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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.

Show MeSH
Proposed mechanism for precise regulation of RasGRP activity. After TCR stimulation, PLCγ1 is activated and initiates DAG signaling. One protein activated by the DAG is RasGRP. DAG accumulation also recruits DGKζ to the RasGRP signaling complex, where it attenuates RasGRP activity by converting the DAG to PA.
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Figure 6: Proposed mechanism for precise regulation of RasGRP activity. After TCR stimulation, PLCγ1 is activated and initiates DAG signaling. One protein activated by the DAG is RasGRP. DAG accumulation also recruits DGKζ to the RasGRP signaling complex, where it attenuates RasGRP activity by converting the DAG to PA.

Mentions: We observed that DGK activity terminated RasGRP activation and that only one DGK isoform, DGKζ, could inhibit. Even an alternatively spliced DGKζ isoform did not significantly affect RasGRP activity, demonstrating the specificity of this regulation. Furthermore, we found that endogenous DGKζ and RasGRP colocalized in A172 cells, indicating that they likely associate with the same signaling complex. Supporting this, we demonstrated that DGKζ and RasGRP coimmunoprecipitated and that deleting a region within the catalytic domain of DGKζ eliminated their interaction. Phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, enhanced the interaction between DGKζ and RasGRP, suggesting that their interaction was facilitated in the presence of DAG. DGKζ 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 PLCγ1, which generates DAG, and DGKζ, which terminates the signal (Fig. 6). This may be a common mechanism to spatially regulate DAG and perhaps other lipid signals.


Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.

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

Proposed mechanism for precise regulation of RasGRP activity. After TCR stimulation, PLCγ1 is activated and initiates DAG signaling. One protein activated by the DAG is RasGRP. DAG accumulation also recruits DGKζ to the RasGRP signaling complex, where it attenuates RasGRP activity by converting the DAG to PA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199204&req=5

Figure 6: Proposed mechanism for precise regulation of RasGRP activity. After TCR stimulation, PLCγ1 is activated and initiates DAG signaling. One protein activated by the DAG is RasGRP. DAG accumulation also recruits DGKζ to the RasGRP signaling complex, where it attenuates RasGRP activity by converting the DAG to PA.
Mentions: We observed that DGK activity terminated RasGRP activation and that only one DGK isoform, DGKζ, could inhibit. Even an alternatively spliced DGKζ isoform did not significantly affect RasGRP activity, demonstrating the specificity of this regulation. Furthermore, we found that endogenous DGKζ and RasGRP colocalized in A172 cells, indicating that they likely associate with the same signaling complex. Supporting this, we demonstrated that DGKζ and RasGRP coimmunoprecipitated and that deleting a region within the catalytic domain of DGKζ eliminated their interaction. Phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, enhanced the interaction between DGKζ and RasGRP, suggesting that their interaction was facilitated in the presence of DAG. DGKζ 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 PLCγ1, which generates DAG, and DGKζ, which terminates the signal (Fig. 6). This may be a common mechanism to spatially regulate DAG and perhaps other lipid signals.

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