From the Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
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ABSTRACT |
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Diacylglycerol kinase (DGK) phosphorylates the
second messenger diacylglycerol to yield phosphatidic acid. To date,
very little is known about the regulation of DGK activity. We have
previously identified the DGK Diacylglycerol kinase
(DGK)1 phosphorylates the
second messenger diacylglycerol (DAG) to yield phosphatidic acid (1).
Because DAG is a physiological activator of protein kinase C (PKC), DGK may act to attenuate PKC activation in response to external stimuli (1-3). To date, nine mammalian DGK isotypes have been cloned (excluding alternatively spliced variants), but surprisingly little is
known about the regulation of these isotypes (for review, see Refs. 4
and 5).
Rho family GTPases (RhoA, Rac, and Cdc42) regulate vital cellular
functions, particularly cytoskeletal reorganization and gene
transcription (6, 7). These small GTPases regulate not only protein
kinases (7) but also lipid-metabolizing enzymes, such as phospholipase
D (PLD) (8) and phosphatidylinositol 5- and 3-kinases (6, 7, 9). In
addition, and interestingly enough, the Rac GTPase has been reported to
form a functional complex with an unidentified DGK in vivo
(10).
We have recently cloned the cDNA of a new DGK isotype, termed
DGK Cells and Plasmids--
COS7-M6 and N1E-115 cells were grown in
Dulbecco's modified Eagle's medium with 8% fetal calf serum and
antibiotics. Cell transfections were all performed with pMT2 expression
vectors using the DEAE-dextran method. Two days after transfection,
cells were used for experiments. DGK Antibodies and Immunofluorescence--
P5D4 monoclonal antibody
directed against VSV-tag was used for Western blotting and
immunoprecipitation of VSV-tagged DGK
For immunofluorescence, cells were fixed in 3.7% formaldehyde in PBS,
permeabilized with PBS containing 0.1% Triton X-100, and subsequently
blocked with 1% bovine serum albumin in PBS for 30 min. Cells were
then stained with primary DGK Cell Fractionation--
Cells were disrupted by sonication in
Hepes (50 mM, pH 8), sucrose (250 mM),
supplemented with protease inhibitors. Cell debris and nuclei were
removed by centrifugation (14,000 × g, 10 min). The
supernatant was centrifuged at 100,000 × g for 1 h to separate cytosol (supernatant) from a pellet (particulate)
fraction consisting of membranes and cytoskeleton. The pellet was
resuspended in 1% Nonidet P-40 lysis buffer and centrifuged
(100,000 × g, 1 h). The supernatant contains
dissolved membranes, and the pellet contains the cytoskeleton.
GST-RhoA Binding and DGK Activity Assay--
N1E-115 cells were
lysed in a buffer containing 150 mM NaCl, 50 mM
Tris-HCl (pH 8.0), 5 mM MgCl2, 1% Nonidet P-40
and protease inhibitors, and incubated with purified GST-fusion protein
(10 µg) at 4 °C. GST-fusion proteins were then collected with
Glutathione-Sepharose beads. The beads were washed four times with
lysis buffer and subjected to SDS-polyacrylamide gel electrophoresis
and immunoblotting with anti-DGK
DGK activity assays were performed as described (11). The enzymatic
product, phosphatidic acid (PA), was separated by TLC using the solvent
system chloroform/methanol/acetic acid (65:15:5, v/v/v).
We first tested whether DGK isotype, which is predominantly
expressed in brain (Houssa, B., Schaap, D., van der Wal, J., Goto, K.,
Kondo, H., Yamakawa, A., Shibata, M., Takenawa, T., and Van
Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422-10428). We now report that DGK
binds specifically to
activated RhoA in transfected COS cells as well as in nontransfected
neuronal N1E-115 cells. Binding is abolished by a point mutation (Y34N)
in the effector loop of RhoA. DGK
does not bind to inactive RhoA,
nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA,
DGK
localizes to the plasma membrane. Strikingly, the binding of
activated RhoA to DGK
completely inhibits DGK catalytic activity.
Our results suggest that DGK
is a downstream effector of RhoA and
that its activity is negatively regulated by RhoA. Through accumulation
of newly produced diacylglycerol, RhoA-mediated inhibition of DGK
may lead to enhanced PKC activity in response to external stimuli.
INTRODUCTION
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Abstract
Introduction
References
(11), and have now considered the possibility that this DGK
might be regulated by Rho GTPases. We report here that DGK
specifically binds to active RhoA but not to Rac or Cdc42. Most strikingly, and unlike other RhoA effectors, DGK
loses catalytic activity when it binds to RhoA.
EXPERIMENTAL PROCEDURES
cDNA was VSV-tagged at the
3'-end. Myc-tagged RhoA, Rac1, and Cdc42 constructs were described
previously (12). Point mutations were introduced into V14-RhoA cDNA
by using QuickchangeTM site-directed mutagenesis kit from
Stratagene and checked by DNA sequencing.
. 9E10 monoclonal antibody
directed against Myc-tag was used for Western blotting and
immunoprecipitation of Myc-tagged small GTP binding proteins.
Anti-DGK
polyclonal antibody #101 was raised against a synthetic
peptide corresponding to the stretch of amino acids 312 to 331 in the
DGK
primary sequence.
antibody #101 and secondary
goat-anti-rabbit antibody conjugated to Texas Red (Molecular Probes
Inc.), each used at 1:100 dilution. After washing, cells were mounted
with Vectashield, and fluorescence was analyzed on a Bio-Rad confocal
microscope (MRC-600).
polyclonal antibody #101.
RESULTS AND DISCUSSION
can bind to Rho family members. To
this end, DGK
was VSV-epitope-tagged and transfected into COS7 cells
together with expression vectors encoding various forms of
Myc-epitope-tagged RhoA, Cdc42, or Rac1. Fig.
1A shows that DGK
co-immunoprecipitates with (wild-type) RhoA but not with Rac or Cdc42.
Wild-type RhoA, when expressed in COS7 cells, is partially active (GTP
bound) as evidenced by its binding to a downstream effector, Rho
kinase.2 To assess how DGK
binding depends on the activation state of RhoA, we used constitutively
activated and inactive versions of RhoA. Fig. 1B shows that
DGK
co-immunoprecipitates with active V14-RhoA but not with inactive
N19-RhoA. Furthermore, it is seen that DGK
fails to co-precipitate
with constitutively active Rac1 (V12-Rac) or Cdc42 (V12-Cdc42). This
argues against DGK
being the unidentified DGK that interacts with
Rac (10).
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Fig. 1.
Specific binding of DGK
to active RhoA in vivo. A, COS7
cells were co-transfected with DGK
(VSV-tagged) and either RhoA,
Rac, or Cdc42 (wild-type; Myc-tagged). Cells were lysed (1% Nonidet
P-40), and the small G proteins were immunoprecipitated with anti-Myc
monoclonal antibody 9E10. Co-immunoprecipitated DGK
was visualized
(upper panel) by Western blotting. B, same
experiment but using the constitutively active mutants V14-RhoA,
V12-Rac, and V12-Cdc42 and the inactive mutant N19-RhoA (all
Myc-tagged). Lower panels show expression controls (total
cell lysates). Positions of Myc-RhoA, Myc-Rac, and Myc-Cdc42 (small G
proteins) and VSV-DGK
in the gel are indicated
(arrowheads). C, endogenous DGK
from N1E-115
neuroblastoma cells binds to active RhoA. GST fusion proteins of active
V14-RhoA or inactive N19-RhoA or GST alone were incubated with N1E-115
cell lysate and then collected through binding to glutathione-Sepharose
beads. Bound DGK
was detected by Western blotting. D,
RhoA effector loop mutation Y34N, but not T37A, abrogates binding to
DGK
; same experimental conditions as in panel
A.
We next examined RhoA binding to endogenous DGK in nontransfected
cells. DGK
is predominantly expressed in brain and neuronal cell
lines, such as N1E-115 neuroblastoma cells (11). Purified GST-RhoA
fusion proteins, immobilized on glutathione-Sepharose beads, were
incubated with N1E-115 cell lysates, and RhoA-bound DGK
was assayed
by Western blotting using a polyclonal antibody against DGK
. As
shown in Fig. 1C, DGK
is pulled down by GST-V14-RhoA but
not with GST-N19-RhoA nor with free GST. The GTP dependence of the
binding was confirmed in a similar experiment, using GST-wild-type RhoA
fusion protein loaded with GTP
S versus GDP
S (data not shown).
We conclude that DGK binds to RhoA in a GTP-dependent
manner but not to other members of the Rho family, suggesting that DGK
is a downstream effector of RhoA. RhoA target molecules bind to
the effector-loop region of RhoA (13). Specific point mutations in this
region have been shown to interfere with effector binding (8, 13-15).
For example, mutations in conserved amino acids Tyr-34 and Thr-37 are
known to abolish binding and activation of PLD (8) and protein kinase N
(14). We therefore mutated these residues in active V14-RhoA to yield
V14/N34- and V14/A37-RhoA and tested whether these mutations affect the
binding of DGK
. Fig. 1D shows that the Y34N mutation
disrupts the binding of V14-RhoA to DGK
, whereas the T37A mutation
has no effect. This suggests that the RhoA-DGK
interaction is
mediated by the RhoA effector loop, in which residue Tyr-34 is critical
for binding.
Activated RhoA is known to localize to the plasma membrane, whereas
inactive GDP-bound RhoA is largely cytosolic (15-18). We investigated
DGK localization by immunofluorescence and cell fractionation
studies. We used DGK
-transfected COS7 cells because the level of
endogenous DGK
in N1E-115 cells was too low to allow proper
detection. Fractionation of cell lysates revealed that the majority of
DGK
is present in the membrane fraction (Fig. 2A). The remainder is found in
the cytosolic and the cytoskeleton fractions. Immunofluorescence
analysis shows the presence of DGK
at the cell periphery (Fig.
2B). In addition, DGK
is present in the cytoplasm and the
perinuclear region, as usual for an overexpression system. A similar
subcellular distribution has been reported for overexpressed RhoA (17).
The presence of DGK
at the cell periphery supports a model in which
DGK
is regulated by RhoA-GTP at the inner side of the plasma
membrane, where the DGK substrate DAG is generated following cell
activation.
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We next investigated how activated RhoA may affect DGK activity.
Epitope-tagged versions of DGK
and V14-RhoA were co-expressed in
COS7 cells, and DGK
was immunoprecipitated either directly, using
anti-VSV monoclonal antibody P5D4, or indirectly, through co-precipitation with RhoA using anti-myc monoclonal antibody 9E10. The
catalytic activity of both pools of DGK
was then tested in an
in vitro kinase assay using
1,2-dioleoyl-sn-glycerol as a substrate. As shown in Fig.
3A, RhoA-bound DGK
is
completely inactive, whereas free DGK
(precipitated with P5D4) is
highly active. Likewise, when bound to active GST-V14-RhoA, endogenous DGK
, pulled down from N1E-115 cell lysates, was completely inactive, in contrast to free DGK
(Fig. 3B). From these results, we
conclude that DGK
is catalytically inactive when physically
associated with active RhoA. In other words, RhoA is a negative
regulator of DGK
activity.
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Contrary to its inhibitory action on DGK, RhoA has been reported to
stimulate the activity of PLD (8, 18). Both DGK and PLD generate PA,
albeit from different lipid sources (DAG and phosphatidylcholine,
respectively). How can one explain the significance of one PA-producing
enzyme (PLD) being activated and the other (DGK) inactivated by RhoA? A
likely possibility is that PLD and DGK and their respective PA products
serve different cellular functions (19) and that these enzymes, their
lipid substrates, and PA products may be located in distinct, spatially separated compartments within the cell. Consistent with this, the PA
pools generated by PLD and DGK differ in fatty acid composition (19,
20), and DGK in stimulated cells does not phosphorylate DAG generated
by sequential PLD/PA phosphohydrolase activities (20, 21) nor DAG that
is randomly generated in the plasma membrane by exogenous phospholipase
C (22).
An important implication of DGK inhibition is that RhoA should be
able to regulate DAG levels and, hence, PKC activity. Interestingly, activated RhoA has recently been reported to associate with PKC
in vivo, important for membrane translocation and activation
of PKC
(15, 23). Similar conclusions have been reached for activated Rho1 and Pkc1 in yeast (24, 25). Combined with these data, our present
results would suggest that RhoA may promote activation of PKC through a
concerted positive action on PKC (at least PKC
) directly and a
negative action on DGK
. Whether RhoA proteins act in larger
signaling complexes in association with both DGK
and PKC, and
whether other DGK isotypes are similarly regulated by Rho family
members, remains to be investigated.
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FOOTNOTES |
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* This work was supported by a grant from the Netherlands Organization for Scientific Research (SON 330-210) and by the Dutch Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: +31-20-5121989;
E-mail: wblit{at}nki.nl.
2 O. Kranenburg, M. Poland, F. P. G. van Horck, and W. H. Moolenaar, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
DGK, diacylglycerol
kinase;
DAG, diacylglycerol;
PKC, protein kinase C;
PLD, phospholipase
D;
VSV, vesicular stomatitis virus;
PBS, phosphate-buffered saline;
GST, glutathione S-transferase;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
GDP
S, guanyl-5'-yl
thiophosphate;
PA, phosphatidic acid.
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REFERENCES |
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