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Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.

Topham MK, Prescott SM - J. Cell Biol. (2001)

Bottom Line: Coimmunoprecipitation of DGK zeta 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 zeta 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 zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.

View Article: PubMed Central - PubMed

Affiliation: The Huntsman Cancer Institute and Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84112, USA.

ABSTRACT
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 zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta 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 zeta 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 zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.

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DGKζ inhibits RasGRP at the level of Ras activation. Elk-1 activity was detected by a luciferase reporter (Stratagene). In all cases, luciferase activity in the cell lysates was determined in triplicate and normalized to β-galactosidase activity, which was included in the transfection. (a) HEK293 cells were transfected with GFP or GFP-RasGRP. 16 h later, a PKC inhibitor (PKCi; 100 nM Ro-32-0432) or control vehicle was added. After 20 min, PMA (0.5 ng/ml) was added for 24 h and then luciferase and β-galactosidase activities in the cell lysates were determined. Shown are the mean and standard deviation. (b) HEK293 cells were transfected with GFP or GFP-RasGRP along with a control vector or DGKζ. 16 h later, one of two PKC inhibitors (a, 200 nM Ro-31-7549; b, 100 nM Ro-32-0432) or control vehicle was added. After 24 h, the cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (c) Raf:ER or a control vector was transfected into HEK293 cells along with DGKζ, ΔATP, or a control vector. 24 h later, estrogen (1 μM) or control vehicle was added for 10 h. The cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (d) Jurkat cells were transfected by electroporation with myc-Ras and either GFP or kinase-dead DGKζ (ΔATP). 48 h later, TCR signaling was activated with anti-CD3 (5 μg/ml, CRIS-7) for up to 4 h. GTP-Ras was affinity-precipitated and then detected by immunoblotting.
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Figure 5: DGKζ inhibits RasGRP at the level of Ras activation. Elk-1 activity was detected by a luciferase reporter (Stratagene). In all cases, luciferase activity in the cell lysates was determined in triplicate and normalized to β-galactosidase activity, which was included in the transfection. (a) HEK293 cells were transfected with GFP or GFP-RasGRP. 16 h later, a PKC inhibitor (PKCi; 100 nM Ro-32-0432) or control vehicle was added. After 20 min, PMA (0.5 ng/ml) was added for 24 h and then luciferase and β-galactosidase activities in the cell lysates were determined. Shown are the mean and standard deviation. (b) HEK293 cells were transfected with GFP or GFP-RasGRP along with a control vector or DGKζ. 16 h later, one of two PKC inhibitors (a, 200 nM Ro-31-7549; b, 100 nM Ro-32-0432) or control vehicle was added. After 24 h, the cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (c) Raf:ER or a control vector was transfected into HEK293 cells along with DGKζ, ΔATP, or a control vector. 24 h later, estrogen (1 μM) or control vehicle was added for 10 h. The cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (d) Jurkat cells were transfected by electroporation with myc-Ras and either GFP or kinase-dead DGKζ (ΔATP). 48 h later, TCR signaling was activated with anti-CD3 (5 μg/ml, CRIS-7) for up to 4 h. GTP-Ras was affinity-precipitated and then detected by immunoblotting.

Mentions: We observed that a mutant, kinase-dead DGKζ 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 DGKζ, we reasoned that DGKζ 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 DGKζ inhibits RasGRP by metabolizing DAG, DGKζ abolished RasGRP activity in the absence of PMA, but did not inhibit PMA-induced RasGRP activity (Fig. 5 a).


Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.

Topham MK, Prescott SM - J. Cell Biol. (2001)

DGKζ inhibits RasGRP at the level of Ras activation. Elk-1 activity was detected by a luciferase reporter (Stratagene). In all cases, luciferase activity in the cell lysates was determined in triplicate and normalized to β-galactosidase activity, which was included in the transfection. (a) HEK293 cells were transfected with GFP or GFP-RasGRP. 16 h later, a PKC inhibitor (PKCi; 100 nM Ro-32-0432) or control vehicle was added. After 20 min, PMA (0.5 ng/ml) was added for 24 h and then luciferase and β-galactosidase activities in the cell lysates were determined. Shown are the mean and standard deviation. (b) HEK293 cells were transfected with GFP or GFP-RasGRP along with a control vector or DGKζ. 16 h later, one of two PKC inhibitors (a, 200 nM Ro-31-7549; b, 100 nM Ro-32-0432) or control vehicle was added. After 24 h, the cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (c) Raf:ER or a control vector was transfected into HEK293 cells along with DGKζ, ΔATP, or a control vector. 24 h later, estrogen (1 μM) or control vehicle was added for 10 h. The cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (d) Jurkat cells were transfected by electroporation with myc-Ras and either GFP or kinase-dead DGKζ (ΔATP). 48 h later, TCR signaling was activated with anti-CD3 (5 μg/ml, CRIS-7) for up to 4 h. GTP-Ras was affinity-precipitated and then detected by immunoblotting.
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Figure 5: DGKζ inhibits RasGRP at the level of Ras activation. Elk-1 activity was detected by a luciferase reporter (Stratagene). In all cases, luciferase activity in the cell lysates was determined in triplicate and normalized to β-galactosidase activity, which was included in the transfection. (a) HEK293 cells were transfected with GFP or GFP-RasGRP. 16 h later, a PKC inhibitor (PKCi; 100 nM Ro-32-0432) or control vehicle was added. After 20 min, PMA (0.5 ng/ml) was added for 24 h and then luciferase and β-galactosidase activities in the cell lysates were determined. Shown are the mean and standard deviation. (b) HEK293 cells were transfected with GFP or GFP-RasGRP along with a control vector or DGKζ. 16 h later, one of two PKC inhibitors (a, 200 nM Ro-31-7549; b, 100 nM Ro-32-0432) or control vehicle was added. After 24 h, the cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (c) Raf:ER or a control vector was transfected into HEK293 cells along with DGKζ, ΔATP, or a control vector. 24 h later, estrogen (1 μM) or control vehicle was added for 10 h. The cells were harvested and luciferase and β-galactosidase activities in the cell lysates were determined. (d) Jurkat cells were transfected by electroporation with myc-Ras and either GFP or kinase-dead DGKζ (ΔATP). 48 h later, TCR signaling was activated with anti-CD3 (5 μg/ml, CRIS-7) for up to 4 h. GTP-Ras was affinity-precipitated and then detected by immunoblotting.
Mentions: We observed that a mutant, kinase-dead DGKζ 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 DGKζ, we reasoned that DGKζ 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 DGKζ inhibits RasGRP by metabolizing DAG, DGKζ abolished RasGRP activity in the absence of PMA, but did not inhibit PMA-induced RasGRP activity (Fig. 5 a).

Bottom Line: Coimmunoprecipitation of DGK zeta 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 zeta 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 zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.

View Article: PubMed Central - PubMed

Affiliation: The Huntsman Cancer Institute and Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84112, USA.

ABSTRACT
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 zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta 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 zeta 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 zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.

Show MeSH