Correspondence to: Stephen M. Prescott, The Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84132. Tel:(801) 585-3401 Fax:(801) 585-6345 E-mail:stephen.prescott{at}hci.utah.edu.
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Abstract |
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Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK
also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK
and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK
in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK
, which controls local accumulation of DAG that would otherwise activate RasGRP.
Key Words: diacylglycerols, diacylglycerol kinase, signal transduction, H-Ras oncogenes, RasGRP protein
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Introduction |
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DAG is a lipid second messenger that transiently accumulates after activation of growth factor receptors and other receptors (
RasGRP was identified as a guanine nucleotide exchange factor (GEF) that is specific for Ras (
We demonstrate here that DGK activity inhibits RasGRP. This regulation appears to be selective and spatially discrete: only one of six DGK isotypes, DGK, inhibited RasGRP. Consistent with this regulation occurring in a signaling complex, we observed that DGK
associated with both RasGRP and H-Ras and that it colocalized with RasGRP in a glioblastoma cell line. Additionally, overexpression of a kinase-dead DGK
prolonged Ras activation after ligation of the T cell receptor (TCR) in Jurkat cells, indicating that RasGRP is regulated by DGK
in vivo. This regulation likely occurs through spatial metabolism of signaling DAG and may represent a general mechanism in which a DGK associates with a protein activated by DAG and regulates its activity through its DGK enzymatic function.
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Materials and Methods |
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Expression Plasmids
Wild-type, and V12- and A15-H-Ras in pEF-Myc were a gift from Dr. Andrew Thorburn (University of Utah). Human DGKs ß, , and
in pSRE were a gift from Dr. Fumio Sakane (Sapporo Medical University, Sapporo, Japan). Cloning of human DGKs
,
2, and
has been published previously (
by creating a unique EcoRI site (Quickchange mutagenesis kit; Stratagene) using the oligonucleotide (5'-GAGGACCAGGAGAATTCTGTGTAG-3') and its complement. The FLAG tag was then inserted by digesting the cDNA with EcoRI and then ligating the annealed sense and antisense oligonucleotides encoding the FLAG epitope tag as described previously (
were generated by digesting the above plasmid with EcoRI and either BsaBI (amino acids 1748), HindIII (amino acids 1605), or XhoI (amino acids 1467) and then a FLAG epitope tag was ligated as above. NH2-terminal hemagglutinin (HA) epitope tags were placed onto DGKs ß,
,
,
,
, and
by cloning the full-length DGK into pHA-cytomegalovirus (CMV; CLONTECH Laboratories, Inc.). A plasmid encoding human RasGRP was constructed by combining two EST clones (EMBL/GenBank/DDBJ accession nos.
Z41118 and
AA283882) and a PCR product isolated from A172 cell cDNA using the oligonucleotides 5'-GATGCAGATGGAAACCTGTGTC-3' and 5'-GTGGCTTTGAAGGTGTTAGTGG-3'. The clone was then ligated in frame into the XhoI-HindIIIdigested pEGFP-C3 vector (CLONTECH Laboratories, Inc.) or pHA-CMV (CLONTECH Laboratories, Inc.) digested with NotI. A second HA epitope tag was subsequently ligated into the pHA-CMV construct using oligonucleotides as described above. The C1 domain of RasGRP was removed by digesting green fluorescent protein (GFP)-RasGRP with XcmI and then religating the cDNA, or a point mutation (C506G) was created in its C1 domain using site-directed mutagenesis (Stratagene) with the oligonucleotide 5'-GAAGCCCACTTTTGGTGACAACTGTGC-3' and its complement.
Cell Lines and Transfection
A172, Cos-7, and HEK293 cells were cultured and transfected as described ( using a Gene Pulser (Bio-Rad Laboratories) at 220 V and a capacitance of 960 microfarads in Opti-MEM (Life Technologies). After 20 min of recovery, the cells were transferred to 10 ml growth medium and then assayed at 48 h.
Antibodies and Immunofluorescence
Two peptides (EEFQELVKAKGEELHC and CGVSPKPDPKTISKHVQ) corresponding to human RasGRP were synthesized, conjugated to keyhole limpet hemocyanin, and injected into rabbits. The antibodies were purified from serum using their affinity peptides. Their specificity was verified by Western blotting extracts from cells transfected with HA-RasGRP. Affinity-purified anti-RasGRP (EEFQ) or affinity-purified anti-DGK (
) using protein labeling kits (Molecular Probes).
Indirect immunofluorescent staining of A172 cells was performed as described previously ( (1:100) or anti-RasGRP (EEFQ, 1:200). To stain actin, Texas red phalloidin (1:200; Molecular Probes) was added with the secondary antibody. To verify the specificity of immunostaining, two volumes of an affinity peptide (1 mg/ml) were preincubated with one volume of the antibodies (0.71.0 mg/ml) for >1 h on ice before immunostaining. For direct immunofluorescence to simultaneously detect both RasGRP and DGK
, the same protocol was used, except that the directly conjugated antibodies, each diluted at 1:50, were combined and incubation of the secondary antibody and phalloidin was omitted.
Immunoprecipitation
DGK-FLAG, V12- or A15-H-Ras, HA-RasGRP, or control plasmids were transfected into HEK293 or Cos-7 cells (500 ng each construct). 48 h later, cells were harvested in IP buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM DTT, 1 mM PMSF, 0.5 mM sodium orthovanadate, and 10 mg/ml each leupeptin, pepstatin, aprotinin, and soybean trypsin inhibitor), allowed to lyse for 10 min, and then centrifuged to remove debris. To immunoprecipitate DGK
, 25 µl monoclonal anti-FLAG M2 (Sigma-Aldrich) or normal mouse IgG (Santa Cruz Biotechnology, Inc.) coupled to agarose beads were added to the supernatants. After incubating for 2 h (4°C), the beads were washed with TBSTM (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1% Tween 20, and 5 mM MgCl2) once, IP wash (50 mM Hepes, pH 7.5, 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, 5 mM MgCl2, and 20 mM NaF) three times, and 5 mM MgCl2 in H2O once. The beads were then used for SDS-PAGE. Anti-Ras (C-20; Santa Cruz Biotechnology, Inc.) was used to immunoblot for H-Ras; anti-RasGRP (EEFQ or CGVS) or anti-HA (CLONTECH Laboratories, Inc.) was used to detect RasGRP; and DGK
was detected with an antibody described previously (
Elk-1 Luciferase Assay
PKC inhibitors were from Calbiochem. Elk-1 activity was determined using the Elk-1 luciferase reporter system (Stratagene) according to the manufacturer's instructions with two modifications. First, HA-DGK or human lysosomal acid lipase/cholesterol ester hydrolase (HLAL) (75150 ng), GFP-RasGRP (150 ng), V12-Ras (50 ng), MEK1 (50 ng), Raf:ER (400 ng), or ß-galactosidase (200 ng) cDNA constructs or control vectors were added to experimental points. Second, the HEK293 cells were maintained in medium with 0.5% serum throughout the experiment. Luciferase activity (Promega) was normalized to ß-galactosidase activity (Tropix). Similar results were obtained when luciferase activity was normalized to total protein in the lysates. DGK activity levels and total DAG and lipid phosphate were also determined in appropriate lysates as described previously (
Affinity Precipitation of GTP-Ras from Cell Homogenates
To measure changes in GTP-Ras induced by a DGK in HEK293 cells, the cells were transfected with GFP or GFP-RasGRP (300 ng), myc-Ras (100 ng), and the DGK or a control protein (800 ng) as described ( in Jurkat cells, the cells were transfected as described above and then, after addition of anti-CD3 (5 µg/ml, Diatek clone CRIS-7), GTP-Ras was detected as described previously (
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Results |
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DGK and RasGRP Physically Associate in the Cell
We showed previously that DGK could regulate cell proliferation by reducing DAG accumulation in the nucleus (
is found outside of the nucleus (
could also regulate growth-promoting DAG signals at the plasma membrane. One way that cells maintain the fidelity of signaling cascades is to organize appropriate signaling proteins into a complex. Such associations allow the activation of necessary effector molecules, while segregating them to avoid "cross-talk" between signaling pathways (
associates with RasGRP and regulates its activity by locally metabolizing DAG.
To determine whether DGK and RasGRP could associate with the same signaling complex, we cotransfected HEK293 cells with cDNA constructs, encoding DGK
with a FLAG epitope tag at its COOH terminus (DGK
-FLAG) and RasGRP with an NH2-terminal HA epitope tag (HA-RasGRP). We immunoprecipitated DGK
using anti-FLAG and detected RasGRP by immunoblotting. In these experiments, RasGRP coprecipitated with DGK
and their association was robust: >20% of HA-RasGRP coprecipitated (Fig 1 a). Alternatively, when we immunoprecipated RasGRP, DGK
coprecipitated (not shown). These experiments indicated that the two proteins associated with the same signaling complex. In additional experiments we could not detect an interaction between DGK
and two other Ras GEFs, Sos1 and RasGRF1, indicating that its association with RasGRP was selective (not shown). By examining mutants of DGK
, we mapped a region near the COOH terminus of the catalytic domain that substantially reduced coprecipitation (Fig 1 b), indicating that a motif in or near this region was necessary for DGK
to interact with the signaling complex.
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To assess whether endogenous RasGRP and DGK associate with the same signaling complex, we used A172 cells, a glioblastoma cell line that we have shown to express DGK
(
coimmunoprecipitated with RasGRP. We found that RasGRP immunoprecipitates had two times (±0.8; n = 3) more DGK activity than control immunoprecipitates where the antibody was preincubated with its affinity peptide. Using another anti-RasGRP antibody for the immunoprecipitation, we similarly found 2.2 times (±1.7; n = 4) more DGK activity in the precipitates compared with control. These data suggested that endogenous RasGRP and DGK
interacted with the same signaling complex in A172 cells. To determine if the presence of DAG regulated their interaction, we compared DGK activity in RasGRP immunoprecipitates from control A172 cells to cells treated with a phorbol ester, phorbol 12-myristate 13-acetate (PMA). We found in these experiments that compared with untreated cells, PMA almost doubled the amount of associated DGK activity (1.9 ± 0.7; n = 3; Fig 1 c). PMA did not enhance RasGRP precipitation (Fig 1 c), indicating that it increased its association with DGK
. Supporting this, we found by Western blotting that PMA treatment significantly enhanced coprecipitation of DGK
(Fig 1 d). These data demonstrate that endogenous DGK
and RasGRP interact and that their association is likely augmented in the presence of DAG.
DGK and RasGRP Colocalize
As an independent test to determine if RasGRP and DGK may interact in vivo, we assessed whether the endogenous proteins colocalized in A172 cells. Consistent with our previous observations, we found by indirect immunofluorescence and confocal microscopy that a fraction of DGK
was in the nucleus of the cells (not shown). We also observed marked localization of DGK
at the periphery of cell extensions, regions that also costained strongly for actin (Fig 2 a). We found that the distribution of RasGRP peripherally in actin-rich regions was very similar to that of DGK
(Fig 2 a). This suggested that the two proteins colocalized. Since both the anti-DGK
and anti-RasGRP antibodies were produced in rabbits, it was difficult to assess colocalization of the two proteins using indirect immunofluorescence. To allow simultaneous detection of both proteins, we cotransfected Cos-7 cells with GFP-RasGRP and DGK
and then immunostained the cells to assess localization of the overexpressed proteins. To augment cell spreading, we allowed them to spread on a surface coated with fibronectin and then immunostained for DGK
. When overexpressed, both proteins distributed throughout the cytoplasm and nucleus. But, consistent with the A172 cell immunostaining, both proteins also localized at the leading edge of spreading cells (Fig 2 b). As overexpression of proteins can lead to aberrant localization, we directly labeled the two antibodies with separate fluorophores, which allowed simultaneous detection of endogenous DGK
and RasGRP in A172 cells. Using confocal microscopy, we observed that DGK
and RasGRP extensively colocalized, most dramatically at cell extensions peripherally and at the leading edge of migrating cells (Fig 2 c). These results, coupled with our immunoprecipitation data, strongly indicated that DGK
and RasGRP associate with the same signaling complex in vivo.
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DGK Binds Selectively to A15-H-Ras
Ras GEFs promote the release of GDP from Ras and facilitate GTP binding. Inactive, mutant Ras proteins, like A15-Ras are thought to exert dominant negative effects because they have a high affinity for Ras GEFs and sequester them from endogenous Ras proteins ( would also associate with A15-Ras. To test this, we cotransfected HEK293 cells with DGK
-FLAG and A15-Ras and then immunoprecipitated DGK
. We found that A15-Ras coprecipitated with DGK
and that the interaction was robust: >20% of the total A15-Ras coprecipitated (Fig 3 a). We also observed the converse: immunoprecipitates of A15-Ras had DGK
activity (not shown). Further, when all three proteins, A15-Ras, RasGRP, and DGK
, were included in the transfection, both RasGRP and A15-Ras coprecipitated with DGK
(not shown), indicating that they associated with the same signaling complex.
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V12-Ras is a constitutively active mutant that, unlike A15-Ras, has a very low affinity for Ras GEFs ( and found that, compared with A15-Ras, it had a very low affinity for DGK
, even though cell lysates had substantially more V12-Ras (Fig 3 b). These results indicated that DGK
preferred to associate with signaling complexes containing inactive Ras. To test whether there is direct binding between H-Ras and DGK
, we incubated recombinant proteins in vitro and found that DGK
coprecipitated both GDP- and GTP-bound H-Ras with equal efficiency (not shown). DGK
's indifference in vitro for GTP-Ras versus GDP-Ras, but preference in vivo for A15-Ras, seemed contradictory. To further probe this issue, we tested the in vivo affinity of DGK
for wild-type H-Ras, which predominantly binds GDP (
's affinity for wild-type H-Ras was similar to its affinity for V12-Ras and much less than that for A15-Ras (not shown). Its preferential association with mutant, inactive H-Ras proteins, which sequester Ras GEFs, suggests that DGK
does not distinguish between GTP- or GDP-Ras in vivo, but instead prefers to associate with signaling complexes enriched in Ras GEFs. Consistent with this, the in vitro binding affinity between recombinant DGK
and H-Ras was much less than the affinity in vivo between DGK
and A15-Ras. So, although DGK
appears to physically, but weakly, bind H-Ras, it prefers to associate with the signaling complex, probably by binding to other proteins in the complex.
DGK Regulates RasGRP Activity
RasGRP has a DAG-responsive C1 domain that is necessary for its transforming activity ( associated with a signaling complex containing H-Ras and RasGRP to regulate the local DAG concentration and thus control Ras activity by regulating RasGRP. We first verified that RasGRP required its C1 domain for activity. Using an Elk-1 luciferase reporter (
could regulate RasGRP, we cotransfected H-Ras with a myc epitope tag (myc-Ras) along with RasGRP and wild-type DGK
or mutant, kinase-dead DGK
into HEK293 cells and then measured GTP-Ras by affinity precipitation (
significantly attenuated Ras activation induced by RasGRP (Fig 4 a). DGK activity was required for the inhibition: the kinase-dead DGK
(
ATP, G355D;
and it still coprecipitated RasGRP with equal efficiency (not shown).
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Regulation of RasGRP by DGK Is Spatially Discrete
The regulation of RasGRP by DGK is compatible with their patterns of tissue expression: both RasGRP and DGK
mRNA are highly expressed in brain and hematopoietic organs. However, DGK isotypes exhibit significant overlap in their expression patterns. In fact, one cell will often express two or three different DGK isotypes, often from different DGK subfamilies (
was due to wholesale metabolism of DAG or if it resulted from selective inhibition by DGK
and not other DGKs. We tested six different DGK isotypes (ß,
,
,
,
, and
) for inhibition by cotransfecting them with myc-Ras and RasGRP; we then used affinity precipitation to detect GTP-Ras. Each of the DGKs had an NH2-terminal HA epitope tag, so their protein expression levels were directly comparable by Western blotting. Although most of the isotypes have significantly higher expression than DGK
, only DGK
significantly attenuated RasGRP activity (Fig 4 b). Comparing in vivo DGK activity is not possible, and it is not clear whether in vitro DGK activity assays accurately reflect in vivo activity. But, using an in vitro assay system incorporating ideal conditions for most of the tested DGK isotypes, we found that activity levels roughly correlated with their protein expression levels in several experiments (not shown). In fact, DGK
often had four to five times more in vitro activity and protein expression than DGK
, but consistently increased RasGRP activity. Further, a DAG lipase (human lysosomal acid lipase), which metabolizes DAG by a different mechanism, did not significantly inhibit RasGRP activity (not shown). These results indicate that the inhibition of RasGRP could not be reproduced by globally manipulating cellular DAG levels and that the inhibition by DGK
must be spatially discrete.
The DGK gene has two splice variants (
2) where the initial 54 amino acids are replaced with a 262amino acid fragment. The alternative splicing appears to alter the subcellular localization of
2 (Topham, M.K., manuscript in preparation) and does not affect in vitro activity levels. Thus,
2 offered a unique opportunity to test whether the inhibition of RasGRP was spatially discrete and selective. Using either affinity precipitation of GTP-Ras or the Elk-1 luciferase system, we found that
2 did not significantly inhibit RasGRP (Fig 4 c), whereas the more common splice variant of DGK
did. Protein expression levels of the splice variants were virtually identical in these experiments as judged by Western blotting using a specific antibody that recognizes both proteins (not shown). This lack of inhibition by
2 was not owing to differences in DGK activity: both proteins had similar in vitro activity levels (Fig 4 c). We considered the possibility that the more common DGK
inhibited RasGRP because it more efficiently metabolized total cellular DAG. However, we found similar total DAG levels in the cell homogenates (Fig 4 C). This discrepancy, differential inhibition of RasGRP but similar total DAG levels, likely reflects technical constraints of the DAG assay. It detects only global cellular DAG. Quantitatively measuring precise, spatial changes in DAG accessible to the RasGRP signaling complex is not technically possible. However, we believe that our assays measuring active Ras indirectly detect these focal changes. We conclude that DGK
selectively inhibits RasGRP by a spatially discrete mechanism.
Inhibition of RasGRP Activity Occurs at the Level of Ras Activation
We observed that a mutant, kinase-dead DGK did not inhibit RasGRP, indicating that DGK activity was required for the inhibition and suggesting that this occurred through localized metabolism of DAG. To assure that the inhibition of Ras activity that we observed resulted from metabolism of DAG by DGK
, we reasoned that DGK
would not affect RasGRP activity induced by phorbol esters, which act as DAG analogues but cannot be metabolized by DGKs. We verified with the Elk-1 luciferase system that PMA, a phorbol ester, increased RasGRP activity. This activation was not reduced by PKC inhibitors (Fig 5 a), which demonstrated that the PMA was likely activating RasGRP. Supporting our hypothesis that DGK
inhibits RasGRP by metabolizing DAG, DGK
abolished RasGRP activity in the absence of PMA, but did not inhibit PMA-induced RasGRP activity (Fig 5 a).
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Activation of PKC isoforms can initiate mitogen-activated protein kinase signaling, but the precise mechanism of activation of the cascade is not clear ( was mediated through RasGRP, rather than PKC.
As an additional test to assure that the level of inhibition by DGK occurred at RasGRP, we reasoned that DGK
would not affect mitogen-activated protein kinase activation initiated downstream of Ras. To activate this signaling cascade without affecting Ras, we used a chimeric cDNA consisting of the hormone-binding domain of the estrogen receptor fused to an oncogenic form of Raf-1 (
did not affect Elk-1 luciferase activity induced by this construct (Fig 5 c). Consistent with this, we also observed that DGK
did not significantly inhibit luciferase activity induced by constitutively active forms of H-Ras (V12-Ras) or MEK1 (not shown). These data demonstrate that DGK
inhibits RasGRP rather than affecting a downstream event.
Kinase-dead DGK Prolongs Ras Signaling in Jurkat Cells
RasGRP is known to activate Ras and DAG is required for its activity. However, little is known of the physiological contexts in which this activation occurs. and RasGRP are overexpressed, the DGK attenuates RasGRP activity. For the endogenous proteins, this is likely a mechanism to terminate Ras signaling. Overexpression of inactive, mutant proteins can interfere with the physiological function of their endogenous, wild-type counterparts. So, to test if endogenous DGK
regulates RasGRP, we determined whether overexpression of mutant, kinase-dead DGK
affected Ras activation. In Jurkat cells, RasGRP signaling is initiated after activation of the TCR (
is expressed in these cells (not shown). To test if kinase-dead DGK
altered Ras signaling, we overexpressed it along with myc-Ras in Jurkat cells and then activated the TCR with an antibody for up to 4 h. Using GTP-Ras affinity precipitation, we consistently observed slightly higher basal GTP-Ras in cells overexpressing kinase-dead DGK
. After activation of the TCR, we found in control cells that GTP-Ras peaked between 5 and 10 min and then gradually declined to basal levels by 1 h. Conversely, in cells expressing kinase-dead DGK
, GTP-Ras peaked for up to 20 min and then gradually declined, but did not reach basal levels for >2 h. The kinase-dead DGK
likely prolonged Ras activation by interfering with the function of endogenous DGK
. We conclude that in Jurkat cells, endogenous DGK
facilitates termination of TCR signaling by regulating the activity of RasGRP.
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Discussion |
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Our observations support a novel mechanism of localized regulation of RasGRP by DGK. We found evidence that these proteins interacted using immunoprecipitations and we observed that they colocalized in a glioblastoma cell line. Interestingly, their localization was at the leading edge of migrating cells, an area of intense actin remodeling. The specific function of RasGRP in this region is not clear, but Ras activity has an integral role in cell motility (
inhibits RasGRP and that this is a highly localized event. The most direct evidence supporting this regulation as precise and spatial is the lack of inhibition by
2, the alternatively spliced DGK
isoform. This variant differs only at the NH2 terminus (
, coupled with our immunoprecipitation and immunofluorescence data, strongly indicates that H-Ras, RasGRP, and DGK
are spatially organized in a regulated signaling complex.
DGK Activity May Inhibit Cell Transformation
Ras activity must be precisely regulated or abnormal cellular proliferation can occur. Supporting this, an estimated 30% of human tumors have an activating mutation of Ras (1, which causes excess DAG, induced a malignant phenotype (
1 was a necessary component of growth factorinduced mitogenesis (
activity could cause inappropriate DAG signaling, leading to malignant changes by activating RasGRP. Indeed, we demonstrated that expression of inactive DGK
in Jurkat cells prolonged Ras activation after TCR ligation. Thus, by attenuating the DAG pool necessary to maintain RasGRP activity, DGK
may have a pivotal role in modulating Ras signaling in some contexts.
Advantages to Formation of a Signaling Complex
Associating with a DGK offers RasGRP three potential advantages in regulating signals. First, DGK activity allows the signal to activate RasGRP to be more spatially precise, because both the production (PLC) and inactivation (DGK) of DAG are controlled in the same location. Second, since the DGK regulates DAG independently of PLC, it offers a safety mechanism to reduce RasGRP activity in cases of abnormally high PLC activity. Third, formation of a signaling complex offers a kinetic advantage. Signaling events mediated through low molecular weight GTP-binding proteins like Ras and G proteins are generally short-lived to allow for rapid subsequent reactivation. Rapid on/off cycling is achieved by associating the GTP-binding protein with its GTPase-activating proteins (GAPs) and GEFs. For example, likely contributes to this mechanism by allowing rapid cycling of the activity of RasGRP. Extending this paradigm, RasGRP may be similarly regulated by associating with both a PLC (GEF) and a DGK (GAP).
DGKs May Have Diverse Roles in Signaling Complexes
Local regulation of DAG signaling by DGK isozymes may be a generalized mechanism to regulate DAG-activated proteins. Supporting this, , negatively regulated synaptic transmission (
Indicating a general role for DGKs as integral partners in signaling complexes, associated with a complex containing RhoA, and
Conclusions
We observed that DGK activity terminated RasGRP activation and that only one DGK isoform, DGK, could inhibit. Even an alternatively spliced DGK
isoform did not significantly affect RasGRP activity, demonstrating the specificity of this regulation. Furthermore, we found that endogenous DGK
and RasGRP colocalized in A172 cells, indicating that they likely associate with the same signaling complex. Supporting this, we demonstrated that DGK
and RasGRP coimmunoprecipitated and that deleting a region within the catalytic domain of DGK
eliminated their interaction. Phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, enhanced the interaction between DGK
and RasGRP, suggesting that their interaction was facilitated in the presence of DAG. DGK
also selectively coimmunoprecipitated with a mutant H-Ras protein, A15-Ras, that binds strongly to Ras GEFs. This suggests a model where the activity of RasGRP, and consequently Ras, is exquisitely regulated by the coordinated activity of PLC
1, which generates DAG, and DGK
, which terminates the signal (Fig 6). This may be a common mechanism to spatially regulate DAG and perhaps other lipid signals.
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Footnotes |
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1 Abbreviations used in this paper: CMV, cytomegalovirus; DGK, DAG kinase; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GRP, guanyl nucleotide-releasing protein (GRP); HA, hemagglutinin; PA, phosphatidic acid; PMA, phorbol 12-myristate 13-acetate; TCR, T cell receptor.
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Acknowledgements |
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We thank A. Thorburn for extensive discussions and T. Crotty, D. Roberts, D. Lim, H. Jiang, and H. Rust for technical assistance.
M.K. Topham was a Howard Hughes Medical Institute Physician Postdoctoral Fellow when this work was performed.
Submitted: 6 September 2000
Revised: 24 January 2001
Accepted: 25 January 2001
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References |
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