-ßII translocation was investigated further by isolation of nuclei
and western blotting of nuclear extracts. Some PKC-ßII was detected in
the nuclear fraction at G1 and S phase but was significantly
increased at G2/M (Fig.
2A). The ratio of PKC-ßII to nuclear lamin B was calculated
to allow for unequal loading of gels and confirmed increased association of
this isoenzyme with the nucleus at G2/M
(Fig. 2B). PKC-ßII
translocation to the nucleus was also confirmed by indirect immunostaining.
Cells in G1, S and G2/M were indirectly immunostained
using an isoenzyme-specific antibody to PKC-ßII and immunofluorescence
analysed by laser scanning confocal microscopy. PKC-ßII immunoreactivity
was present in the cytosol and nucleus at G1 and S and the fraction
of the isoenzyme associated with the nucleus increased during G2/M
(Fig. 2C). Counterstaining of
cells with propidium iodide allowed for quantification of the degree of
coincidence between FITC-immunofluorescence (i.e. PKC) and PI (i.e. DNA)
staining. This analysis showed that association of PKC-ßII with the
nucleus was highest during G2/M, with a 21.6±3% increase in
nuclear PKC-ßII compared with G1. Our data thus confirm the
nuclear translocation of PKC-ßII during G2/M phase transition
(Goss et al., 1994).
|
DAG mass in nuclei at different stages in the cell cycle
The DAG content of whole U937 cells at G1, S and G2/M
stages of the cell cycle and nuclei isolated from these cells, was measured
using a mass conversion assay. DAG mass in whole cells was very similar across
the cell cycle, with only a modest increase in DAG at G2/M
(388±42 pmoles/106 cells) compared with G1 phase
cells (351±48 pmoles/106 cells), confirming published data
(Thompson and Fields, 1996).
Nuclear DAG levels were significantly greater at G2/M compared with
nuclei from cells in G1 or S phase
(Fig. 3A). The change in
nuclear DAG levels can account for the small increase in whole cell DAG seen
at G2/M and is therefore unlikely to result from contamination of
nuclear preparations with whole cells. These data are expressed as pmoles of
DAG per 106 cells or nuclei and could potentially be influenced by
the different size of the cells as they progress though cell cycle. However,
expressing DAG mass as a ratio of total phospholipid gave the same trend, with
a lesser but significant increase in nuclear DAG at G2/M
(5.77±0.6 pmol DAG/pmol phospholipid) relative to G1
(3.34±0.3 pmol DAG/pmol phospholipid; P<0.05).
|
Changes in DAG molecular species in nuclei during cell cycle
Measurement of changes in DAG mass are of limited value in interpreting
differential changes in the activation status of specific PKC isoenzymes
through the cell cycle, as eight of the known isoenzymes are responsive to
DAG. In addition, measurements of DAG mass could mask significant changes in
individual molecular species and although most DAG species can activate PKC in
vitro, we have already shown that polyunsaturated DAGs are probably the
physiological activators of PKC in vivo
(Wakelam, 1998).
Silver nitrate TLC analysis of nuclear DAG (following [32P]-phosphorylation and diazomethane methylation to form dimethyl [32P]-phosphatidate) revealed that mono- and di-unsaturated DAGs were the major unsaturated species in the nucleus (Fig. 3B). There was a fall in the level of monounsaturated species during G2/M as a percentage of total nuclear DAG, although the absolute mass did not change significantly. However, a corresponding and significant increase in polyunsaturated species was seen during G2/M, specifically in the tetra-unsaturated DAGs, both as a percentage of total nuclear DAG (Fig. 3B) and in absolute mass suggesting that physiologically relevant DAGs were generated at the nucleus through the cell cycle. To obtain further information on the nature of these DAG species, nuclear lipid extracts were 3,5-dinitrobenzoyl derivatizated and then separated by HPLC. The major nuclear DAG species identified were 14:0/16:0, 14:0/18:1n-9, 16:0/16:0, 16:0/18:1n-9, 16:0/18:2n-6, 18:0/18:0, 18:0/18:1n-9, 18:0/18:2n-6 and 18:1n-9/18:1n-9, while the predominant polyunsaturated species was shown to be the tetraunsaturated 1-stearoyl, 2-arachidonyl glycerol (18:0/20:4n-6; SAG). The mass of this DAG doubled during G2/M (2.05-fold increase compared with G1 levels), correlating with the nuclear translocation of PKC-ßII. The actions of SAG on PKC-ßII were therefore investigated further.
PA changes
Since termination of DAG signalling is believed to be through the action of
diacylglycerol kinase (DAGK), resulting in the formation of PA, this lipid was
also analysed for cell-cycle-dependent changes. Using LC-MS, both whole cell
and nuclear PA levels were found to remain essentially constant across the
cell cycle at 110±25 pmoles/106 cells and 21±6
pmoles/106 nuclei. Similarly, no obvious cell-cycle-dependent
changes were observed in the species profiles. The major nuclear PA structures
were 16:0/16:0 and 16:0/18:1n-9, representing approximately 20 and 40 mol% of
total diacyl species, respectively, while the major polyunsaturated species
was 18:0/20:4 at approximately 5 mol%. Whole cell PA had a slightly more
unsaturated species profile compared with that for nuclei, with less 16:0/16:0
but more 18:1n-9/18:1n-9 and 18:0/18:1n-9.
Effect of 1-stearoyl, 2-arachidonyl glycerol on PKC isoenzyme
activity in vitro
To determine whether the elevation of SAG in the nucleus at G2/M
might contribute to the selective activation of PKC-ßII seen during cell
cycle we tested the ability of SAG to activate PKC isoenzymes using an in
vitro kinase assay and recombinant human PKC isoenzymes. The results
(Fig. 4) show that SAG could
activate classical PKCs in vitro, with PKC- responding as well as
PKC-ßII to SAG. In addition, SAG was found to be less efficient in the
activation of the novel PKC isoenzyme PKC-
(Fig. 4). SAG is therefore a
potent activator of PKC and shows some specificity for classical PKC
isoenzymes.
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Discussion |
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The data reported here address the question of selective activation of PKC
isoenzymes during cell cycle and are novel in several respects. Unlike the
majority of reports in the literature concerning cell cycle changes in lipid
generation (Divecha et al.,
1997) or PKC activation (Goss
et al., 1994
), we have not used pharmacological agents to arrest
cells at a particular stage of the cell cycle. Such studies have synchronised
proliferating cells either by serum starvation, or with agents such as
hydroxyurea, aphidocolin or nocodazole. These extreme methods will almost
certainly affect cell processes, including signalling pathways. More
importantly, following release from growth arrest, cells do not remain
synchronised and subtle changes in lipid generation during each cell cycle
stage could be missed due to lack of synchronisation. Therefore in the studies
reported here, cells were separated into the different stages of the cell
cycle by centrifugal elutriation, which causes minimal cellular perturbation.
In addition, DAG mass and individual DAG molecular species were analysed in
whole cells and nuclei isolated from cells at G1, S and
G2/M. The data reveal distinct patterns of DAG species generation
and PKC isoenzyme activation through the cell cycle.
Our results show that PKC-ßII was most active in the G2/M
phase, accompanied by increased nuclear localisation. These observations
confirm the findings of Fields and co-workers, who have reported the
association of PKC-ßII with the nucleus at G2/M
(Thompson and Fields, 1996)
and its role in mitosis as a mitotic lamin kinase
(Goss et al., 1994
). Their
studies were performed on cells that were synchronised by treatment with
aphidocolin and then harvested at regular intervals to assess nuclear
localisation of PKC-ßII. By the time the population taken to be
G2/M were harvested 8 hours later, the cells were no longer
synchronised. Although their G2/M fraction contained approximately
50% of cells in G2/M, as assessed by FACS analysis, a significant
proportion of cells were in S phase
(Hocevar and Fields, 1991
).
However, the data reported here show that although PKC-ßII showed
increased membrane association during S phase, significant translocation to
the nucleus only occurred during G2/M.
Although the studies of Fields and co-workers have determined the
involvement of PKC-ßII in the regulation of cell cycle, they have not
identified mechanisms to fully explain the differential activation and nuclear
translocation of this isoenzyme during cell cycle. The process of selective
activation of PKC isoenzymes, eight of which are responsive to the
physiological activator diacylglycerol, is fundamental to the PKC signalling
pathway, but remains poorly understood. Murray and Fields identified a nuclear
factor that stimulated PKC-ßII activity to levels 3-6 times greater than
those achieved by optimal concentrations of calcium, DAG and PS
(Murray et al., 1994) and have
shown subsequently that this factor is phosphatidylglycerol, PG
(Murray and Fields, 1998
).
However, levels of PG at the nuclear membrane do not change through the cell
cycle and are thus insufficient alone to mediate the selective nuclear
targeting and activation of PKC-ßII during G2/M. Our data
suggest that activation of PKC-ßII through cell cycle may also involve
the generation of polyunsaturated DAG species, specifically 1-stearoyl,
2-arachidonyl glycerol. DAG species rich in stearate and arachidonate indicate
phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2
(Divecha et al., 1991
) and a
nuclear PI-PLC activity has been identified that is active during
G2 (Sun et al.,
1997
). Moreover, inhibition of nuclear PI-PLC led to a decrease in
nuclear DAG and cell cycle arrest in G2
(Sun et al., 1997
). SAG
generated at the nucleus is therefore likely to arise from the hydrolysis of
PtdIns(4,5)P2. Although the increase in tetraunsaturated
species at G2/M were modest, we propose that SAG acting together
with PG, could mediate the targeting of PKC-ßII to the nucleus at
G2/M. Increases in nuclear polyunsaturated DAG during
G2/M have also been observed in the human keratinocyte Hakat cell
line (T.R.P., unpublished), demonstrating that this phenomenon is reproducible
and not unique to the hematopoietic cells.
In an attempt to pick out the signalling DAG from the nonsignalling
background we applied the methodology of D'Santos et al.
(D'Santos et al., 1999), which
uses the endogenous nuclear DAGK to specifically
[32P]-phosphorylate the nuclear DAG, the [32P]-PA can
then be analysed by silver nitrate TLC. A short (5 minute) incubation might be
expected to phosphorylate the signalling DAG at a faster rate than
nonsignalling DAG since, in vivo, one or more DAGKs must have the capacity to
rapidly and selectively phosphorylate signalling DAG and thus terminate DAG
signalling. Unfortunately, while exogenous DAG could be phosphorylated by the
endogenous nuclear DAGK, indicating an active kinase, only trace amounts of
endogenous nuclear DAG were phosphorylated, far too little for species
analysis (data not shown). This suggests differences in nuclear DAGK activity
and/or regulation between the U937 cells used here and the murine
erythroleukemia (MEL) cells used in the D'Santos work. A complementary LC-MS
approach to investigate nuclear PA species changes failed to detect
reproducible differences over the cell-cycle; however, changes may not be
detectable since our earlier work suggested that polyunsaturated PA formed
from signalling polyunsaturated DAG may be metabolised more rapidly than the
PA derived from other sources, thereby preventing accumulation of signalling
DAG-derived PA (Pettitt et al.,
1997
). An alternative explanation is that the DAG may be
metabolised in the nuclei through a different pathway, for example the action
of CTP:phosphocholine cytidyltransferase activity directly generating PC. The
consequence of this would be that the nuclear DAG is not available for
phosphorylation.
Current models of classical PKC isoenzyme activation suggest that
association with membranes induces clustering of acidic phospholipids such as
PS and PG (Bazzi and Nelsestuen,
1991) and the role of DAG is to increase affinity of PKC for the
membrane (Murray and Fields,
1998
). The role of diacylglycerol in the activation of novel PKCs
is less clear, as recent studies have shown that DAG enhanced association of
PKC-
with PS-containing micelles, but had a much lesser effect on
PKC-
(Medkova and Cho,
1998
). The inability of SAG to induce membrane association of
PKC-
, or significant activation in vitro, would concur with these data
and support the suggestion that DAG may not be a key activator of the novel
PKCs in vivo (Medkova and Cho,
1998
). As SAG was able to activate PKC-
in vitro,
generation of this DAG alone would not be expected to produce the selective
translocation and sustained activation of PKC-ßII seen during
G2/M. However, if our results are considered together with those of
Murray and Fields (Murray and Fields,
1998
), we can propose a model in which increased generation of SAG
at the nucleus during G2/M induces the initial association of
PKC-ßII with the nuclear membrane, followed by clustering of PG that then
causes firm PKC binding and enhanced activation. Targeting of PKC isoenzymes
to distinct intracellular sites is crucial for their role in cell regulation
and the translocation and docking mechanisms involved appear to vary. PG,
working in concert with SAG, may thus represent a lipid membrane anchor for
PKC-ßII at the nucleus.
In conclusion, we propose that the generation of different DAG molecular species at specific sites within the cell contributes to specific PKC isoenzyme activation and translocation during cell cycle and possibly other cell processes influenced by PKC. Activation by DAG coupled with association with isoenzyme-specific docking elements, such as PG, will add a further level of selectivity to PKC activation.
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Acknowledgments |
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