From the Sealy Center for Oncology and Hematology and
the Departments of § Human Biological Chemistry and Genetics
and ¶ Pharmacology, University of Texas Medical Branch,
Galveston, Texas 77555-1048
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ABSTRACT |
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A major mechanism by which protein kinase C (PKC)
function is regulated is through the selective targeting and activation of individual PKC isotypes at distinct subcellular locations. PKC
II is selectively activated at the nucleus during
G2 phase of cell cycle where it is required for entry into
mitosis. Selective nuclear activation of PKC
II is
conferred by molecular determinants within the carboxyl-terminal
catalytic domain of the kinase (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 previously described a
lipid-like PKC activator in nuclear membranes, termed nuclear membrane
activation factor (NMAF), that potently stimulates PKC
II activity through interactions involving this domain
(Murray, N. R., Burns, D. J., and Fields, A. P. (1994)
J. Biol. Chem. 269, 21385-21390). We have now
identified NMAF as phosphatidylglycerol (PG), based on several lines of
evidence. First, NMAF cofractionates with PG as a single peak of
activity through multiple chromatographic separations and exhibits
phospholipase sensitivity identical to that of PG. Second, purified PG,
but not other phospholipids, exhibits dose-dependent NMAF
activity. Third, defined molecular species of PG exhibit different
abilities to stimulate PKC
II activity. 1,2-Dioleoyl-PG
possesses significantly higher activity than other PG species,
suggesting that both fatty acid side chain composition and the glycerol
head group are important determinants for activity. Fourth, in
vitro binding studies demonstrate that PG binds to the
carboxyl-terminal region of PKC
II, the same region we
previously implicated in NMAF-mediated activation of PKC
II. Taken together, our results indicate that specific
molecular species of nuclear PG function to physiologically regulate
PKC
II activity at the nucleus.
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INTRODUCTION |
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Protein kinase C (PKC)1 is a family of serine/threonine kinases involved in the transmission of a wide variety of extracellular signals (1, 2). The individual PKC family members are classified according to their cofactor requirements (3, 4). Classical, or calcium-dependent PKC isotypes require calcium, diacylglycerol (DAG), and phosphatidylserine (PS) for activation. The novel PKCs require DAG and PS, but not calcium, whereas the atypical PKCs do not require DAG or calcium, but appear to require PS for activation. PKC isotypes exhibit tissue- and cell type-specific patterns of expression, suggesting specialization of function. Indeed, accumulating evidence indicates that PKC isotypes serve distinct, nonoverlapping functions in cellular physiology (reviewed in Refs. 1, 3, and 5).
An important mechanism by which PKC function is regulated is through
the targeting of PKC isozymes to distinct subcellular locations (1, 5).
In human leukemia cells, which express PKC ,
II, and
, we have found that PKC
II is selectively activated at the nucleus during the G2 phase of cell cycle (6, 7). At
the nucleus, PKC
II directly phosphorylates the nuclear
envelope polypeptide lamin B at sites involved in mitotic nuclear
lamina disassembly (8-10). Inhibition of nuclear PKC
II
activity leads to cell cycle arrest in G2 phase,
demonstrating the importance of nuclear PKC
II
activation in the entry of cells into mitosis (7). In contrast, PKC
and PKC
are not observed at the nucleus, and we have demonstrated
that they play key roles in leukemia cell differentiation and
survival/apoptosis, respectively (11, 12).
Given its involvement in cell cycle progression, we investigated the
mechanisms underlying the selective nuclear translocation and
activation of PKC II. Using chimeric PKC molecules,
produced by exchanging the regulatory and catalytic domains of PKC
and
II, we determined that the catalytic domain of PKC
II contains molecular determinants that are important
for selective nuclear targeting of the enzyme (13). In related
biochemical studies, we examined the mechanism by which PKC
II is activated at the nucleus (14). We found that
component(s) within the nuclear membrane selectively stimulate PKC
II activity 3-6-fold above the level achieved in the
presence of optimal concentrations of calcium, DAG, and PS (14). This
nuclear membrane activation factor (NMAF) was shown to be soluble in
nonionic detergents and organic solvents, and to be insensitive to
protease treatment, suggesting that it is a lipid (14). In the present
study, we identify NMAF as phosphatidylglycerol (PG) based on the
fractionation profile of NMAF and the ability of purified PG to
activate PKC
II. Interestingly, individual purified PG
species vary in their ability to activate PKC
II,
suggesting that the nuclear membrane contains specific PG species that
serve to potently stimulate PKC
II activity. Finally, we
demonstrate that PG binds to the carboxyl-terminal region of PKC
II, consistent with the role of this region in nuclear
activation of PKC
II (14). Our data indicate that
nuclear membrane PG is an important physiologic regulator of PKC
activity at the nucleus.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Isolation of Nuclear Membrane Extracts-- Human promyelocytic (HL60) leukemia cells were maintained in suspension culture in Iscove's medium supplemented with 10% calf serum as described previously (7, 14). Nuclear envelopes were prepared as described previously (7) and subjected to two-phase extraction in chloroform/methanol/water (8:4:3) to separate lipids from nonlipid constituents (15). The organic (lower) phase was isolated and dried under N2 gas for subsequent analysis.
Thin Layer and Silica Column Chromatography of Nuclear Membrane
Extracts--
Preparative TLC was used to fractionate nuclear membrane
extracts into major lipid classes based on the migration of purified lipid standards (Avanti Polar Lipids). Nuclear membrane extracts, isolated as described above, were resuspended in chloroform, spotted on
Diamond K6F Silica Gel 60 TLC plates (Whatman) and developed in
chloroform/methanol/water (65:25:4). Developed plates were air-dried,
and the migration of phospholipid standards was determined by staining
with 0.1% 8-anilino-1-naphthalene sulfonic acid ammonium salt (Sigma)
in water, pH 7.0, and visualized under short wave UV light (16). The
TLC plates were partitioned based on the migration of purified
standards, and the silica from each section was scraped from the plate
and recovered. Phospholipids from each section were eluted into
chloroform/methanol (3:1), and the resulting extracts were dried under
N2 gas and stored at 20 °C until analyzed for activity
as described below.
In Vitro PKC Activity Assay--
The ability of nuclear membrane
extracts, column fractions, and purified phospholipids to stimulate PKC
II activity was assessed in a standard in
vitro PKC assay (14) using purified recombinant baculovirus-expressed human PKC
II as the source of
enzyme (8). Briefly, PKC activity was measured by following
incorporation of [32P]phosphate from
[
-32P]ATP into histone H1 (Sigma). Reactions were
performed under optimal activating conditions (100 µM
CaCl2, 20 µM dioleoylglycerol, 40 µg/ml PS,
and 10 µg of histone H1) in the absence and presence of nuclear
membrane extracts, column fractions, or purified phospholipids. All
assays were carried out for 15 min at room temperature, conditions under which [32P]phosphate incorporation is linear (14).
Unless otherwise stated, nuclear membrane extract or column fractions
from 107 cell equivalents were assayed in each reaction.
The kinase reactions were terminated by addition of SDS sample buffer
and the samples boiled for 5 min prior to SDS-polyacrylamide gel
electrophoresis analysis. Histone phosphorylation was quantitated by
phosphorimaging analysis of the gels as described previously (14).
Phospholipase Digestion of Nuclear Membrane Extracts and Purified
Phosphatidylglycerol--
The sensitivity of nuclear membrane extracts
and purified PG to lipase digestion was assessed using
phosphatidylinositol-specific phospholipase C (PI-PLC)
(Sigma), phosphatidylcholine (PC)-PLC (Calbiochem), and
phospholipase A2 (PLA2) (Calbiochem). Nuclear membranes or purified PG were suspended in appropriate buffers for the
individual lipases (PI-PLC: 10 mM Tris, pH 7.4, 144 mM NaCl, 0.02% bovine serum albumin, 100 µM
CaCl2; PC-PLC: 6 mM imidazole, pH 7.4, 150 mM NaCl, 1 mM CaCl2;
PLA2: 50 mM Tris, pH 7.4, 10 mM
CaCl2, 100 mM KCl) along with 4-5 units of
lipase activity and the mixture incubated at 37 °C for 30 min
(17-19). Reactions were terminated by addition of chloroform and
methanol in the ratio of 8:4:3 (chloroform/methanol/digestion mixture).
After two-phase extraction, the organic phase was isolated, dried under N2 gas, and stored at 20 °C until analysis.
Quantitative Measurement of PG Mass-- The amount of PG in nuclear membrane extracts and column fractions was assessed using the specific, enzymatic PG assay described by Jones and Ashwood (20). Briefly, membrane extracts or column fractions were incubated with phospholipase D, glycerokinase, and glycerol-3-phosphate oxidase to generate hydrogen peroxide from PG. Hydrogen peroxide formed in this step was quantitated after addition of 4-aminoantipyrine, 3,5-dichloro-2-hydroxybenzenesulfonic acid, and peroxidase, by following generation of red chromogen at its absorption maximum of 510 nm. A blank was generated for each unknown using the same reaction conditions in the absence of phospholipase D. Absorbance is directly proportional to the quantity of PG in the sample. The total mass of PG in each sample was determined using a standard curve generated with purified PG (Avanti Polar Lipids). Specificity of the assay was confirmed using other purified phospholipid classes, none of which gave absorbance above background at 100 µg/ml phospholipid.
Lipid Vesicle Binding Assay--
A fusion protein between
glutathione S-transferase (GST) and the carboxyl terminus of
PKC II (amino acids 576-673; GST-PKC
II
CT) was generated by polymerase chain reaction of the carboxyl-terminal fragment of the human PKC
II cDNA using the
following primers: forward 5'-CGGGATCCCACTGATGACCAAACACC-3' and reverse
5'-CCCTCGAGGATTAGCTCTTGACTTCG-3'. These primers allowed introduction of
a 5'-BamHI and a 3'-XhoI restriction site to
facilitate directional cloning of the polymerase chain reaction product
into the pGEX-5X-3 expression vector (Amersham Pharmacia Biotech). GST
fusion protein was expressed in Escherichia coli and
purified on glutathione affinity columns according to the
manufacturer's protocol (GST gene fusion system, Amersham Pharmacia
Biotech). Purified GST or GST-PKC
II CT was mixed with sonicated lipid vesicles (250 µg of lipid) or buffer alone (50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM
MgSO4, and 0.5 mM CaCl2) for 15 s and then filtered through 100-kDa molecular mass cutoff
filters (Amicon) according to Rebecchi et al. (21). Lipid
vesicles contained dioleoyl-PG, PC, or both as indicated in the figure
legend. In some instances, PS and DAG (20 and 8 mol %, respectively)
were included in place of PC as indicated in the text. Lipid vesicles were quantitatively retained by the filter as determined by TLC analysis. Filtrate (unbound) and bound fractions were resolved by
SDS-polyacrylamide gel electrophoresis and the presence of GST fusion
protein determined by immunoblot analysis with anti-PKC
II (Santa Cruz) and anti-GST antibody (PharMingen) and
chemiluminescence. Quantification was by densitometric analysis of the
developed films.
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RESULTS |
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Initial Characterization of Nuclear Membrane Activation
Factor--
Based on our initial observation that NMAF activity is
solubilized from nuclear envelopes by either nonionic detergent or organic solvent extraction, we hypothesized that NMAF may be a lipid
(14). Further characterization demonstrated that NMAF activity
fractionated completely into the organic phase of a two-phase (Folch)
extraction and that NMAF activity is largely resistant to heat
inactivation by boiling for 5 min, supporting the hypothesis that NMAF
is lipid and not protein. In addition, NMAF exhibits the same
stimulatory effect on PKC II activity in both our
standard vesicle assay and a mixed micelle assay (22), indicating that NMAF activity is not the result of an artifact inherent to the assay
system (data not shown). These data, along with our previous observation that NMAF activity is resistant to exhaustive protease treatment (14), provide convincing evidence that NMAF is lipid and not
protein.
Thin Layer Chromatographic Fractionation of NMAF--
In order to
characterize the lipid component(s) of nuclear membranes responsible
for NMAF activity, nuclear membrane extracts were resolved into major
lipid classes by TLC. TLC plates were spotted with nuclear membrane
extract in chloroform and developed using a solvent system that allows
resolution of the major phospholipid classes. The developed TLC plates
were then divided into regions based on the migration of lipid
standards, scraped, and eluted as described under "Experimental
Procedures." Individual TLC fractions were then assayed for NMAF as
measured by the ability to stimulate PKC II activity as
described previously (14). NMAF was quantitatively recovered as a
single peak of activity in two consecutive fractions (F5 and F6) from
the TLC plate (Fig. 1). Comparison of the
migration of NMAF activity with purified phospholipid standards reveals that NMAF comigrates with several major phospholipid classes. Specifically, PG, PC, and phosphatidylethanolamine (PE) all migrated within the region of the TLC containing NMAF activity. In contrast, NMAF activity was completely resolved from other major phospholipid classes, including phosphatidic acid, PS, phosphatidylinositol (PI),
and lysophospholipids, and from the neutral lipid metabolite DAG.
These data suggest that NMAF may correspond to PG, PC, or PE.
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Ability of Individual Phospholipids to Activate PKC
II--
Our previous studies indicated that NMAF is not
PS or DAG, since NMAF activates PKC
II in the presence
of maximal concentrations of these cofactors (14). Substantiating this
finding, PS and DAG are clearly resolved from NMAF activity after TLC
separation (Fig. 1). The migration of NMAF on TLC plates suggested that
NMAF might correspond to one of the three phospholipids PG, PC, or PE.
Therefore, we directly determined the ability of these phospholipids to
activate PKC
II (Fig. 2).
As can be seen, PG was able to stimulate PKC
II activity
in a dose-dependent fashion above the level induced by the
conventional PKC activators, DAG, PS, and calcium. In contrast, PC and
PE showed little or no stimulatory activity at concentrations up to 250 µg/ml, demonstrating that the NMAF-like activity exhibited by
phospholipid addition is specific for PG.
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PG Is Present in Nuclear Membrane Extracts and Cofractionates with NMAF Activity-- Given the ability of PG to stimulate PKC activity, we next assessed whether PG is present in nuclear membrane extracts and whether endogenous PG cofractionates with NMAF activity. For this purpose, we subjected nuclear membrane extracts to fractionation by silica column chromatography (Fig. 4A). Samples were loaded onto silica columns in chloroform, and lipid constituents were eluted into solvents of increasing polarity. Individual fractions were collected and assayed for the ability to stimulate PKC activity (Fig. 4A). NMAF activity was retained on the column and eluted as a single peak in 1:1 chloroform/methanol (CHCl3/CH3OH). Purified PG standard also eluted as a single peak in 1:1 CHCl3/CH3OH, further suggesting that PG corresponds to NMAF (data not shown). Therefore, we directly assessed the levels of PG in the silica column fractions from nuclear membrane extracts using a specific enzymatic assay for PG mass (20). Nuclear membrane PG elutes specifically in 1:1 CHCl3/CH3OH along with NMAF activity (Fig. 4B). From the quantitative PG assay, we calculate that nuclear membranes contain ~0.39 µg of PG/107 cells. This level of PG corresponds to a concentration of ~10 µg/ml in our standard assay. These data confirm the presence of PG in the nuclear membrane and demonstrate that nuclear PG comigrates with NMAF activity.
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Individual Species of PG Differ in Ability to Activate PKC
II--
Having identified NMAF as PG, we wished to
determine whether the fatty acid side chain composition of individual
PG species is important for PKC stimulatory activity. Therefore, we
assessed the ability of individual purified molecular species of PG to activate PKC
II (Fig. 5).
The PG preparation used in the studies shown in Figs. 2 and 3 is a
mixture of PG species with a complex distribution of fatty acid side
chain constituents (Avanti Polar Lipids catalog). The two predominant
side chain constituents of this PG mixture are C16:0 palmitic acid
(34%) and C18:1 oleic acid (31%). Therefore, we compared the ability
of defined PG species containing these two fatty acid side chain
constituents to stimulate PKC
II activity (Fig.
5A). 1,2-Dipalmitoyl-PG and the mixed fatty acid species
1-palmitoyl-2-oleoyl PG exhibited activities comparable with or weaker
than the original mixture. In contrast, 1,2-dioleoyl-PG was more
potent, exhibiting about 1 log higher activity than the original
mixture.
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The Carboxyl-terminal Region of PKC II Selectively
Binds PG--
Our previous results demonstrated that NMAF-mediated
activation of PKC
II is isozyme selective and suggested
that the carboxyl-terminal region of PKC
II is important
for this activation (14). Therefore we devised a lipid vesicle binding
assay to directly assess whether the carboxyl-terminal region of PKC
II binds to PG. In this assay, PG vesicles were
incubated with a GST fusion protein containing the carboxyl terminus of
PKC
II (GST-PKC
II CT). Vesicle-bound protein was separated from unbound protein by centrifugation through a
100-kDa cutoff filter and the bound fraction analyzed for the presence
of the GST fusion protein (Fig. 7). Under
these conditions, GST-
II CT bound to PG-containing
vesicles (Fig. 7A). Binding was selective for GST-PKC
II CT, since little or no binding of GST to
PG-containing vesicles was detected (Fig. 7B). Likewise, binding of GST-PKC
II CT was selective for PG, since PC
vesicles did not bind GST-PKC
II CT (Fig. 7A, lane
3). Binding to PG vesicles was dependent on the PG content of
mixed vesicles containing different proportions of PG and PC,
indicating that binding is both concentration-dependent and
saturable (Fig. 7C). The presence of PS and DAG did not
influence binding to PG (data not shown), consistent with the fact that PS and DAG bind to the C2 region within the regulatory domain of PKC
II, which is not present in the GST-PKC
II CT fusion protein. In conclusion, our data
demonstrate that PG binds selectively to the carboxyl-terminal region
of PKC
II. These data are consistent with our finding
that this region of PKC
II contains important determinants involved in the nuclear translocation and activation of
PKC
II (13, 14).
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DISCUSSION |
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Protein kinase C function is regulated by multiple mechanisms,
including the tissue- and cell type-specific expression of individual
PKC isotypes. In addition, PKC isotypes exhibit intrinsic differences
in cofactor requirements and sensitivities to lipid second messengers.
Appropriate intracellular targeting of PKC also appears to be critical
for proper PKC isotype function in vivo. Intracellular
targeting can be achieved through specific interactions of PKC with a
growing family of docking proteins. For instance, members of the
protein kinase A anchoring protein family appear to serve a role in
PKC-mediated signaling at the postsynaptic density (23). Protein kinase
A anchoring proteins target PKC and other signaling molecules to the
same intracellular compartment through simultaneous binding to distinct
binding sites on the protein kinase A anchoring protein (23). Still
other PKC-binding proteins serve as receptors for activated protein kinase C and play functional roles in PKC targeting and translocation events (24, 25). It will be of interest to determine whether similar
mechanisms aid in the targeting of PKC II to the nucleus of human leukemia cells. In this paper, we provide direct evidence that
specific lipid components within the nuclear membrane play a key role
in PKC signaling by stimulating PKC activity at the nucleus.
In previous studies, we demonstrated that PKC II is
selectively translocated to the nucleus of human leukemia cells in
response to proliferative stimuli (1, 7-9). Nuclear PKC
II translocation and activation is cell cycle-regulated,
occurring during the G2 phase of cell cycle (6, 7). At the
nucleus, PKC
II phosphorylates sites on the nuclear
envelope polypeptide lamin B that are involved in the process of
mitotic nuclear lamina disassembly (6, 7, 9). Inhibition of nuclear PKC
activity leads to cell cycle arrest in G2 phase prior to
mitosis, demonstrating that nuclear PKC activity is required for cell
cycle progression through the G2/M phase transition (7).
Using PKC chimera, we demonstrated that nuclear translocation of PKC
II is dependent upon the carboxyl-terminal catalytic
domain of the enzyme (13). At the nucleus, PKC
II activity is stimulated by a component within the nuclear membrane, termed NMAF, that serves to potently activate the enzyme (14). An
active component of NMAF has now been identified.
Identification of NMAF as Phosphatidylglycerol--
Based on
several lines of evidence, we have shown that NMAF corresponds to PG.
First, NMAF is a heat-stable, lipophilic activity that comigrates with
PG on thin layer and silica column chromatographies. Second, purified
PG, but not other phospholipids, exhibits NMAF-like activity. Third,
NMAF and PG exhibit similar sensitivities to phospholipases. Fourth, PG
is present in the nuclear membrane in sufficient quantities to
stimulate PKC II activity. Fifth, specific PG species
exhibit different activities indicating that the selectivity of NMAF
activity lies not only in the glycerol head group, but also in the
fatty acid side chain constituents. 1,2-Dioleoyl PG was found to be
significantly more potent at stimulating PKC
II activity
than the other PG species tested. The selectivity for oleic acid is
stereospecific since 1,2-dielaidoyl-PG, which is identical to
1,2-dioleoyl PG except for the orientation of the
9 double bond in
the C18:1 fatty acid chain, exhibits activity that is about one log
lower than 1,2-dioleoyl-PG. These results argue against the possibility
that PG causes a nonspecific membrane effect, such as a change in
membrane charge density, leading to PKC activation. Whereas it is
possible that the nuclear membrane contains other lipid components that
contribute to NMAF activity, we provide convincing evidence that PG has
sufficient activity and is present in appropriate quantities to account
for NMAF activity.
Effects of Acidic Phospholipids on PKC Membrane Binding and Kinase
Activity in Vitro--
A number of studies have demonstrated that the
phospholipid environment of the target membrane can influence PKC
activity in vitro. Utilizing defined vesicle and/or mixed
micelle assays, the requirement for calcium, DAG, and phospholipids in
PKC membrane binding and activation has been investigated. In the
presence of calcium and DAG, PKC exhibits an identical sigmoidal
dependence on PS for membrane binding and activation (27). DAG
increases the affinity of PKC for PS, but not for other acidic
phospholipids (28). Though PE and PG can reduce the amount of PS
required for maximal binding and activity, they cannot replace the
requirement for PS (28). Based on these studies, it has been suggested
that PKC exhibits a dual requirement for acidic phospholipid, a
specific requirement for PS binding and nonspecific electrostatic
interactions with other acidic phospholipids (28). Our data also
indicate that PG, in addition to PS, can influence PKC activity.
However, our data suggest that, like the specific requirement for PS,
the interactions involving PG are specific. Furthermore, our binding studies demonstrate that PG binds to the carboxyl-terminal region of
PKC II, a site distinct from the binding site for PS in
the regulatory domain. In addition, our data indicate that specific PG
species can differentially influence PKC activity, indicating that
specific lipid-PKC interactions underlie the stimulatory effects of
PG.
Phosphatidylglycerol Is a Physiologic Activator of Nuclear PKC
II--
The effects of phospholipid composition and
membrane fluidity on PKC activity have been assessed by selective
enrichment of rat liver membranes with various phospholipids (29).
Addition of PG, PS, PE, or dioleoyl-PC to membrane can lead to enhanced activation of PKC, with PG being the most effective activator (29). Our
data are consistent with these findings and indicate that nuclear
membrane PG is a physiologically relevant regulator of PKC activity.
However, the exact mechanism by which PG stimulates PKC
II activity remains to be fully elucidated. One
potential mechanism stems from the observation that PKC interacts well
with acidic phospholipids (28, 30). Bazzi and Nelsestuen (30) demonstrated that calcium-dependent binding of PKC to
phospholipid vesicles induces clustering of acidic phospholipids
including PG. They suggested that PKC may induce certain acidic
phospholipids to form microdomains within physiologic membranes.
Therefore, the presence of specific phospholipids, such as certain PG
species, may influence PKC activity by forming clustered subdomains
that serve to enhance the membrane binding and catalytic activity of PKC. Likewise this clustering may influence substrate selection through
enhanced interactions of PKC and substrate at the membrane surface.
Future studies will focus on determining the mechanisms by which
nuclear PG stimulates PKC
II activity, the fatty acid side chain composition and PKC stimulatory activity of individual nuclear PG species, and the potential involvement of nuclear PG in the
temporal and spatial regulation of nuclear PKC
II
activity.
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ACKNOWLEDGEMENTS |
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We thank Bin Sun for technical assistance in the construction and expression of GST fusion proteins and Drs. Robert Chapkin and Jon Teng for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA56869 (to A. P. F.).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.
Leukemia Society of America Scholar. To whom correspondence
should be addressed: Sealy Center for Oncology and Hematology, University of Texas Medical Branch, Medical Research Bldg., Rm. 9.104, 301 University Blvd., Galveston, TX 77555-1048. Tel.: 409-747-1940; Fax: 409-747-1938; E-mail: afields{at}marlin.utmb.edu.
1 The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; PS, phosphatidylserine; NMAF, nuclear membrane activation factor; PG, phosphatidylglycerol; PI-PLC, phosphatidylinositol-specific phospholipase C; PC, phosphatidylcholine; PLA2, phospholipase A2; GST, glutathione S-transferase; PE, phosphatidylethanolamine.
2 N. R. Murray and A. P. Fields, unpublished results.
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REFERENCES |
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