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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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RasGRP coimmunoprecipitates with DGKζ. (a) DGKζ with or without a FLAG epitope tag was cotransfected into HEK293 cells along with HA-RasGRP. 48 h later, anti-FLAG antibodies were used to immunoprecipitate DGKζ. Normal mouse IgG was used as a control. After SDS-PAGE of the pellets or 10% of the lysates, RasGRP was detected by immunoblotting with anti-HA. The blot was then stripped and reprobed for DGKζ. (b) HA-RasGRP was transfected along with a control vector or progressive COOH-terminal truncations of DGKζ containing FLAG epitope tags. The DGK proteins were immunoprecipated with anti-FLAG and then coimmunoprecipitation of HA-RasGRP was detected by immunoblotting with polyclonal anti-HA. The blot was stripped and then the DGKs were detected using anti-DGKζ. The three DGK constructs shown encode amino acids 1–748, 1–605, or 1–467 with FLAG epitope tags at their COOH termini. (c) A172 cells, either control or treated with PMA (90 ng/ml, 30 min), were lysed and then RasGRP was immunoprecipitated using an affinity purified antibody. To verify the specificity of the immunoprecipitation, the antibody was preincubated with its affinity peptide or a control peptide before the immunoprecipitation. The precipitates were then used either for Western blotting to detect RasGRP (top) or for DGK activity assays (bottom). (d) RasGRP was immunoprecipitated from control or PMA-treated A172 cells as described in the legend to c and then the precipitates were used for Western blotting to detect coimmunoprecipitation of DGKζ.
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Figure 1: RasGRP coimmunoprecipitates with DGKζ. (a) DGKζ with or without a FLAG epitope tag was cotransfected into HEK293 cells along with HA-RasGRP. 48 h later, anti-FLAG antibodies were used to immunoprecipitate DGKζ. Normal mouse IgG was used as a control. After SDS-PAGE of the pellets or 10% of the lysates, RasGRP was detected by immunoblotting with anti-HA. The blot was then stripped and reprobed for DGKζ. (b) HA-RasGRP was transfected along with a control vector or progressive COOH-terminal truncations of DGKζ containing FLAG epitope tags. The DGK proteins were immunoprecipated with anti-FLAG and then coimmunoprecipitation of HA-RasGRP was detected by immunoblotting with polyclonal anti-HA. The blot was stripped and then the DGKs were detected using anti-DGKζ. The three DGK constructs shown encode amino acids 1–748, 1–605, or 1–467 with FLAG epitope tags at their COOH termini. (c) A172 cells, either control or treated with PMA (90 ng/ml, 30 min), were lysed and then RasGRP was immunoprecipitated using an affinity purified antibody. To verify the specificity of the immunoprecipitation, the antibody was preincubated with its affinity peptide or a control peptide before the immunoprecipitation. The precipitates were then used either for Western blotting to detect RasGRP (top) or for DGK activity assays (bottom). (d) RasGRP was immunoprecipitated from control or PMA-treated A172 cells as described in the legend to c and then the precipitates were used for Western blotting to detect coimmunoprecipitation of DGKζ.

Mentions: To determine whether DGKζ and RasGRP could associate with the same signaling complex, we cotransfected HEK293 cells with cDNA constructs, encoding DGKζ with a FLAG epitope tag at its COOH terminus (DGKζ-FLAG) and RasGRP with an NH2-terminal HA epitope tag (HA-RasGRP). We immunoprecipitated DGKζ using anti-FLAG and detected RasGRP by immunoblotting. In these experiments, RasGRP coprecipitated with DGKζ and their association was robust: >20% of HA-RasGRP coprecipitated (Fig. 1 a). Alternatively, when we immunoprecipated RasGRP, DGKζ 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 DGKζ and two other Ras GEFs, Sos1 and RasGRF1, indicating that its association with RasGRP was selective (not shown). By examining mutants of DGKζ, 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 DGKζ to interact with the signaling complex.


Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.

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

RasGRP coimmunoprecipitates with DGKζ. (a) DGKζ with or without a FLAG epitope tag was cotransfected into HEK293 cells along with HA-RasGRP. 48 h later, anti-FLAG antibodies were used to immunoprecipitate DGKζ. Normal mouse IgG was used as a control. After SDS-PAGE of the pellets or 10% of the lysates, RasGRP was detected by immunoblotting with anti-HA. The blot was then stripped and reprobed for DGKζ. (b) HA-RasGRP was transfected along with a control vector or progressive COOH-terminal truncations of DGKζ containing FLAG epitope tags. The DGK proteins were immunoprecipated with anti-FLAG and then coimmunoprecipitation of HA-RasGRP was detected by immunoblotting with polyclonal anti-HA. The blot was stripped and then the DGKs were detected using anti-DGKζ. The three DGK constructs shown encode amino acids 1–748, 1–605, or 1–467 with FLAG epitope tags at their COOH termini. (c) A172 cells, either control or treated with PMA (90 ng/ml, 30 min), were lysed and then RasGRP was immunoprecipitated using an affinity purified antibody. To verify the specificity of the immunoprecipitation, the antibody was preincubated with its affinity peptide or a control peptide before the immunoprecipitation. The precipitates were then used either for Western blotting to detect RasGRP (top) or for DGK activity assays (bottom). (d) RasGRP was immunoprecipitated from control or PMA-treated A172 cells as described in the legend to c and then the precipitates were used for Western blotting to detect coimmunoprecipitation of DGKζ.
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Figure 1: RasGRP coimmunoprecipitates with DGKζ. (a) DGKζ with or without a FLAG epitope tag was cotransfected into HEK293 cells along with HA-RasGRP. 48 h later, anti-FLAG antibodies were used to immunoprecipitate DGKζ. Normal mouse IgG was used as a control. After SDS-PAGE of the pellets or 10% of the lysates, RasGRP was detected by immunoblotting with anti-HA. The blot was then stripped and reprobed for DGKζ. (b) HA-RasGRP was transfected along with a control vector or progressive COOH-terminal truncations of DGKζ containing FLAG epitope tags. The DGK proteins were immunoprecipated with anti-FLAG and then coimmunoprecipitation of HA-RasGRP was detected by immunoblotting with polyclonal anti-HA. The blot was stripped and then the DGKs were detected using anti-DGKζ. The three DGK constructs shown encode amino acids 1–748, 1–605, or 1–467 with FLAG epitope tags at their COOH termini. (c) A172 cells, either control or treated with PMA (90 ng/ml, 30 min), were lysed and then RasGRP was immunoprecipitated using an affinity purified antibody. To verify the specificity of the immunoprecipitation, the antibody was preincubated with its affinity peptide or a control peptide before the immunoprecipitation. The precipitates were then used either for Western blotting to detect RasGRP (top) or for DGK activity assays (bottom). (d) RasGRP was immunoprecipitated from control or PMA-treated A172 cells as described in the legend to c and then the precipitates were used for Western blotting to detect coimmunoprecipitation of DGKζ.
Mentions: To determine whether DGKζ and RasGRP could associate with the same signaling complex, we cotransfected HEK293 cells with cDNA constructs, encoding DGKζ with a FLAG epitope tag at its COOH terminus (DGKζ-FLAG) and RasGRP with an NH2-terminal HA epitope tag (HA-RasGRP). We immunoprecipitated DGKζ using anti-FLAG and detected RasGRP by immunoblotting. In these experiments, RasGRP coprecipitated with DGKζ and their association was robust: >20% of HA-RasGRP coprecipitated (Fig. 1 a). Alternatively, when we immunoprecipated RasGRP, DGKζ 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 DGKζ and two other Ras GEFs, Sos1 and RasGRF1, indicating that its association with RasGRP was selective (not shown). By examining mutants of DGKζ, 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 DGKζ to interact with the signaling complex.

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
Related in: MedlinePlus