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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 and DGK colocalize. (a) A172 cells were immunostained for DGKζ (top) or RasGRP (bottom). Phalloidin was used to identify actin filaments. To assure that the immunostaining was specific, the antibodies were preincubated with their affinity peptide before staining the cells. Several areas of intense staining common to actin and DGKζ or RasGRP are indicated by the arrows. (b) Cos-7 cells were cotransfected with GFP-RasGRP and DGKζ. 24 h later, they were suspended and then allowed to spread for 30 min on glass slides coated with fibronectin. The cells were then immunostained to detect DGKζ (red), nuclei were counterstained (blue), and immunofluorescence images were obtained. (c) To view migrating cells, A172 cell monolayers were wounded with a pipet tip 12 h before immunostaining. Using antibodies directly conjugated with separate fluorophores, the cells were immunostained to detect RasGRP (green) and DGKζ (red) and then viewed by confocal microscopy (Bio-Rad Laboratories), and digital images were obtained. One representative cell is shown migrating into the wounded area, with arrows indicating overlapping localization that was also apparent in most cells. The boxed area is magnified in the lower panels to demonstrate colocalization at the leading edge. Bars, 10 μm.
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Figure 2: RasGRP and DGK colocalize. (a) A172 cells were immunostained for DGKζ (top) or RasGRP (bottom). Phalloidin was used to identify actin filaments. To assure that the immunostaining was specific, the antibodies were preincubated with their affinity peptide before staining the cells. Several areas of intense staining common to actin and DGKζ or RasGRP are indicated by the arrows. (b) Cos-7 cells were cotransfected with GFP-RasGRP and DGKζ. 24 h later, they were suspended and then allowed to spread for 30 min on glass slides coated with fibronectin. The cells were then immunostained to detect DGKζ (red), nuclei were counterstained (blue), and immunofluorescence images were obtained. (c) To view migrating cells, A172 cell monolayers were wounded with a pipet tip 12 h before immunostaining. Using antibodies directly conjugated with separate fluorophores, the cells were immunostained to detect RasGRP (green) and DGKζ (red) and then viewed by confocal microscopy (Bio-Rad Laboratories), and digital images were obtained. One representative cell is shown migrating into the wounded area, with arrows indicating overlapping localization that was also apparent in most cells. The boxed area is magnified in the lower panels to demonstrate colocalization at the leading edge. Bars, 10 μm.

Mentions: As an independent test to determine if RasGRP and DGKζ 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 DGKζ was in the nucleus of the cells (not shown). We also observed marked localization of DGKζ 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 DGKζ (Fig. 2 a). This suggested that the two proteins colocalized. Since both the anti-DGKζ 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 DGKζ 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 DGKζ. 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 DGKζ and RasGRP in A172 cells. Using confocal microscopy, we observed that DGKζ 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 DGKζ and RasGRP associate with the same signaling complex in vivo.


Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.

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

RasGRP and DGK colocalize. (a) A172 cells were immunostained for DGKζ (top) or RasGRP (bottom). Phalloidin was used to identify actin filaments. To assure that the immunostaining was specific, the antibodies were preincubated with their affinity peptide before staining the cells. Several areas of intense staining common to actin and DGKζ or RasGRP are indicated by the arrows. (b) Cos-7 cells were cotransfected with GFP-RasGRP and DGKζ. 24 h later, they were suspended and then allowed to spread for 30 min on glass slides coated with fibronectin. The cells were then immunostained to detect DGKζ (red), nuclei were counterstained (blue), and immunofluorescence images were obtained. (c) To view migrating cells, A172 cell monolayers were wounded with a pipet tip 12 h before immunostaining. Using antibodies directly conjugated with separate fluorophores, the cells were immunostained to detect RasGRP (green) and DGKζ (red) and then viewed by confocal microscopy (Bio-Rad Laboratories), and digital images were obtained. One representative cell is shown migrating into the wounded area, with arrows indicating overlapping localization that was also apparent in most cells. The boxed area is magnified in the lower panels to demonstrate colocalization at the leading edge. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 2: RasGRP and DGK colocalize. (a) A172 cells were immunostained for DGKζ (top) or RasGRP (bottom). Phalloidin was used to identify actin filaments. To assure that the immunostaining was specific, the antibodies were preincubated with their affinity peptide before staining the cells. Several areas of intense staining common to actin and DGKζ or RasGRP are indicated by the arrows. (b) Cos-7 cells were cotransfected with GFP-RasGRP and DGKζ. 24 h later, they were suspended and then allowed to spread for 30 min on glass slides coated with fibronectin. The cells were then immunostained to detect DGKζ (red), nuclei were counterstained (blue), and immunofluorescence images were obtained. (c) To view migrating cells, A172 cell monolayers were wounded with a pipet tip 12 h before immunostaining. Using antibodies directly conjugated with separate fluorophores, the cells were immunostained to detect RasGRP (green) and DGKζ (red) and then viewed by confocal microscopy (Bio-Rad Laboratories), and digital images were obtained. One representative cell is shown migrating into the wounded area, with arrows indicating overlapping localization that was also apparent in most cells. The boxed area is magnified in the lower panels to demonstrate colocalization at the leading edge. Bars, 10 μm.
Mentions: As an independent test to determine if RasGRP and DGKζ 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 DGKζ was in the nucleus of the cells (not shown). We also observed marked localization of DGKζ 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 DGKζ (Fig. 2 a). This suggested that the two proteins colocalized. Since both the anti-DGKζ 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 DGKζ 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 DGKζ. 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 DGKζ and RasGRP in A172 cells. Using confocal microscopy, we observed that DGKζ 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 DGKζ and RasGRP associate with the same signaling complex in vivo.

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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